ANALYSIS OF MURINE HOMEOBOX GENES

ANTHONY GRAHAM

Thesis for the Degree of Doctor of Philosophy

Division of Eukaryotic Molecular Genetics National In s titu te fo r Medical Research The Ridgeway, M ill H ill London NW7 1AA

Univeristy College London Gower Street London WC1E 6BT

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i i The work in this thesis is original except where stated. The sequences used for comparisons in Chapter 3 were not determined by the author but were either from publications from other laboratories or from other members of the laboratory.

Anthony Graham: ABSTRACT The work in this thesis is consistent with , and strengthens, suggestions that murine homeobox genes play a role in the establishment of regional specification. It is shown that there is a correlation between the physical order of members of the Hox 2 cluster and their order of expression along the rostrocaudal axis, such that each more 3' gene is expressed more rostrally. Such a correlation has been previously shown for the Drosophila homeotic genes o f the ANT-C and BX-C, where i t was clear that these genes were involved in the establishment of regional identity along the rostrocaudal axis. It has also been demonstrated that the relationship between the murine and Drosophila clusters extends further than aspects of their expression patterns. Sequence comparisons suggest that the murine Hox clusters and the Drosophila homeobox clusters arose from a common ancestral cluster that existed prior to the divergence between the protostomes and thejdeuterostomes, jheref0re the corresponding utilisation of ordered region specific expression of members of homeobox clusters in flie s and mice together with their conservation would suggest that these genes are involved in the establishment of rostrocaudal regional specification in these organisms, and by inference in all other higher metazoans. I t is also demonstrated that the Hox 2 genes show s p a tia lly and temporally dynamic patterns of expression in the transverse plane of the developing central nervous system. Since a ll of the Hox 2 genes e xh ib it similar expression patterns it is fe lt they are reflecting events during ontogeny of the spinal cord. The observed patterns correlate with the timing and location of the b irth of the major classes of neurons. I t is suggested that the Hox 2 genes are conferring rostrocaudal positional information on each class of newly born neurons. These genes also e xh ib it a s trik in g dorsal restriction in their expression within the developing spinal cord which does not appear to correlate with morphology and eventually breaks down. ACKNOWLEDGEMENTS Firstly, I would like to thank my external supervisor Dr. Robb Krumlauf for all of his excellent help, encouragement and guidance during the course of this work. I also thank Dr. David Latchman (University College, London) for efficiently fu lfillin g his role as my University supervisor. This research was carried out at the National Institute for Medical Research and was supported by the MRC. I wish to acknowledge the assistance provided by the NIMR photography section. I also thank many of the past and present members of NIMR, particularly o f the laboratories of Molecular Embryology and Embryogenesis, fo r a ll of their help and friendship. Particular thanks to a few gurus - Jonathan Cooke, / Peter Holland, Malcolm Maden and DennisSummerbell who have been an invaluable source of information and discussion . Jeremy Green and Peter Vize have been wonderful proof readers, spotting many a ridiculous mistake, and who have also come up with more than a few smart ideas. Extra thanks go to Nancy Papalopulu, my long suffering lab mate and friend, who has helped immensely throughout the period of this work both technically and intellectually and without whom I don't know what I would have done. I also wish to thank Pal Njolstad ,the zebrafish man, who has generously supplied much unpublished information. Cheers Big Man, by the way - Sheila, Kathleen, Gerard, P h ilip , Brian ,Carol and Dan and not to forget Sandy for many an interesting biological discussion. Finally thanks to mum and dad for everything for the past many years.

iv CONTENTS Page No. T itle Page i

Dedication ii

Abstract iii

Acknowledgements iv

Contents v

List of Figures x

Abbreviations xiii

References 110

Chapter One : Introduction 1

1.1 Regional specification. 1

1.2 A summary of mouse embryogenesis. 3 1.2.1 Pre-implantation development. 4 1.2.2 Gastrulation and the generation of the body plan. 5

1.3 Strategies towards a molecular understanding of murine development. 9

1.4 Regional specification in Drosophila embryogenesis. 11 1.4.1 The genetics of anteroposterior specification. 11 1.4.2 The genetics of dorsoventral specification. 14 1.4.3 Neurogenesis. 15

1.5 The homeobox. 16

Chapter Two : Material and Methods 19

v 2.1 Buffers and stock solutions. 19

2.2 Bacterial media and antibiotics. 22

2.3 Animals. 22

2.4 Cloning and DNA methods. 22 2.4.1 Bacterial strain. 22 2.4.2 The production of cells competent for transformation. 23 2.4.3 Bacterial transformation. 23 2.4.4 Standard DNA methods. 23 2.4.5 Restriction digests. 24 2.4.6 Agarose gel electrophoresis. 24 2.4.7 Isolation of DNA fragments from gels. 24 2.4.8 Sub-cloning and ligations. 24 2.4.9 Isolation of plasmid DNA- mini prep. 25 2.4.10 Isolation of plasmid DNA- maxi prep. 25 2.4.11 Southern b lo t. 26 2.4.12 Colony screen. 26

2.5 RNA methods. 27 2.5.1 In vitro transcription. 27 2.5.2 Agarose formaldehyde gels. 28 2.5.3 Northern blot. 28 2.5.4 In situ hybridisation. 28

2.6 Sequence comparisons. 30 2.6.1 Sequences used fo r comparisons. 30

2.7 The Hox 3.1 probe 31

Chapter Three: The murine and Drosophila clusters are evolutionary related. 32

3.1 Introduction. 32

3.2 The mouse Hox clusters are related, possibly by duplication. 35 vi 3.3 S im ila ritie s between mouse Hox complexes and those of other vertebrates. 38

3.4 The mouse and Drosophila homeobox gene complexes have common features of organisation. 39

3.5 A common ancestor fo r the mouse and Drosophila homeobox gene complexes. 41

Chapter 4 : Expression of Hox 2 genes along the rostrocaudal axis of the mouse embryo. 45

4.1 General features of murine homeobox gene expression. 45

4.2 Relationship between the clustering of homeoboxes and their expression. 48

4.3 The Hox 2 probes used in in situ hybridisation. 50

4.4 Ordered expression of Hox 2 genes in the central nervous system (CNS). 51

4.5 Correlation between gene order and expression is also evident in other ectodermal derivatives. 56

4.6 Ordered differential expression of Hox 2 genes in mesodermal derivatives. 60

4.7 Discussion: Position of genes within a cluster reflect domains of expression along the rostrocaudal axis for both mouse and Drosophila. 65 4.7.1 Ordered expression in ectoderm. 65 4.7.2 Hox 2 gene expression in the hindbrain and rhombomeres. 66 4.7.3 Hox 2 gene expression in the mesoderm. 68 4.7.4 The anterior boundaries in mesoderm and ectoderm are not coincident. 70 4.7.5 Correlation between gene order and expression is a general aspect of homeobox clusters. 70 vi i Chapter Five : Dynamic Hox 2 gene expression during development of the central nervous system. 72

5.1 Introduction 72

5.2 Hox 2 genes are dorsally restricted at 12.5 days 73

5.3 Hox 2 genes are expressed uniformly across the nerve cord at 10.5 days. 79

5.4 Strong lateral expression of Hox 2 genes at 11.5 days. 79

5.5 Expression of Hox 2 genes reappears ve n tra lly at 14.5 days. 83

5.6 Hox 3.1 is expressed in stripes. 83

5.7 Discussion 86 5.7.1 Hox 2 gene expression correlates with the time and place of the birth of the major classes of spinal neurons. 87 5.7.2 The Hox 2 genes and Hox 3.1 must be responding to d iffe re n t signals. 88 5.7.3 Re-expression ve n tra lly and the sharp dorsal demarcation in the expression patterns. 89

Chapter Six : General discussion 92

6.1 Positions of genes in a cluster reflect domains of expression in the rostrocaudal axis for both mouse and Drosophila. 92

6.2 Evolution of homeobox gene clusters and the evolution of the body plan. 93

6.3 Relationships between the expression patterns of genes 95 from related murine Hox clusters.

6.4 Hox 2 gene expression and the vertebrate head. 96 vi i i 6.5 Hox genes are involved in the interpretation of regional 97 cues.

6.6 Differences between Drosophila and mouse development that 97 could preclude the use of identical genetic mechanisms.

6.7 Molecules that could organise Hox gene expression in vertebrates. 99 6.7.1 Growth factors and regional specification in Xenopus embryogenesis. 99 6.7.2 Retinoic acid and rostrocaudal regional specification. 100 6.7.3 Retinoic acid and homeobox gene expression. 101

6.8 Other roles fo r homeobox genes. 102

6.9 Homeobox target genes. 103

6.10 future experimental approaches to homeobox gene function. 104 6.10.1 Tissue grafts and Hox gene expression. 104 6.10.2 Generation of gain of function mutants. 105 6.10.3 Generation of loss of function mutants. 108

ix LIST OF FIGURES Chapter 3

3.1 -The organisation of the Hox 2 cluster of 9 murine homeobox genes and a diagrammatic representation of the Hox 2.6 gene structure.

3.2 -Gap alignment to identify regions of identity between different protein sequences.

3.3 -Amino acid sequence comparisons o f the mouse and Drosophila homeodomains showing the existence of related subgroups.

3.4 -A schematic alignment of the Hox 1 and Hox 2 clusters.

3.5 -Comparison between three related murine homeodomain proteins.

3.6 -Generalised structure showing characteristic features and conserved regions of a typical vertebrate homeodomain protein.

3.7 -Diagrammatic representation of the relationship between Hox 2.6 and the Drosophila Dfd.

3.8 - Representation of the relationship between Drosophila and murine homeobox gene clusters.

3.9 - A possible scheme for the evolution of the Drosophila and murine clusters from a common ancestral cluster.

Chapter 4

4.1 - In situ hybridisation of the Hox 2.2 gene in the 12.5 day mouse embryo.

4.2 - Schematic representation of the fate map of a Drosophila blastoderm stage embryo.

4.3 - Comparisons of the patterns of expression for Hox 2.5, Hox 2.4 and Hox 2.2 in the 12.5 day mouse embryo. x 4.4 - Anterior boundaries of expression in the central nervous system for six members of the Hox 2 cluster as revealed by in situ hybridisation.

4.5 - Patterns of expression of the Hox 2 genes that show the most a nterior lim its o f expression in the central nervous system.

4.6 - Diagrammatic representation of the anterior boundaries of expression in the central nervous system fo r the Hox 2 clu ste r, and their correlation with gene position in the cluster.

4.7 - Differential expression of members of Hox 2 in the vagal/glossopharyngeal nerve complex.

4.8 - Expression of Hox 2 genes in some neural crest derivatives.

4.9 - Anterior boundaries of expression of three members of Hox 2 in the prevertebrae.

4.10 - Differential expression of members of Hox 2 in the lung of the 12.5 day mouse embryo.

4.11 - Expression patterns of members of the Hox 2 clu ste r in the stomach and gut of a 14.5 day mouse embryo.

4.12 - Mesonephric tubules of the 10.5 day mouse embryo displaying expression of the Hox 2.4, Hox 2.2 and Hox 2.6 genes.

4.13 - Expression of the Hox 2 genes in the metanephric kidney, developing gonad and adrenal gland of a 14.5 day embryo.

Chapter 5

5.1 - Expression patterns of four members of Hox 2 at five different points along the rostrocaudal axis of a 12.5 day mouse embryo.

5.2 - The patterns of expression for seven members of the Hox 2 cluster xi in the 10.5 day nerve cord.

5.3 - Changes in expression pattern of Hox 2.2 in the spinal cord between 10.5 and 11.5 days of gestation.

5.4 - Transverse sections of the 11.5 day, mouse nerve cord probed with Hox 2 genes.

5.5 - Expression of three Hox 2 genes at three different points along the rostrocaudal axis of the 14.5 day mouse embryo.

5.6 - A comparison between the expression patterns of the Hox 2.5 and the Hox 3.1 genes in the caudal 12.5 day mouse spinal cord.

xii ABBREVIATIONS

ANT-C Antennapedia Complex ATP Adenosine triphosphate BSA Bovine Serum Albumin BX-C Bithorax Complex cDNA Complementary Dexoribonucleic acid CNS Central Nervous System DEPC Diethyl Pyrocarbonate DNA Deoxyribonucleic acid DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid ES Embryonal Stem EtBr Ethidium Bromide FGF Fibroblast Growth Factor I CM Inner Cell Mass b-Me b-Mercaptoethanol mRNA Messenger Ribonucleic Acid MOPS Morpholinopropanesulfonic Acid NaPB Sodium Phosphate Buffer O.D. Optical Density Poly A Polyadenylic Acid Poly C Polycytidylic Acid RNA Ribonucleic Acid SDS Sodium Dodecyl Sulphate SSC Standard Sodium C itrate TAE Tris Acetate EDTA TBE Tris Borate EDTA TE Tris-EDTA TESPA 3-Ami nopropyltri ethoxys i 1ane TGF Transforming Growth Factor TRIS Tris(hydroxymethyl)methyl amine t-RNA Transfer Ribonucleic acid UTP Uridine Triphosphate XTC-MIF XTC-Mesoderm inducing facto r

xiii CHAPTER ONE - INTRODUCTION

1.1 Regional Specification A core problem in development is that of regional specification: how the correct structures are formed in the correct place at the correct time. The process of cellular differentiation, which determines the synthesis of macromolecules that characterise a p a rtic u la r ce ll type, and that of ce ll movement, which is concerned with a change in form, are thought to be secondary (Wolpert, 1971). The role of regional specification would be in ensuring that the appropriate cellular differentiation occurs in the right place. For example the cellular differentiation of the cartilage is most probably the same in the forelimb and hindlimb but its spatial organisation is d iffe re n t (Wolpert, 1971). This is also observed with the process of ce ll movement where again the importance of regional specification would be in determining what cells w ill move or generate the forces necessary for a given change in shape (Wolpert, 1971). The body plan/parts of an animal are not specified all at once but are thought to re su lt from a hierarchy of developmental decisions (Slack, 1983). I t has been suggested that, during embryogenesis, at any given point there are a small number of decisions available to a c e ll, each of which is irre v e rs ib le , under normal conditions. For example, fo r c e lls to be competent to form neural tissue they would have to decide to be ectoderm, rather than mesoderm or endoderm, and then to be neural rather than epidermal. It is fe lt that the outcome of any decision w ill not generally be a terminally differentiated state but rather a new state of determination which, if the decisions are irreversible, would result in a reduction in potency as compared with the previous state (Slack, 1983). One could break regional specification into two independent processes: an in stru ctive event during which the positional information is imparted, and, an interpretation by the competent tissue (Wolpert, 1971). Slack (1983) has suggested that there are two general processes which could account fo r the instructive signal: cytoplasmic localisation, and induction. Cytoplasmic lo ca lisa tio n would be the process where, as the embryo divides, each of the daughter cells receive different cytoplasms and/or different membranes that cause them to undertake different paths of development. In contrast induction would involve, a competent tissue becoming d iffe re n tia lly determined in response to a chemical signal or interaction with ce lls or matrix from other regions of the embryo. The interpretation would be the process through which the positional information is made apparent and would probably directly or

1 indirectly involve differential gene activation/repression (Kauffman, 1973, Wolpert and Stein, 1984). An important implication of regional specification is that cells of the same histological type could be in different states of determination i.e. they would be non-equivalent (Lewis and Wolpert, 1976). Again using the vertebrate limb as an example these authors have suggested that c e lls in the fore limb and hind limb can both differentiate as cartilage, however despite the similar ca rtila ge phenotype the c e lls would be non-equivalent in terms o f th e ir positional values. I t is suggested that th is difference would eventually manifest its e lf in growth or surface properties such as between the wrist, and the radius and ulna (Lewis and Wolpert, 1976). The theory of non-equivalence also implies that cells carry internal records of past external influences and that these records may govern the response of ce lls to present external influences. In support of this Wolpert and Stein (1984) cite an experiment in which a piece of prospective thigh region was transplanted to the tip of the wing bud. This transplanted material developed into a toe and even induced the overlying ectoderm to form a covering o f scales and claws. This experiment was interpreted as demonstrating that the transplanted material carried a leg label and remembered its histo ry when i t was transplanted to the wing bud. The nature of these records is thought of in terms of a gene control network. This network would confer identity on the cells through different combinations of genes which may be on or o ff (Lewis and Wolpert, 1976). In terms of regional specification one could think of the differential activity of such a network as being part of the interpretation machinery of the cells which responded to the positional information. The record of non­ equivalence that each cell possesses could be in the combinations of gene a c tiv ity /in a c tiv ity which remain a fte r the in it ia l positional information has been imparted. It is interesting to consider the evolutionary aspects of regional specification, such as the phylogenetic origin of the apparatus of regional specification and its degree of conservation. It is felt that there will be some degree of generality in the biochemical machinery underlying regional specification which may be in the positional signal(s) and/or in the apparatus of interpretation (Slack, 1983). A number of lines of evidence support this idea. Firstly, a mouse polarising region grafted to the anterior portion of a chick limb bud results in the specification of additional digits. It seems possible that the signal for organising the anteroposterior axis of the limb may be conserved in a ll amniotes (Fallon and Crosby, 1977). The neural tube which arises from dorsal ectoderm in a ll vertebrates is another case in that

2 there would seem to be evidence from both chick and amphibia that i t is formed as a re s u lt o f induction from the underlying mesoderm (Spemann, 1938, Grabowski, 1957). These examples have dealt with vertebrates which represent only about 5% of the known species of animal and as such are a reasonably small sampling. It is interesting to speculate whether general mechanisms of regional sp e cifica tio n would be conserved across much wider evolutionary distances. The s im ila ritie s in early vertebrate development argue that there are highly conserved general mechanisms. However is there any common ground between insects and vertebrates? The comparisons between insects and vertebrates is intriguing since these animals represent the tips of the two major higher metazoan lineages, the protostomes and thejdeuterostomes respectively. These two lineages derive their names from the origin of their definitive mouths. The protostomes derive th e ir d e fin itiv e mouth from the blastopore while the deutrostomes form th e ir blastopore poste rio rly and the mouth forms as a new structure (Balinsky, 1981). The protostomes tend to e xh ib it spiral cleavage which involves unequal cleavage and results in the separation of small upper micromeres from larger lower micromeres. This process of spiral cleavage is used to generate a fixed or predetermined cell fate (Balinsky, 1981). The deutrostomes exhibit radial rather than spiral cleavage. It is also generally stated that the protostomes and the deutrostomes form their coeloms in different manners (Clark, 1964). The former tending to form its coelom through cavitation of a solid mass of mesoderm. Such a coelom is termed a schizocoelom. The deutrostomes tend to from the mesoderm and the coelom together as pouches from the original gut cavity of the gastrula. The wall becomes the mesoderm and the separated cavity persists as the coelom. This type of coelom is called an enterocoelom. Since the mouse and Drosophila represent two extremes in developmental strategies i t would be interesting to compare them. Common aspects in th e ir developmental processes would predict that any such process would have existed prior to the sp lit between the protostomes and deutrostomes. By extrapolation they could be common to all higher metazoans, and provide important clues to general developmental strategies for regional specification. 1.2 A Summary o f Mouse Embryogenesis The work in this thesis has focused upon trying to identify genes that are important in vertebrate and p a rtic u la rly mammalian embryogenesis and fo r that purpose the mouse has been employed. The mouse is the best characterised vertebrate with regards to genetics, cell and molecular biology and therefore offers advantages that one does not fin d in other vertebrate systems. Through

3 reverse genetic manipulation of the mouse genome, using traditional transgenic technology or via embryonal stem (ES) cells, the capability now exists to alter the developmental patterns of gene expression by overexpressing or removing gene a c tiv ity in the mouse embryo. The phenotypes that may re sult from these sort of experiments could provide approaches to c r itic a l processes and decisions in early embryogenesis. Thus the mouse offers many advantages to workers who are interested in the genetics of vertebrate and particularly mammalian development. 1.2.1 Pre-implantation development The early stages of mouse development are not concerned with the specification of the body plan but with the segregation of extra-embryonic tissue from embryonic tissue. The extra-embryonic tissues are necessary fo r the support and nutrition of the embryo (Rugh, 1968, Theiler, 1972). The segregation of the ce lls which w ill produce the embryo depends on events during cleavage. There is evidence to suggest that at the 8-16 and 16-32 cell stage, cells differentiate according to their position such that the outer c e lls w ill be directed to form the trophectoderm, which is an extra-embryonic tissue, while the inner cells w ill contribute to the embryo. (Tarkowski and Wroblewska, 1967, Hillman et a l., 1972). Hillman and co-workers demonstrated that while a fo u r-ce ll blastomere could form both embryonic and extra- embryonic tissue i f one placed a labelled fo u r-c e ll blastomere on the outside of another four ce ll embryo then th is c e ll tended to remain outside and to contribute to the extra-embryonic tissues. At the eight cell stage the blastomeres of the mouse embryo undergo a dramatic shape change which is termed compaction wherein the spherical appearance the blastomeres becomes flattened (Lewis and Wright, 1935). The physical isolation of the cells, either inside or outside, is associated with compaction of the embryo at the eight- sixteen-cell stage (Ducibella and Anderson, 1975). This involves polarisation o f the blastomeres o f the embryo and the formation of tight junctions between cells at the periphery of the embryo (Ducibella and Anderson, 1975). The polarisation of the c e lls is demonstrable in many ways; localisation of m icrovilli to the apical surface, increased adhesiveness at the basolateral surface and nuclear displacement (Reeve, 1981a, Reeve and Ziomek, 1981b). The polarisation of the blastomeres precedes compaction and w ill occur i f compaction is prevented (Ziomek and Johnson, 1980, Kimber and Surani, 1981). Elements of the p o la rity in the eight ce ll blastomere seem to be conserved through the next ce ll d ivisio n (Johnson and Ziomek 1981). The asymmetry would be carried through ce ll d ivisio n by orienting the plane of cleavage approximately perpendicular to the axis of

4 p o la rity . This can produce two 16-cell stage blastomeres one of which is polarised and has an apical surface with m icrovilli and while the other has no polarity and is adhesive at all points (Johnson et al., 1986). The embryo is called a morula from compaction to the 32-cell stage when a fluid fille d blastocoel begins to form in the interior (Rugh, 1968, Theiler, 1972). This activity expands the embryo into a blastocyst which contains an outer layer of cells, the trophectoderm, and a clump of cells attached to one point of its interior, the inner cell mass (ICM). The outside polar cells of the sixteen cell stage are those that contribute to the trophectoderm while those that are internal and apolar produce the progenitors of the inner ce ll mass (Tarkowski and Wroblewska, 1967). The c e lls o f the ICM are s t i l l to tip o te n t at th is stage and give rise to both embryonic and extra-embryonic tissues (Winkel and Pedersen, 1988). The outer cells of the ICM delaminate to produce a layer of cells, one cell thick, that is called the hypoblast (Rugh, 1968, Theiler, 1972). The other internal cells of the ICM give rise to the epiblast from which all embryonic tissues are formed. This has been demonstrated by reconstituting trophectoderm of one glucose phosphate isomerase variant with the ICM of another variant and allowing the embryos to develop. This study revealed that the e n tire foetus is derived from the ICM (Papaioannou, 1982). In rodents, after implantation, the epiblast becomes suspended on a cylindrical column of tissue which protrudes into the expanding blastocyst cavity. This structure is referred to as the egg cylinder. 1.2.2 Gastrulation and the generation of the body plan It is now, at about six days of gestation, that the embryo begins to gastrulate and this w ill terminate about three and a half days later. Gastrulation is a morphological transformation whereby the multilayered larval form is created, by rearrangement of tissues, from an e a rlie r monolayered form. It is during gastrulation that the body plan of the animal is laid down and the anteroposterior axis of the embryo firs t becomes visible. It is fe lt that the anteroposterior axis is specified prior to gastrulation and is a consequence of the orientation of the implanting embryo with respect to its uterine environment (Smith 1985). By analogy with chick embryos, i t is possible that the anteroposterior polarity is firs t established in the hypoblast and later induced to the epiblast (Mitrani and Eyal-Giladi, 1981, Mitrani et al., 1983). In birds and mammals the meso-endodermal lineages delaminate from the epiblast and involute through the primitive streak, which is like a fault line, lying along the anteroposterior axis at the posterior end of the

5 epiblast. In the mouse, cell division rates rise dramatically in an area just anterior of the centre of the midline, the proliferative zone, and there is a reorientation of cell division at the posterior end (Snow 1977). This marks the beginning of the primitive streak. The new tissue is inserted between the epiblast and the hypoblast. At th is stage i t would be more appropriate to term the epiblast the ectoderm. The mouse starts gastrulation with about 500-600 cells and within 24 hours i t has produced some 7000-8000 ectodermal and 6000-7000 mesodermal ce lls (Snow, 1977). Proliferation would seem to be important in murine gastrulation since mitotic inhibitors arrest gastrulation. This is in contrast to Xenopus which can continue gastrulation in the presence of such in h ib ito rs (Cooke, 1973). As gastrulation proceeds the meso-endodermal c e lls extend between the epiblast and hypoblast to form a complete layer that matches the area covered by the ectoderm. Most regions of the epiblast seem to be piuripotent up to the eighth day, although there may be regional differences such that cells from anterior regions prefer to colonise ectoderm while those from distal and posterior regions contribute more to mesoderm (Beddington, 1982, Tam and Beddington, 1987). Cells that emerge from the anterior part of the primitive streak contribute mainly to the notochord, gut and paraxial mesoderm while cells from the middle region give rise to la te ra l mesoderm and from the caudal portion to extra-embryonic mesoderm (Snow, 1981, Tam and Beddington, 1987). While the heart has been anatomically described as forming from a group of cells at the most anterior extremity of the embryonic axis i t has also been shown that i f a portion of the distal tip of the 7 day old egg cylinder is removed heart development is abolished (Rugh, 1968, Theiler, 1972, Snow, 1985). The notochord is laid down along the mid-line of the embryo and the paraxial mesoderm lies on either side. The early differentiation of the paraxial mesoderm is characterised by the sequential condensation of epithelial balls of mesoderm, called somites, at the cranial end. The mesoderm that gives rise to the somites is termed presomitic and i t has been suggested that there exists a prepattern of somitomeres in this tissue that condense into the somites (Tam et al., 1982, Tam, 1988). In more cranial regions the paraxial mesoderm that emerges from the p rim itive streak forms somitomeres, of which there are thought to be seven, and does not condense in to somites (Meier and Tam, 1982). Adjacent to the paraxial mesoderm lies the intermediate mesoderm which is the precursor of the mesonephros (Saxen et a l., 1986,). I t has been suggested, in the rat, sheep and in a primate that the intermediate mesoderm also gives rise to cells of the somatic part of the gonad and this is

6 thought to also be true in the mouse (Paranko, 1987, Zamboni and Upadhyay, 1982, Yoshinga et a l., 1988). The la te ra l plate mesoderm is arranged in two layers, one that lies beside the hypoblast, the splanchnic mesodermal plate, and one which lie s ju s t beneath the ectoderm, the somatic mesodermal plate. The splanchnic plate is associated with the endoderm and contributes to the mesenchymal portions of many visceral organs such as the lung, stomach and gut while the somatic mesoderm contributes to the outer regions of the body such as the limbs (Balinsky, 1981). It is important to note that there is a general rostral to caudal temporal sequence in maturation. For example, while there are somites condensing in rostral portions of the embryo gastrulation is s till occurring more caudally with cells s till passing through the primitive streak (Rugh, 1968, Theiler, 1972). The ectoderm forms both epidermis and neural tissue with the neural plate forming along the midline after the primitive streak has regressed (Rugh, 1968, Theiler, 1972). In avian and Xenopus embryos neural tissue becomes induced by the underlying axial/paraxial mesoderm and th is is probably also true of the mouse (Slack 1983). Neurulation involves the neural plate thickening, and the lateral folds rising up and closing to produce the neural epithelium (Martins-Green, 1988). In the mouse the firs t fusion of the neural folds occurs at the level of the th ird or fourth somite and proceeds ro s tra lly and caudally (Sakai, 1989). After this in itia l event there are also a number of other secondary sites of fusion (Sakai, 1989). In the chick i t has been suggested that changes in cell shape are important events in the formation and shaping of the neural epithelium (Schoenwolf and Powers, 1987, Smith and Schoenwolf, 1987) and it is fe lt that the same events may also occur in the mouse (Martins-Green, 1988). Again there is a ro stra l to caudal maturation, with the neural fold having formed in anterior regions while the plate has not yet been specified in posterior regions (Rugh, 1968, Theiler, 1972). It seems likely that positional information w ill be imparted by the mesoderm onto the ectoderm, although no direct experiments have been carried out on the mouse which would address th is question. Grafting experiments in avian embryos would seem to suggest that as in amphibia, i t is the mesoderm that imparts positional information to the ectoderm (Hara, 1978). This was demonstrated by recombining competent ectoderm with axial mesoderm from d iffe re n t regions along the rostrocaudal axis. The ectoderm was induced to form neural tissue and the regional characteristics of the induced tissue varied and was dependent upon the s ite of o rig in of the mesoderm (Hara, 1978). The neural primordium is also the source of the neural crest ce lls which

7 are mesectodermal c e lls which form many diverse derivatives (Le Douarin, 1982). The neural crest cells migrate from the margin of the neural groove, in cranial regions, and from the dorsal part of the neural tube in the trunk (Smits-van Prooije et al 1988). In trunk regions these ce lls form the neurons and g lia of the peripheral nervous system, pigment and adrenomedullary c e lls . The cranial neural crest has a greater plasticity and can also form cartilage and bone in the face (Le Douarin, 1982). The eight day mouse embryo has an S shape with its dorsal side lying towards the placenta. This is transformed into a C shape embryo by day 9.5 with the ventral surface lying towards the placenta. This turning is initiated at about the level o f the eighth somite and from here i t spreads both caudally and ro s tra lly (Poleman et a l., 1987). The mechanism that lie s behind th is axial rotation is unknown although i t has been suggested that asymmetric mitotic activity in the neural tube may be involved (Poelmann et al 1987). 9.5 days of gestation also marks the time of the closure of the caudal neuropore. The primitive streak that probably persists to this time is replaced by the caudal eminence or end-bud (Rugh, 1968, Theiler, 1972). This caudal eminence gives rise to notochord, hind gut, caudal somites and the neural cord. Once the caudal neuropore has closed neural material is not la id down as a plate but as a cord. The cavity of the already formed spinal cord extends into the neural cord. This process is termed secondary neurulation (Schoenwolf,. 1984). The somites break down to form the dermatome, myotome and sclerotome from about 9 days onwards, and again th is progresses in a ro stra l to caudal sequence (Rugh, 1968, Theiler, 1972). The dorsolateral portion of the somites produces the dermatome which contributes to the dermis, and the myotome which contributes to the skeletal muscle. The sclerotome, which is derived from the ventral medial part of the somite, condenses around the notochord and forms the pre-vertebrae which become the backbone of the animal. The notochord degenerates as development proceeds. At th is stage one can also id e n tify the fore limb buds, the hind limb buds have not yet formed, and many of the major organs are beginning to differentiate(R ugh, 1968, Theiler, 1972). The lung and stomach forming from the foregut, and the hindgut forms caudally (Sorokin, 1965, Bryden et a l., 1973, Rugh, 1968, Theiler, 1972). The mesenchymal components of these organs are probably derived from the splanchnic mesodermal plate. Epithelial-mesenchymal interactions are very important in the development of these organs. For example in the lung i t is the mesenchyme at the tip of the developing alveoli that induces branching. This mesenchyme w ill induce branching on other regions of the lung i f transplanted (Sorokin, 1965). Neurogenesis in the mouse occurs between about 9 and 14 days of gestation

8 (Wentworth, 1984a, 1984b). I t has been noted that there is a general ro stra l to caudal sequence in the maturation o f the neurons in the rodent nervous system (Altman and Bayer, 1984). These authors also describe a general ventral to dorsal gradient in the maturation of the major classes of neurons. The f i r s t major class of spinal neurons to be born are the motor neurons of the ventral horn while the inter-neurons o f the dorsal horn are among the la s t born. At day 15 of gestation, all the major organ systems are established, the animal has formed a ll 65 somites and looks rodent-like (Rugh, 1968, Theiler, 1972). The remainder o f the gestation period which in to ta l lasts fo r about 20-21 days is concerned with the d iffe re n tia tio n o f the c e ll types of each organ system and with the growth of the embryo.

1.3 Strategies Towards a Molecular Understanding of Murine Development This description of murine embryogenesis has hardly touched upon the mechanisms or molecules that could be involved in the regional specification that is morphologically obvious. Yet clearly one would wish to move beyond such descriptive studies and to begin to define important molecules that are involved in mammalian development. There are a number of approaches that are currently being attempted in the hope of isolating such molecules. One approach is to try and clone classically identified genes that e xh ib it interesting phenotypes such as Brachyury , which affects the development of the notochord, or rachiterata , which affects the pre-vertebrae. In the case of Brachyury , long range walking and cloning of chromosomal regions has lead to the identification of the gene and analysis of this gene is providing insight into the formation of mesoderm (Hermann pers. comm.). This approach has the obvious advantage that once the gene is cloned the mutant phenotypes already e xist therefore aiding both fu rth e r genetic and molecular studies. Another approach is to work with molecules that have been shown to be important in other biological processes suchas c e llu la r transformation. The large range of growth factors would be examples of this class and molecules related to the fibroblast growth factors and the transforming growth factors such as XTC-MIF and Vgl have been shown to have important roles in Xenopus embryogenesis (Slack et a l., 1988, Smith, 1989). I t has also been recently shown that the proto-oncogene c-kit maps to the cla s s ic a lly defined mouse|iOCus, white (Chabot et al., 1988, Geissler et al., 1988). This gene is involved in the development of stem c e lls in the haemopoetic, neural crest and germ ce ll lineages. A number of groups have attempted to create random recessive mutants

9 through insertional mutagenesis in the hope of inactivating important developmental genes (Gridley et a l., 1987). This approach has used DNA micro­ injection into fertilised eggs, retroviral infection of early embryo, blastocysts and midgestation embryos. One then breeds the animals to homozygosity and looks fo r an interesting developmental phenotype. One re tro v ira lly induced mutation that exhibits an embryonic lethal phenotype, mov 13, has been shown to disrupt the al (I) collagen gene (Schnieke et al., 1983). Embryonic stem (ES) c e lls are derived from the early embryo and can be maintained in culture as undifferentiated pluripotent stem cell lines. In culture one can manipulate these cells and then reintroduce the cells back into the embryo where they retain the ability to contribute to all the tissue of the animal including the germ line. Thus it is possible to mutate a gene in tissue culture and introduce that mutation into the animal. Using this technology one could produce a range of mutations ranging form large disruptions to subtle changes in the coding sequence. One approach towards identifying developmentally interesting genes has been to attempt to tag these genes with reporter genes. This can be most effectively achieved using ES cells since transfection of the reporter construct into plates of cells allows one to amass many different integration events. The different transfected cells can be stored and analysed at a later date. The developmental pattern of expression of a given clone of transfected cells is then analysed by reintroducing the modified ES cells into the blastocyst and thereby creating a chimaeric animal. If the expression pattern proves to be interesting the sequences flanking the integrated reporter can be cloned out and the endogenous gene examined. In th is strategy the integration of the vector should create a mutation (Rossant and Joyner 1989) which can then be passed through the germ lin e of the animal and bred to homozygosity. However, the approach which has yielded the broadest spectrum of developmental control genes is based on conservation in protein and nucleic acid motifs between different species. This involves searching for murine genes that are structurally related to genes which have been identified as being developmentally important in other organisms. One obviously hopes that i f a murine cognate is found then th is gene w ill be important in murine embryogenesis. This means, fo r example, taking Caenorhabiditis elegans or Drosophila melanogaster genes and looking fo r cognate genes in the murine genome.

10 1.4 Regional Specification in Drosophila Embryogenesis The fruit fly Drosophila melanogaster has been intensively used in genetic research for over 70 years and it is in this organism that we have the best understanding of the genetic basis of development. Because of its short life cycle, it is possible to screen large numbers of embryos for mutations affecting the basic body plan. Many important genes have been identified by mutant screens that selected for defects in the pattern of the cuticle (Nusslein-Volhard and Weischaus, 1980, Weischaus et a l., 1984, Jurgens et a l., 1984, Nusslein-Volhard et a l., 1984). Drosophila is also the organism in which molecular biology has made the biggest impact in the understanding of the events underlying pattern formation. The embryology of Drosophila melanogaster has been described in d e ta il many times before, such as in Campos-Ortega and Hartenstein (1985), as has the molecular genetics of embryonic pattern formation in Drosophila, such as in Akam, (1987) and Ingham, (1988). I w ill only present an outline of the important events in the specification of Drosophila embryonic pattern. The Drosophila egg is ellipsoid in shape and polarised so that the future axes, the anteroposterior, dorsoventral and le ft- r ig h t axes, of the embryo can be distinguished. The early stages of Drosophila embryogenesis are d iffe re n t from that of many animals in that the zygotic nucleus undergoes a series of rapid, synchronous nuclear divisions that are not accompanied by cytokinesis. This leads to the formation of a syncytium, i n which 512 nuclei are located in a common cytoplasm. The subdivision into c e lls is delayed. A small group of nuclei move into the cortical cytoplasm at the posterior pole of the egg and become surrounded by plasma membranes. These ce lls form the pole ce lls which later become the germ cells. Most of the remaining nuclei colonise the cortical cytoplasm at the periphery of the egg. They undergo another four divisions at this site until the actual cleavage of the cytoplasm occurs and the plasma membranes separating the nuclei form. This monolayer of cells around the periphery of the egg is called the blastoderm. Many of the signals that progressively establish the regional specification of the Drosophila embryo act at the scyncitial blastoderm stage. 1.4.1 The genetics of anteroposterior specification The f ir s t acting genes are maternal genes and i t is these genes that are responsible for establishing the polarity of the egg. Genes of the bicoid (bed) group are responsible for determining the anterior structures while those of the oskar (osk) group specify posterior regions and the terminal structures rely on the genes of the torso group (Fronhofer et a l., 1986, Lehman and Nusslein-Volhard, 1986, Nusslein-Volhard et a l., 1987). The

11 anterior determinant would seem to be the product of the bed gene which acts in a positive instructive fashion to organise the anterior pattern (Fronhofer et a l., 1986,). Contrastingly the gene of the osk group that may be the posterior determinant, the nanos (nos) gene, is fe lt to have a permissive role in the posterior pattern (Hulskamp et al., 1989, Irish et al., 1989) These maternal genes interact with the zygotic genome to activate the gap genes which are responsible for the primary subdivision of the embryo. These genes are so named because mutations in these genes cause deletions in particular parts of the body, which creates a gap in the anteroposterior pattern (Nusslein-Volhard and Wieschaus, 1980). There are three principal members of this class, hunchback (hb), Kruppel (Kr) and knirps (kni) (Nusslein-Volhard and Wieschaus, 1980). hb is expressed in two broad domains, one extending from the anterior pole to 50% of the egg length (EL) and the other from the posterior pole to 25% of EL (Tautz, 1988). Kr is expressed in a broad band in the middle of the embryo and kni is expressed both ju s t anterior and posterior of the Kr band (Tautz and P fie fle , 1989). In bed mutant embryos there is no zygotic expression of hb but Kr is expressed more a n te rio rly than normal and in a broader domain (Tautz, 1988). The Kr domain of expression is also extended in the absence of the posterior determinants (Gaul and Jackie, 1987). These results have been interpreted as illustrating that the genes of the bed and osk groups act to repress Kr in both anterior and posterior regions. The activation of hb by bed has been shown to occur in a concentration dependent fashion (Struhl et a l., 1989 , Driever et a l., 1989b). The bed protein contains a DNA binding domain and it has been shown that there are a number o f bed binding sites upstream of the hb promoter, which exhibit different affinities for this protein (Berleth et al., 1988, Dreiver and Nusslein-Volhard 1989a, Struhl et al., 1989, Dreiver et a l. 1989b). The hb gene besides being zygotically activated in anterior regions also displays a maternal transcript that is located throughout the egg (Tautz et al., 1987). This maternal expression does not normally persist in posterior regions but in mutant embryos of the osk class this expression does persist. It has been shown that ectopic expression of hb in posterior regions acts to suppress abdominal segmentation and therefore acts lik e mutants from the osk group (Hulskamp et a l., 1989, Struhl, 1989a). I t is thought that the role of the nos gene is in clearing this posterior expression of hb (Hulskamp et a l., 1989, Irish et al., 1989, Struhl, 1989a). This is supported by the observation that hb,nos double mutants develop a normal abdomen (Hulskamp et a l., 1989, Iris h et a l., 1989).

12 Analysis of the expression of the gap genes in different gap gene mutant embryos provides evidence for bilateral regulatory interactions amongst the gap genes (Jackie et al., 1986). Thus this type of interaction with mutual repression between adjacent domains results in the sharpening and generation of stable boundaries. These interactions are likely to be at the transcriptional level since hb, Kr and kni encode proteins that have motifs characteristic of many transcription factors (Rosenberg et al., 1986, Tautz et a l., 1987, Nauber et a l., 1988). The gap genes act to cause position specific expression of the pair-rule genes, which fu rth e r subdivide the embryo (Frasch and Levine, 1987b). There are eight members of the pair-rule class of genes, all of which show transient expression in seven or eight stripes during cellularisation of the blastoderm (Akam, 1987). Of these genes runt and hairy would seem to be the firs t to act and to have a major role in setting up the striped pattern of the other pair rule genes (Ingham and Gergen, 1988c). These two genes are in it ia lly expressed uniformly but by the beginning of c e llu la ris a tio n they are expressed in two complementary series o f seven stripes around the embryo and may act to interpret the positional information established by the maternal and gap genes (Howard, 1988). It is not clear exactly how these patterns are established but there would seem to e xist an interaction between the gap genes and between the runt and hairy gene products themselves and this is supported by the analysis o f gene expression in mutant embryos (Ingham and Gergen, 1988c). Once established the complementary stripes of runt and hairy serve as the driving force for the refinement of the spatial patterns of the other pair-rule genes. The pair-rule genes will act to define the domains of expression of the segment p o la rity genes, such as engrailed (en) and wingless (wg), that are the f ir s t evidence of the parasegmental organisation (Martinez-Arias and Lawrence, 1985). The en stripes represent the anterior lim it of a parasegment and the wg stripes are localised to the posterior ce lls of each parasegment (Ingham et a l., 1985, Baker, 1987). I t has been demonstrated that alternate en stripes require the combined a c tiv ity of d iffe re n t sets of p a ir-ru le genes. In odd numbered stripes, the en expressing ce lls are those which express both even- skipped (eve) and paired (prd), while in the even-numbered stripes they are those that express fushi-tarazu (ftz) and odd-paired (Ingham et a l., 1988b). In contrast to en, wg expression appears to be repressed by the eve and ftz proteins (Ingham et al., 1988b). At the end of the blastoderm stage the eve and ftz bands narrow so that they are separated by a single cell, and it is th is ce ll that expresses wg. I t is also at th is stage that each parasegment primordium is programmed

13 to follow a unique differentiation pathway and this depends on the selective action o f the homeotic genes of the Antennapedia (ANT-C) and Bithorax (BX-C) complexes (Akam, 1987) . The term homeotic comes from the mutant phenotypes of these genes which cause one segment type to be transformed to that of another homologous structure (reviewed in Gehring and Hiromi, 1986). The homeotic genes are firs t transcribed at the blastoderm stage at the same time as the pair rule genes and are affected by the maternally acting genes and gap genes, which cause broad regional differences in the expression o f the homeotic genes (Harding and Levine, 1988). For example the PI promoter of the Antennapedia (Antp) gene depends absolutely upon Kr while the p2 promoter is acted upon by a combination of osk, hb and ftz (Irish et al., 1989). This pattern is then refined by the p a ir-ru le genes themselves. For example i t has been demonstrated that ftz, which is involved in the correct regulation of en, is also necessary for the expression of Sex combs reduced (Scr), Antennapedia (Antp) and Ultrabithorax (Ubx) (Ingham and Martinez-Arias, 1986). It is clear that the homeotic genes act to modulate th e ir own transcription and i t has been demonstrated that the Deformed (Dfd) gene exhibits autoactivation (Kuziora and McGinnis 1988) and i t has also been shown that the more p o ste rio rly expressed homeotic genes w ill act to repress the more a n te rio rly expressed genes, such as the repression of (Ubx) by Abdominal-B (Abd-B) and abdominal-A (abd-A) (Hafen et a l. 1984, Harding et a l. 1985, Struhl and White 1985). These interactions eventually result in defined domains of homeotic gene expression which allow these genes to specify parasegment character. For example Scr, Antp, and Ubx exhibit enhanced levels of expression in the primordia of parasegments 2, 4 and 6 respectively and these are reflected in the mutant phenotype of these genes (Martinez-Arias et a l., 1987, C arroll et a l., 1986a, Akam and Martinez-Arias, 1985). The Scr mutants show a transformation of the la b ia l segment, which is p a rtly derived from parasegment 2, whereas Antp mutants have a transformation of the second thoracic segment, which has a contribution from parasegment 4 (Kaufman et a l., 1980, Wakimoto and Kaufman, 1981). S im ila rly the f ir s t abdominal segment which is derived from parasegment 6 is transformed in a Ubx mutant (Sanchez-Herrero et a l., 1985). 1.4.2 The genetics of dorsoventral specification The dorsoventral polarity is also thought to be established through a hierarchy of interactions that start with maternally encoded gene products. The firs t identified maternally acting gene was dorsal (Nusslein-Volhard, 1979). This gene exhibits an extreme phenotype in which the embryo becomes

14 completely dorsalised (Santamaria and Nusslein-Volhard, 1983). Of the other maternal genes that have been id e n tifie d Toll seems to be acting early in the generation of dorsoventral polarity. Rescue of Toll mutant embryos can be effected with cytoplasm taken from any part of the w ild type egg (Anderson et a l., 1985). Yet in contrast to other mutants of th is class, such as dorsal, the site of injection defines the position at which the ventral most structure w ill form. As with dorsal the Toll RNA, is not localised but is uniformly d istrib u te d in the early embryo (Hashimoto et a l. 1988). The Toll protein is thought to become modified or localised in the ventral region of the embryo, and i t is thought that th is may be dependent upon Toll activity itself (Levine 1988a). Levine (1988a) suggests that i t is the Toll product which is d ire c tly or indirectly involved in the localisation of the dorsal activity to ventral regions of the embryo and that it is this activity that may play a key role in the differentiation of dorsoventral pattern elements. Again, as with the anteroposterior network, it seems that the zygotic genes act to interpret and further subdivide the maternal dorsoventral inform ation. A number of such genes have been id e n tifie d . For example Zerknult (zen) and decapentaplegic (dpp) are two such genes which have null mutants which cause the loss o f dorsal derivatives and e xh ib it a ventralised phenotype (Wakimoto et a l., 1984, Iris h and Gelbart, 1987). Contrastingly the elim ination of mesoderm is the observed phenotype of mutants in the twist and snail genes, while the single-minded mutants wipe out both the neuronal and non-neuronal derivatives o f the ventral most ectoderm (Simpson, 1983, Thomas et al., 1988). The patterns of expression of some of these genes is in itia lly quite broad and becomes defined as gastrulation proceeds (Ingham 1988a). A number of these genes contain regions that would encode DNA binding domains. zen contains a homeo domain and snail has a zinc finger motif (Rushlow et al., 1987, Boulay et a l., 1987). Therefore these genes may exert th e ir effects through transcriptional regulation. Other zygotically acting genes such as dpp display identity to growth factor like molecules and could therefore be involved in cell-cell signalling (Padgett et al., 1987) . Thus while there is much less molecular detail about the sequence of events in the establishment of dorsoventral polarity it is obvious that it shows parallels with the establishment of anteroposterior polarity and that it is separate from the anteroposterior polarity. 1.4.6 Neurogenesis Many of the segmentation and homeotic genes are not only expressed during early embryogenesis but are also expressed at la te r times in larva l and imaginal structures (e.g. Dfd and Scr, Martinez-Arias et al., 1987). The

15 embryonic nervous system, in p a rtic u la r, shows high levels of expression of many of these genes. The segmentation genes eve, e/ 7 , and ftz show expression in a specific subset o f neurons in every segment of the developing CNS (Frasch et a l., 1987a, Dinardo et a l., 1985, Carroll and Scott, 1985). These genes seem to be involved in neuronal determination and it has been shown that ftz and eve are components o f the mechanism co n tro llin g ce ll fate during neuronal development (Doe et a l., 1988b, Doe et a l., 1988c). I t was also demonstrated that the segmentation genes also interact with each other in the developing nervous system, although not in the same fashion as was observed in the blastoderm (Doe et a l. 1988c). In the central nervous system loss of ftz affects the expression of eve while the opposite situation is found in the blastoderm. This would suggest that these genes are responding to d iffe re n t cues in the nervous system as apposed to those in the blastoderm. The homeotic genes are expressed at th e ir highest levels in embryonic nervous system (Levine et a l., 1983, Akam and Matinez-Arias, 1985, Chadwick and McGinnis, 1987, Kuriowa et a l., 1985, Martinez-Arias et a l., 1987). In this system they also appear to be involved in conferring positional specification. Thus they are not expressed in every segment of the nervous system but exhibit higher levels of expression in specific parasegments and some genes are expressed at lower levels in the surrounding parasegments. Mutations in these genes affect those regions that exhibit the high levels of expression of the genes. As has been described for the early embryo the homeotic genes in the central nervous system s t i l l e xh ib it cross regulatory interactions, such as the repression of anterior gene expression by posterior genes.

This summary o f Drosophila development cle a rly points out the use o f a hierarchy of decisions, through gene activation, to specify the pattern of the Drosophila body. Genes for the anteroposterior axis in itia lly act to subdivide the embryo t ill the parasegments are defined and then finally through the action of the homeotic genes these metameric units are given an id e n tity . The hierarchical action of genes is also evident in the establishment of the dorsoventral axis.

1.5 The Homeobox As is evident from the discussion above there are many interesting developmental genes in Drosophila, that have been identified by classical genetics. One particularly interesting class of genes are the homeotic genes which can cause the transformation of one body segment into another. These

16 genes act to interpret the anteroposterior positional information and confer id e n tity on the parasegment primordia. There was obviously a great deal of interest in cloning these genes and analysing their sequences. The molecular analysis of these genes revealed a common sequence that was present in two of the homeotic genes , Antp and Ubxt and in one segmentation gene (ftz). This common element, which was termed the homeobox because i t was discovered in homeotic genes, is a conserved 183 base pair sequence of DNA that is present w ithin the protein coding regions of these genes (McGinnis et a l., 1984, Scott and Weiner, 1984). Subsequent analysis has shown that the homeobox is present in a ll the homeotic genes of the ANT-C and BX-C complexes and in genes such as bed and en that have been discussed above. Sequence analysis has shown that the protein domain that is encoded by the homeobox, the homeodomain, is structurally related to the prokaryotic DNA binding domains that have the helix-turn-helix motif (Gehring, 1987). It has been shown that the homeodomain is a DNA binding domain and homeodomain proteins are tra n scrip tio n a l regulators (Levine and Hoey, 1988b). Further molecular characterisation of genes from Drosophila, vertebrates, and other organisms has shown that there are actually quite of range of homeobox type sequences. The major classes would include those related to one of Antp, en, eve or caudal (cad) and a new class that contain the POU-homeodomain. The POU homeodomain has been found in three mammalian tran scrip tio n factors P it-1 , Oct-1 and Oct-2 and one Caenorhabiditis gene unc-86y th e ir homeodomains while being related to the Drosophila homeodomains are actually more sim ila r to each other (Bodner et a l., 1988, Ingraham et a l., 1988, Sturm et a l., 1988, Clerc et a l., 1988, Finney et a l., 1988). This has been termed the POU homeo domain

fo r the P it-1 , Oct-1 and Oct-2 and unc -86 genes (Herr et a l., 1988). These genes are also related by virtu e of a second region the POU-domain, that is about 75 amino acids in length. These different classes of homeodomains differ from each other at many points over the 61 amino acids but can be grouped together by considering th e ir amino acid changes in the most conserved region of the homeo domain, the helix-turn-helix region. This thesis w ill largely concern it s e lf with homeoboxes/homeodomains of the Antp type and unless otherwise stated the term homeobox/homeodomain shall refer to those of the Antp type. An in itia l study of the phylogenetic distribution of homeoboxes suggested that the presence of th is sequence may correlate with a segmented body plan (McGinnis, 1985). Yet i t was subsequently shown that homeoboxes were evolutionarily more widespread than was previously appreciated with homeoboxes being found in organisms of widely separate phyla from non-segmented

17 invertebrates to mammals (Holland and Hogan, 1986, Costa et a l., 1988). Of the genes in which the homeobox was firs t discovered, only ftz is a segmentation gene while the others are responsible for anteroposterior specification (Slack, 1984). Later studies of other Drosophila homeotic genes uncovered patterns of expression that suggested roles in positional information independent of the process of segmentation (Hoey et al., 1986). Thus the specification of positional values along the anteroposterior axis of an animal may be a general role for homeotic genes. There have been many homeobox genes cloned from the mouse genome, and there are at least 27 such genes. As with Drosophila these genes are found in clusters in the genome and exhibit temporally and spatially restricted patterns of expression during development. Aspects of the structure and expression of these genes w ill be dealt with later in this thesis. The expression patterns of these genes would be consistent with them having a role in important developmental processes. However it is possible that the conservation o f the homeobox sequence between mouse and Drosophila represents evolutionary/developmental economy, in that if one has a useful protein structure one keeps employing that structure although not necessarily for the same purpose. Alternatively conservation could represent true conservation of function. In the latter case one would predict that the murine homeobox genes are also acting to interpret anteroposterior positional information. If this were true and given that Drosophila and mammals represent the peaks of the two major coelornate lineages, the protostomes and the deutrostomes respectively, one would be implying that there has existed common biochemical elements for the interpretation of anteroposterior positional information since before the sp lit between these two major lineages.

18 CHAPTER TWO - MATERIALS AND METHODS

2.1 Buffers and Stock Solutions

Alkaline SDS 0.2M NaOH, 1% SDS.

Blue ju ice (X10) 50% glycerol, 20mM T ris .C l, 20mM EDTA (pH 8.2), 0.1% Bromophenol blue.

Denaturing solution 0.5M NaOH, 1.5M NaCl.

Denhardts solution (100X) 2% (w/v) BSA fra ctio n V, 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrolidone. Filter sterilised and stored at -20°C.

DEPC-treated water 0.1% (v/v) DEPC was added and then autoclaved.

DTT, 1M 1M DTT in lOmM NaOAc (pH 5.2). Sterilised by filtration. Stored at -20°C.

EDTA,500mM pH adjusted to 8.0. Autoclaved. For RNA work this solution was also treated with DEPC prior to autoclaving.

El ution buffer lOmM Tris.C l (pH 7.6), ImM EDTA (pH 8.0), 0.1% SDS.

Hybridisation buffers In situ 50% deionised formamide, 0.3M NaCl, 20mM Tris.C l (pH 8.0), 5mM EDTA, lOmM

NaP04 (pH 8.0), 10% Dextran sulphate, IX Denhardts, 0.5mg/ml yeast RNA.

Riboprobe pre-hyb 50% deionised formamide, 5X SSC, 5X

Denhardts, 50mM NaPB (pH 6 . 8 ), 250ug/ml

19 Salmon sperm DNA, lOOug/ml tRNA, lOug/ml poly A,poly C, 0.2% SDS.

Riboprobe hyb 50% deionised formamide, 5X SSC, IX

Denhardts, 20mM NaPB (pH 6 . 8 ), lOOug/ml Salmon sperm DNA, lOOug/ml tRNA, lOug/ml poly A,poly C, 0.2% SDS, 10% Dextran sulphate.

Ligase buffer (X5) 250mM Tris.C l (pH 7.6), 50mM MgCl2, 25% (w/v) polyethylene glycol 8000, 5mM DTT

Lysis buffer (mini-prep) 8 % sucrose, 2% TritonX-100, 50mM EDTA (pH 8.0), 50mM Tris.Cl (pH 8.0).

Lysis buffer (maxi-prep) 50mM glucose, 25mM Tris.Cl (pH 8.0), lOmM EDTA (ph 8.0).

MOPS (X20) 0.4M morpholinopropanesulfonic acid (MOPS) (pH 7), 50mM NaOAc, lOmM EDTA

(pH 8 ). Filter sterilised and stored at 4°C.

Neutralising solution(southern) 0.5M Tris.Cl (pH 7.6), 3.0M NaCl. (colony screen) 0.5M Tris.Cl (pH 7.6), 1.5M NaCl.

PBS (X10) 1.3M NaCl, 70mM Na 2HP04, 30mM NaH2 P04. pH was adjusted to 7 and autoclaved. For RNA work i t was treated with 0.1% DEPC and then autoclaved.

Probe hydrolysis buffer 80mM NaHC 0 3 » 120mM Na2C0 3 » lOmM DTT (pH 10.2). Filter sterilised and stored at -20°C.

Probe neutralising buffer 0.2M NaOAc (pH 6.0), 1% (v/v) acetic acid, lOmM DTT. Filter sterilised and stored at -20°C.

20 Restriction enzyme buffers Low s a lt (X10) lOOmM Tris.C l (pH 7.6), lOOmM MgCl2, lOmM DTT. Medium s a lt (X10) 500mM NaCl, lOOmM Tris.C l (pH 7.6), lOOmM MgCl2, lOmM DTT. High s a lt (X10) 1M NaCl, 500mM Tris.C l (pH 7.6), lOOmM MgCl2, lOmM DTT. Very high s a lt (X10) 1.5M NaCl, 500mM Tris.C l (pH 7.6), lOOmM MgCl2, lOmM DTT. EcoRl buffer (X10) 1M NaCl, 1M Tris.C l (pH 7.6), lOOmM MgCl2. Smal buffer (XIO) 200mM KC1, lOOmM Tris.C l (pH 8.0), lOOmM MgCl2, lOmM DTT. All restriction enzyme buffers were filtered sterilised and stored at - 20°C.

Saline (XIO) 8.3% (w/v) NaCl. Treated with 0.1% DEPC and then autoclaved.

Sodium acetate, 3M pH adjusted to 5.2 with glacial acetic acid. Autoclaved. For RNA treated with 0.1% (v/v) DEPC then autoclaved.

Sodium phosphate buffer (NaPB) 1M Na2HP04 mixed with 1M NaH 2 P04 u n til

pH 6 . 8 . Autoclaved.

SSC (X20) 3M NaCl, 0.3M Na c itra te pH adjusted to 7. For RNA treated with 0.1% DEPC and autoclaved.

Transcription buffer (XIO) 400mM Tris.C l (pH 7.6), 60mM MgCl2, 20mM spermidine, 200mM NaCl.

Tris.Cl ,2M ph adjusted to 7.6 and 8.0. For RNA work th is solution was made up w ith DEPC treated water.

Tris-acetate (TAE) XIO 40mM Tris-acetate, ImM EDTA (pH 8.2).

21 Tris-borate (TBE) XIO 900mM Tris base, 900mM boric acid, 20mM EDTA (pH 8.2).

TE lOmM Tris.C l (appropriate pH), ImM EDTA (pH 8.0). Autoclaved.

2.2 Bacterial Media, and A n tib io tics

Media L-agar 1% (w/v) bacto tryptone, 0.5% (w/v) bacto yeast extract, 0.5% (w/v) NaCl 1.5% (w/v) bacto-agar.

L-broth As L-agar but omit bacto agar.

Antibiotics A m p icillin Dissolved at 50mg/ml in sterile water filtered sterilised and stored at -20°C. Added to media and agar to a fin a l concentration of 50-100ug/ml.

Tetracycline Dissolved at 20mg/ml in 50% (v/v) ethanol , filte r sterilised and stored at -20°C. Added to media and agar at a

final concentration at 20ug/ml.

2.3 Animals The mice used in this study were of the CBA inbred strain of mus musculus, maintained by NIMR biological services division. The age of the embryos was determined from the day on which the vaginal plug was found. The day on which the plug was found was called day 0 of pregnancy. At noon the next day the embryos are 1.5 day p.c.

2.4 Cloning and DNA Methods

2.4.1 Bacterial strain The only bacterial strain used in this work was the JM 109 strain of Escherichia Coli K12. The strain is RecAl, enc/Ai, thi, HsdR17t supE44, re /A i,

22 lambda', del(lac-proAB), (F 't traD36, proAB, lacI^Z-del M15).

2.4.2 The production of cells competent for Transformation This method is basically that of Maniatis et al (1982). Throughout all steps in this procedure solutions and centrifuge bottles were sterile and chilled on ice. Bacterial cells were streaked out, for single colonies, on an L-agar plate and incubated at 37°C. After overnight incubation a single colony was used to inoculate two 5ml 1-broth aliquots which were again grown overnight. Each of two flasks containing 500ml of L-broth were inoculated with one of the 5ml overnight cultures. The flasks were then incubated in a shaker incubator at 37°C until the O.D .550 of the culture reached 0.45 - 0.55. When the c e lls had reached th is point the cultures were chilled on ice, to restrict further growth. The cells were then pelleted by centrifugation at 5k for 5-8 minutes. The supernatant was poured off and each p e lle t was gently resuspended in 125ml of 0.1M MgC^. The two MgCl 2 suspensions were combined into a single centrifuge bottle and again pelleted as above and then resuspended in 250ml 0.1M CaCl 2 solution. They were le ft for 20 minutes on ice and then again pelleted. Again the supernatant was discarded and the c e lls were resuspended in 25ml o f a CaCl 2/glycerol solution. This solution consisted of 21.5ml of 0.1M CaCl 2 and 3.5ml o f glycerol. 250ul aliquots of these cells were quick frozen and stored at -70°C.

2.4.3 Bacterial transformation

An aliquot of CaCl 2 treated bacterial cells was thawed slowly on ice. Typically not more than 1 - O.lug of DNA, be it a closed plasmid or the product of a ligation reaction, was used to transform cells. The DNA which was dissolved in 50ul of TE was added to lOOul of c e lls . These were incubated on ice fo r 20-30 minutes and then heat shocked at 42°C fo r 2 minutes. This mix was inoculated into 5ml of L-broth and incubated, with shaking, at 37°C for 60-90 minutes. Various amounts of the 5ml culture, ranging from lOOul to 500ul to the remainder were plated on L-agar plates containing the appropriate selectable marker. Plates were then incubated overnight at 37°C. Single colonies were picked the next day fo r subsequent processing, as required.

2.4.4 Standard DNA methods Routine procedures involved in DNA manipulation ( e.g. phenol: chloroform extraction and ethanol precipitation) were performed as described in Maniatis et al (1982). DNA samples were stored at -20°C.

23 2.4.5 Restriction digests All restriction enzymes were used according to the manufacturers instructions. In this lab one of seven restriction enzyme buffers was used depending on the enzyme. These buffers are low (L), medium (M), high (H) and very high (VH) salt buffers. There are also specific buffers for EcoRl (Rl), and Smal (Sma).

2.4.6 Agarose gel electrophoresis R estriction digests, or other DNA samples, were analysed by electrophoresis through agarose gels. The percentage of each gel varied depending on the size o f DNA fragments that were to be analysed. The gels consist of agarose dissolved in either IX TBE or IX TAE and run in the same buffer. TAE was used as a gel running buffer when a slow run was required i.e. for southern blotting or precise mapping. The restriction digest or DNA solution was added to the gel in blue juice. DNA fragments were visualised by staining w ith the fluorescent dye, ethidium bromide which fluoresces strongly under u ltra -v io le t lig h t. This dye was e ith e r included in the gel at 0.5ug/ml or after electrophoresis the gel was bathed in a solution containing the ethidium bromide at the same concentration. Fragment sizes were determined by running DNA markers of known molecular weight. The set of fragments that were commonly used can be obtained from BRL as the lkb ladder.

2.4.7 Isolation of DNA fragments from gels If a fragment was to be isolated from a gel the gel was stained after electrophoresis. The fragment of appropriate size was cut out of the gel using a sharp scalpel. The gel slice was placed in a dialysis bag that was clipped at one end with a dialysis clip and contained 0.5X TBE. The slice was allowed to f a ll to the bottom of the bag and the rest of the buffer was removed. This le ft just enough buffer to cover the slice. The bag was immersed in a shallow layer of 0.5X TBE in an electrophoresis tank and electrophoresed at 100 volts fo r 2-3 hours. The p o la rity o f the current was then reversed fo r 40 seconds. The flu id from the bag was removed and the bag was then washed with a small volume of 0.5X TBE. The wash was added to the bag flu id . The 0.5X TBE, that now contains the DNA, was passed over a column of packed si 1 iconised glass wool. The DNA was then cleaned by phenol: chloroform extraction and ethanol precipitation.

2.4.8 Sub-cloning and ligations Fragments of interest were subcloned in pKS vectors (Stratagene) for

24 subsequent am plification and to allow in vitro transcription. Vector DNA was digested with the appropriate enzymes, the digestions checked on a gel and then the DNA was phenol: chloroform extracted and ethanol precipitated. DNA fragments to be subcloned were isolated from the gel as described above. "Sticky end" ligations generally used lug of vector DNA and 1 to 3 molar equivalents of insert DNA. The ligation reaction volume was lOOul and contained IX ligase buffer, ImM ATP and lu of T4 DNA ligase. The ligation was carried out for six hours at room temperature and the products were used to transform bacteria.

2.4.9 Isolation of plasmid DNA - Mini-prep A single colony was inoculated into a 5ml L-broth culture, containing the appropriate selection, and grown overnight in a shaking incubator at 37°C. The cells were pelleted by centrifugation at 3k for 10 minutes. The pellet was resuspended in 700ul o f ly s is buffer . This suspension was transferred to a micro-centrifuge tube and to this 60ul of a lysozyme solution, lOmg/ml in lOmM

T ris (ph8 .0), was added. These tubes were incubated at room temperature fo r 10 minutes. The tubes were then placed in a vigorously boiling water bath for 40 seconds. After, they were spun immediately in a microcentrifuge for 7 minutes. The glutinous pellets were fished out with a toothpick and isopropanol was added to the top of the tube. The tubes were vortexed and then spun for 15 minutes. The alcohol was sucked o ff and the pellet was redissolved in 200ul of TE and then phenol: chloroform extracted and ethanol precipitated. The pellet was redissolved in 20ul o f TE.

2.4.10 Isolation of plasmid DNA - Maxi-prep Large scale isolation of plasmid DNA was used for high purity preparations. A 5ml overnight L-broth culture, which had been inoculated with a single colony, was added to 500ml of L-broth and grown overnight with shaking at 37°C. In both instances the L-broth contained the appropriate selection for the plasmid. This 500 ml culture was centrifuged at 8 k fo r 10 minutes and the supernatant discarded. The pellet was resuspended in 50ml of lysis buffer. 2.5ml of 40mg/ml lysozyme in ly s is buffer was added and incubated at room temperature for 10 minutes. This solution was then mixed with 100ml of alkaline SDS and le f t on ice fo r 5 minutes. 75ml of 3M KOAc was added to the preparations , mixed and incubated on ice for 20 minutes. Following centrifugation, at 8 K for 10 minutes, the supernatant was poured through a cheese cloth filte r. This flow through was mixed with 0.6 volume of

25 isopropanol and put at -20°C for half an hour. The DNA was recovered by centrifugation at 8 K for 10 minutes. The DNA pellet was redissolved in 15ml TE and then ethanol precipitated. This pellet was redissolved in 2ml of TE and the volume then remeasured. To th is solution CsCl 2 was added to a fin a l concentration of 1.15g/ml. The final total volume was made up to 5ml with

CsCl2 > with a refractive index of 1.393, and also included 300ul of EtBr. This tube was heat sealed. The sample was centrifuged over-night at 55K in a Beckman vti65.2 rotor. The banded plasmid was taken o ff the gradient and transferred to a new ultra-centrifuge tube. To this was added 75ul of EtBr and

the volume was made up to 5ml with the above CsCl 2 solution. The tube was heat

sealed and spun at 55k fo r 6 hours. The band was harvested and the EtBr was removed by isoamyl extraction. The volume was expanded to 15ml with TE and the DNA was ethanol precipitated. The pellet was redissolved in 1ml of TE and the

OD250 was determined. The DNA was then re-ethanol precipitated and fin a lly dissolved in TE at a concentration of lug/ul.

2.4.11 Southern blot Southern hybridisation was employed in the characterisation of cloned DNA sequences. R estriction digests o f DNA were run on agarose gels, as described above. Gels were soaked in denaturing solution twice for th irty minutes and then in neutralising solution, again twice for thirty minutes. Gels were placed on a sheet o f Whatman 3mm paper, that was pre-soaked in 20X SSC, which was supported over, but in contact with, a reservoir of 20X SSC (Maniatis et al 1982). A sheet of genescreen, pre-wetted in water and 2X SSC, was placed on top of the gel, followed by two pieces of whatman 3mm paper, again pre-wetted in 2X SSC. On top of this was a stack of level tissues and placed on top of the tissues was a 1kg weight. Transfer was allowed to proceed fo r 30 minutes for DNA fragments at high concentration and overnight for fragments at low concentration. After transfer the lanes of the gel were marked and the filte r

rinsed briefly in 6X SSC. The f i l t e r was then baked at 80°C fo r two hours. The dry filte r was now ready to be probed which was invariably with a riboprobe. Filters were usually pre-hybridised for 1 to 4 hours in riboprobe pre­ hybridisation buffer at the hybridisation temperature. Filters were incubated overnight at 65°C for riboprobes in hybridisation buffer.

2.4.12 Colony screen This method was used to identified true recombinants in the antibiotic resistant progeny of a bacterial transformation. After the bacterial colonies have grown to a reasonably small diameter, i.e. 0 . 1- 0 . 2mm, the plate is

26 removed from the incubator. A piece of dry nitro-cellulose filte r is carefully placed on top of the plate. The filte r is left in place for a couple of minutes and then pealed o ff. The f i l t e r is placed on whatman 3mm f i l t e r paper with the adherent bacterial colonies face up. A second identical filte r is then placed directly on top of the firs t filte r and fla t pressure is applied. Both filte rs , while s till stuck together, are marked by making needle holes. This enables orientation to be determined afterwards. The two f ilt e r s are separated and both transferred to new L-agar plates, with the appropriate selection. These plates are grown overnight at 37°C. There are now two copies of the original plate, each on nitro-cellulose filte rs. One of these filters is kept as a master copy. The other filte r is placed for 5 minutes on filte r paper soaked in 10% SDS, then 5 minutes on filte r paper soaked in denaturing solution and finally for 5 minutes on filte r paper soaked in neutralising solution. The filte r is then washed in a solution o f 2X SSC, 0.1% SDS. The f i l t e r is then baked at 80°C for two hours. The filte r was then probed with a riboprobe, as appropriate. The filte rs are pre-hybridised and hybridised as above. After probing the positive colonies are id e n tifie d by comparing the signal on the autoradiograph to the master plate, using the marks to align the two.

2.5 RNA Methods

2.5.1 In vitro Transcription Riboprobes were synthesised essentially as described by Melton et al (1984) using either 32P or 35$ labelled TP, as appropriate. The reactions were carried out in IX transcription buffer and lOmM DTT. The other three ribonucleotides were used at a final concentration of 0.75mM and 25 units of RNasin was included in each 20ul reaction. Either 20 units of T7 or T3 or 10 units of SP 6 RNA polymerases were used , as required, to transcribe from lug of linearised DNA template. The transcriptions were at 37°C for 60-90 minutes. Afterwards the DNA template was removed using DNase at a final concentration of 0.025 g per ul and incubating at 37°C for a further 10 minutes. At this step t-RNA, at 1.025ug/ul, was added. The reaction was stopped by the addition of a h a lf volume of stop buffer containing 250mM EDTA, 3mM NaOAc. Unincorporated nucleotides were removed by column chromatography over A1.5m agarose beads. The column was washed in e llu tio n buffer and the excluded peak was pooled.

P32 labelled probes fo r southern or colony hybridisations were used without further treatment.

27 oc S'53 labelled probes fo r in situ hybridisation were reduced to an average size of 50-150 bases by limited alkaline hydrolysis (Cox et al., 1984). The peak fraction was alcohol precipitated and the pellet then redissolved in 50ul lOmM DTT. To th is an equal volume of hydrolysis buffer was added and the probe incubated at 60°C fo r a time period that was dependent upon the probe length. The time required to reduce a probe to an average size o f 100 bases was calculated from the following formula: time in minutes* (original transcript size in kb - 0.1)/ (original transcript size in kb x 0.011) (Cox et al., 1984). After hydrolysis an equal volume of neutralising buffer was added and the probe was ethanol precipitated. The pellet was redissolved in lOmM DTT at 1x10® d.p.m/ul. The hydrolysed product of this reaction was analysed by electrophoresis through an agarose formaldehyde gel followed by transfer to genescreen and then visu a lisa tio n by autoradiography.

2.5.2 Agarose-formaldehyde gels RNA is electrophoresed through denaturing agarose-formaldehyde gels. Agarose was melted in water and cooled to 55°C before the addition o f IX MOPS and formaldehyde to 2.1M, from a 12.33M stock. RNA samples were prepared in IX MOPS, 2.1M formaldehyde, 50% formamide and IX blue ju ice and were heated at 65°C fo r ten minutes. The samples were then cooled on ice fo r 15 minutes and then loaded into the w ells. The gel was run in IX MOPS at 60-70V fo r 4-6 hours.

2.5.3 Northern blot After electrophoresis the agarose-formaldehyde gel was soaked in 50mM NaOH, 0.1M NaCl then in 0.1M Tris.C l (pH 7.6) and fin a lly in 2X SSC in each case fo r 20 minutes. The RNA was transferred to genescreen soaked in 2X SSC as described above by b lo ttin g in 20X SSC overnight. The lanes were marked and the filte r was briefly rinsed in 6X SSC. The wet f i l t e r was placed in Saran wrap and exposed to U.V. lig h t fo r 5 minutes at 600uWatts/cm^. The f i l t e r was then baked fo r two hours at 80°C.

2.5.4 In situ hybridisation This technique was used to visualise gene transcripts in situ in the embryo. Microscope slides were cleaned by passing them through 10% HC1 in 70% ethanol then water and fin a lly 95% ethanol. The slides were dried in an oven for 15 minutes at 150°C. The dry slides were then placed in a 2% solution of TESPA in acetone fo r 10 minutes. The slides were then washed twice in acetone

28 and once in water. They were dried at 50°C. Embryos were fixed overnight at 4°C in 4% (w/v) paraformaldehyde. They were then washed for 30 minutes, on ice, firs tly in PBS and then in saline. The samples were then placed in ethanol: saline (50:50) for 15 minutes followed by 70% ethanol for 2 times 15 minutes. The embryos were dehydrated by passing them through 80%, 95%, 100% and 100% ethanol fo r 30 minutes at each step and then put in toluene twice for thirty minutes. The toluene was replaced with p a ra ffin wax and the embryos kept at 60°C. The wax was changed three times at twenty minute intervals. The embryos were transferred to a p la s tic mould containing wax and allowed to cool. The embedded embryos were stored at 4°C.

6 um sections were cut on a microtome and the sections were lowered on to a 50°C water bath. Creases were allowed to unfold for 2 minutes before the sections were lifte d onto subbed slides. The excess water was drained and the slides were placed on 37°C hotplate for 1 hour. The slides were then allowed to dry at room temperature overnight in a box containing desicant. The sections were either used now or stored at 4°C. Where possible in the following steps all solutions were DEPC treated and diluted from stock into DEPC treated water. The sections were dewaxed in xylene, twice fo r 10 minutes. They were then rehydrated by being passed through an alcohol series i.e . 100%, 100%, 95%, 80%, 70%, 50% and 30% ethanol. The slides were washed in saline and then PBS for 5 minutes each and then fixed in 4% (w/v) paraformaldehyde for 20 minutes. They were washed twice fo r 5 minutes in PBS. The slides were placed horizontal with the sections facing upwards and a proteinase K solution (20ug/ml) was overlain. The proteinase K digestion was allowed to go for 7 minutes before the slides were washed in PBS for 5 minutes. The sections were refixed in 4% (w/v) paraformaldehyde for 5 minutes and then acetylated in fresh 0.25% (v/v) acetic anhydride in 0.1M triethanolamine.HCl (pH 8.0) for 10 minutes. The slides were washed for 5 minutes, firs tly in PBS and then in saline. The sections were dehydrated by passing them through an alcohol series (as above). They were air dried and used for hybridisation. The probes used in in situ hybridisation were in vitro transcribed from linearised DNA and were labelled with S ^ UTP. The probe was diluted ten fold in hybridisation buffer to give a final activity of 1x10^ d.p.m./ul. About 15ul of hybridisation mix was applied d ire c tly to the sections, spread with a strip of parafilm and a clean coverslip was lowered on top. The sections were hybridised for 14-16 hours at 55°C in a box saturated with 50% formamide and 1.25X SSC. A fte r hybridisation the coverslips were dislodged by

29 soaking in a solution of 5X SSC, lOmM DTT for 20 to 30 minutes.The slides were then washed in 50% formamide, 2X SSC, 0.1M DTT at 65°C fo r 30 minutes. This was followed by two 10 minutes washes in 0.5M NaCl, 10 mM T ris , 5mM EDTA at 37°C and then a 30 minutes incubation, at the same temperature and in the same buffer, with RNase A at 20ug/ml. After the RNase treatment the slides were washed in the same buffer fo r 15 minutes before being rewashed in 50% formamide, 2X SSC and lOmM DTT fo r 30 minutes. Slides were fin a lly washed in 2x SSC and then 0.1X SSC fo r 15 minutes each and dehydrated through an alcohol series: 30% ethanol, 0.25M NH^OAc; 50% ethanol, 0.25M NH 4OAC; 70% ethanol,

0.25M NH4OAc, 80% ethanol, 0.25M NH 40Ac, 90% ethanol, 0.25M NH 40Ac, 100% ethanol, 100% ethanol. The slides were air dried and then used for autoradiography. Slides were dipped in Ilfo rd K5 gel form emulsion dilu te d 2:3 with 1.7% (v/v) glycerol, at 43°C, gelled on a glass plate on ice for 15 minutes and then air dried at room temperature for 2 hours. Slides were then dried in a desiccated box at room temperature, for 6-14 hours, and the box placed at 4°C fo r the exposure period. Slides were exposed fo r between 5 and 9 days. At 20°C the slides were developed fo r 2 minutes in Kodak D19 developer, stopped in 1% acetic acid, 1% glycerol in water for 1 minute and fixed in 30% sodium thiosulphate for 5 minutes. The slides were then washed in water, twice for 10 minutes. Sections were stained with 0.02% (w/v) toludine blue for 1 minute, dehydrated through an alcohol series, cleared in xylene and mounted under a clean glass coverslip using Permount. Sections were examined and photographed, under bright fie ld and dark ground illu m in a tio n , using an Olympus Vanox-T microscope.

2.6 Sequence Comparisons Sequences were compared using the software from the U niversity of Wisconsin Genetics Computer Group (UWGCG) (Devreux et a l., 1984). The "Gap" programme was used to find the optimal alignment between two predicted amino acid sequences. This programme finds optimal aligment by inserting gaps to optimise the number of alignments and uses the algorithm o f Needleman and Wunsch (1970).

2.6.1 Sequences Used fo r Comparisons The sequences used fo r the comparisons in figure 3.3 are from the sources in parenthesis: Hox 2.5 and Hox 2.4 (Graham et a l., 1989); Hox 2.6 and Hox 2.7 (Graham et al., 1988); Hox 2.1 (Krumlauf et al., 1987); Hox 2.2 (Hart et al., 1987); Hox 2.3 (M e ijlin k et a l., 1987); Hox 1.7 (Rubin et a l., 1987); Hox 1.1,

30 Hox 1.2, Hox 1.4 and Hox 1.5 (Duboule et a l., 1986); Hox 1.3 (Odenwald et a l., 1987); Hox 1.6 (Baron et a l., 1987); Hox 3.1 and Hox 3.2 (B rier et a l., 1988); Hox 6.1 (Sharpe et a l., 1988); Hox 4.1 (Lonai et a l., 1987); Hox 5.1 (Featherstone et a l., 1988); Hox 5.2 and Hox 5.3 (Dolle and Duboule, 1989); Abd-B (Regulski et al., 1985); abd-A (Akam et al., 1988); Ubx and Antp (Scott and Weiner, 1984); Scr (Kuriowa et a l., 1985); Dfd (Regulski et al., 1987); zenl (Rushlow et a l., 1988); lab /(F90-2) (Hoey et al., 1986, Mlodzik et al., 1988).

2.7 Hox 3.1 probe The Hox 3.1 probe that was used in part of th is work was a g if t from Dr. Stephen Gaunt and is described in Gaunt (1988).

31 CHAPTER THREE - THE MURINE AND DROSOPHILA CLUSTERS ARE EVOLUTIONARY RELATED 3.1 Introduction I t became apparent as murine homeobox sequences were being cloned that these motifs were clustered close together on the genome (Colberg-Poley et a l., 1985, Hart et al., 1985). Currently it seems that the mouse genome contains at least 27 "Antp like" genes. These genes are organised into four clu ste rs, termed Hox 1, Hox 2, Hox 3 and Hox 5 and are located on chromosomes

6 , 11, 15 and 2 respectively (Bucan et a l., 1986, Hart et a l., 1985, B rier et a l., 1986, Featherstone et a l., 1988). The Hox clusters do not appear to map at the same place as any previously described mouse loci. The clusters were numbered in order of their discovery, thus Hox 1 was the firs t cluster to be discovered. Although there have been reports of a Hox 4 cluster (Lonai et al.,

1987) and a Hox 6 clu ste r (Sharpe et a l., 1988) these clusters have been reassigned to existing clusters. Hox 4 is part of Hox 5 and Hox 6 is part of Hox 3 (Schughart et al., 1989). Each member of the clusters is designated by decimal numbers, such as Hox 2.6. The decimal numbers were in itia lly meant to reflect the linear order of the Hox loci along the chromosome (Martin et a l., 1987). The Hox 2 clu ste r contains at least 9 members to date, 8 areshown in figure 3.1. This figure also shows the overlapping cosmid clones that span the cluster and that eight members have been cloned within a yeast a rtific ia l chromosome (YAC). As with other murine homeobox clusters the Hox 2 cluster is contained within a relatively small piece of DNA, with the 9 genes lying in a 150 kb stretch. A ll of these genes, and indeed other murine genes, share a common genomic structure and th is is illu s tra te d with the Hox 2.6 gene. Typically these genes are small. The Hox 2.6 gene comprises o f two exons within 3.2 kb with the homeobox being located in the second exon (Figure 3.1). The finding of the homeobox in the last exon is also typical of the Drosophila homeobox genes, although in some cases i t w ill be s p lit by an intron (e.g. Hoey et a l., 1986). The f ir s t exon also contains a conserved region, the hexapeptide, that is found in many homeobox genes from both mice and flie s . While some of the Drosophila homeobox genes are enormous, such as Antennapedia which is greater than 100 kb (Schneuwly et a l., 1986), the murine genes tend to be re la tiv e ly small at around 3 kb. The major tra n scrip t of the Hox 2.6 gene is 2.4 kb and contains a long 3' untranslated region. This large untranslated region contains an AUUUA motif which may be involved in post- transcriptional control (Shaw and Kamen, 1986). The difference in gene size between Drosophila and mouse is also reflected in the size of the proteins with many of the fly proteins containing runs of repetitive amino acids and

32 cos 5.4 cos H15 5 kb cosH9 cos 3.1

probes

n— rm — rr i r III I .I |ll_ E E E E E " E E EE EEE| (/>ra 5 ’ 3’ I ~ I I 4 = t 4=3 YAC

2.5 2.4 2.3 2.2 2.1 2.6 2.7 2.8

WtfJ (A II H / X

Hexa / | HOX2.6 100 bp Peptide, Homeobox AUG COOH

Figure 3.1 - The organisation of the Hox 2 cluster of murine homeobox genes and a diagrammatic representation of the Hox 2.6 gene structure. The overlapping cosmid clones and YAC clone that span much of the cluster are illustrated. The predicted Hox 2.6 coding sequence is shown.

Figure 3.2 - Gap aligment to identify regions of identity between different protein sequences. A-Sequence comparisons between murine Hox genes from the same clu ste r, Hox 2.1 vs Hox 2.6, and from the Hox 1 and Hox 2 clusters, Hox 2.3 vs Hox 1.1, Hox 2.2 vs Hox 1.2, Hox 2.1 vs Hox 1.3 and Hox 2.6 vs Hox 1.4 B- Sequence comparisons between members of the murine Hox 2 clu ste r and homeobox genes from the zebrafish, Hox 2.1 vs zf21 and Hox 2.2 vs zf22, and homeobox genes from Xenopus, Hox 2.3 vs XIH 2. In a ll comparisons the bars represent id e n titie s between the two sequences that are being compared.

33 M

2.1 I I I I I I I I III I I 2.6

70 41 60 M 1*6 (26 1*0 164 I I I 700 I I I I I I 1 1 I I ------2.3 I ttl lllllllll IIIHIM ii i mini m i i in ii i it in 111 i i mi i ii ii i i i i ii minimi imiimiiiniim nnninnin niniimiiinninn i i in linn III lllll 1;) i i i i i i i i i i i 26 40 60 I I I I I 120 140 166 t«0 200 720

2.2 I II II I 1.2

2.1 I II I 1.3

2.6 IIIUIIIOMIIIOW HttlllllHIIIII

1.4

B

2.1 ^6505657 n i Zf2.1

34 being considerably larger (e.g. Regulski et a l., 1987). Conceptual translation of the Hox 2.6 sequence produces a 250 amino acid protein with an in frame hexapeptide and homeodomain. Like many of the other homeodomain proteins, Hox

2 .6 is rich in prolines and in serines and as such has many potential sites for post-translational modification. 3.2 The mouse Hox clusters are related, possibly by duplication I t has been noted that the genes of Hox 1 and Hox 2 share sim ila r intergenic spacing and all are transcribed in the same 5' to 3' direction (Graham et a l., 1988). There are many examples of genes in Hox 2 being more related to genes in Hox 1 than to other Hox 2 genes. I f one compares the predicted amino acid sequences from two genes from the same clu ste r, Hox 2.6 and Hox 2.1 for example, the only notable region of identity is at the homeodomain. This is shown in figure 3.2A where amino acid sequences of a number of genes have been compared and id e n titie s are shown by bars and the homeodomain is marked by a lin e . In th is comparison i t is clear that the greatest density of bars is at the homeodomain. Yet i f one compares members of Hox 2 with members of Hox 1 i t is clear that the genes f a ll into Hox 1 - Hox 2 pairs. Comparisons of the homeodomains allows one to separate generally ^he genes into pairs with a Hox 1 member and a Hox 2 member (Figure 3.3). One can clearly see that these pairs show high levels of sequence identity with each other, sharing common specific amino acid changes when compared to Antp. The pairs are Hox 2.5/1.7, 2.3/1.1, 2.2/1.2, 2.1/1.3, 2.6/1.4, 2.7/1.5 and

2 .9 /1 .6 . Where fu ll amino acid sequence is available i t can be demonstrated that those genes id e n tifie d as being related through homeodomain comparisons also exhibit related regions outside the homeodomain. This is shown for Hox 2.3 and 1.1, for 2.2 and 1.2, for 2.1 and 1.3 and for 2.6 and 1.4 (Figure 3.2A). Clearly there is a greater density of bars at the N-termini and just upstream of the homeodomain at the hexa-peptide. There is also extended identity around the homeodomains themselves. Taken together these observations would support the idea that Hox 1 and Hox 2 are related by a duplication of a cluster of at least 7 homeobox genes as is illustrated in figure 3.4. No mouse Hox 1 counterparts have yet been found for Hox 2.4 or 2.8. Further characterisation w ill reveal the existence, or not, of these and other genes and help define the duplication, and the extent of divergence between the two clusters. Recent information would seem to suggest that while a large portion of Hox 1 and Hox 2 are related through duplication the whole clusters do not appear to be related in such a manner. I t appears that the Hox 1 clu ste r is more extensive than Hox 2 and possesses more 5'members than Hox 2 (P.Gruss

35 Hox5.3 GREK-CP—KH------L—M--- E—L—SKSVN—D—V ------L— MS

Hox2.5 SRKK-CP—K------L—M----D—H-V-RL-N-S---V------M- -M— Hoxl.7 TRKK-CP—KH------L—M----D—Y-V-RL-N----- V------M- -I — Hox3.2 TRKK-CP—K------L—M----D—Y-V-RV-N----- V------M- -M— Hox5.2 TRKK-CP—K------L—M----D—Y-V-RI-N----- V------M- -MS- Abd-B VRKK-KP-SKF------L—A-VSKQK-W-L-RN-Q----- V------N- -NSQ

HOX2.4 -R------S------HOX3.1 -RS-----S------L— p----k ----VS---G-----V------N Hox2.3 Hoxl.1

Hox2.2 HOX1.2 HOX 6.1 -R----I-S------

abd-A -R------F----- Ubx -R------

Hox2.1 G—A-TA------Hoxl.3 G—A-TA------Scr T—Q-TS------

HOX2.6 P— S-TA--- Q-V------Y------V------S------DH- Hoxl. 4 P— S-TA----Q-V------T----S-- V------DH- Hox5.1 P— S-TA----Q-V------T----P------DH- Dfd P— Q-TA---H-I------Y------T-V-S------D—

Hox2 .7 S— A-TA— SA-LV------C-P—V-M-NL-N-S------Y— DQ- Hoxl. 5 S TA P-LV------M-P—V-M-NL-N------Y— DQ- Hox4 .1 S— A-TA— SA-LV------FV-P—VQM-NL-N-S------Y— DQ- ZenZl---- L—S-TAF-SV-LV---N—KS-M—Y-T----- QR-S-C V------F— DIQ

Hox2 . 8--- SR-L-TA—NT-L------K—C-P—V---L-D----- V-V------H-RQTQ

Hox2 . 9 PGGL-TNF-TR-LT"------K—S-A—V---AT-G-N-T-V------Q—RER Hoxl. 6 PNAV-TNS-TK-LT------K AA-V---AS-Q-N-T-V------Q— RE- Labial--- NNS—TNF-NK-LT------A----- NT-Q-N-T-V------Q— RV-

Figure 3.3 - Amino acid sequence comparisons of the mouse and Drosophila homeodomains showing the existence of related subgroups. The Antp sequence at the top was used as a basis for all comparisons. Dashes represent identical amino acids. The separation into subgroups is based on the changes shared by the various members. References fo r the sequences used in these comparisons are detailed in material and methods.

Mouse 2.5 2.4 2.3 2.2 2.1 2.6 2.7 2.8 2.9 Hox 2

1.7 1-1 1.2 1.3 1.4 1.5 1.6 Hox 1 ® • — o — © — © -

Figure 3.4 - A schematic alignment of the Hox 1 and Hox 2 clusters. Hox

1 is located on chromosome 6 and Hox 2 on chromosome 11.

36 .6 1 MAMSSFLINSNYVDPKFPPCEEYSQSDYLPSDHSPGYYAGGQRRESGFQPEAAFGRRAPCTVQRY 65 I I I I I : : : I I I I I I I I I I I I I I I II II II .1 1 MAMSSYMVNSKYVDPKFPPCEEYLQGGYLGEQGAD YYGGGAQGADFQPSGLYPRPDFGEQPFGG 64

.6 66 AACRDPGPPPPPPPPPPPPPPGLSPRAPVQPTAGAL LPEPGQRSEAVSSSPPPPPCAQNPLH 127 I I II II : 'II I: III I .1 65 GGPGPGSALPARGHGQEPSGPGSHYGAPGERCPAPPPAPLPGARACSQPTGPKQPPPGTALKQPA 129

.6 128 PSPSHSACKEPWYPWMRKVHVSTVNPNYAGG EP KRSRTAYTRQQVLELEKEFHYNRYLTRRRRV 192 IIIMMMI : INI III I I I I I I I I I I I I I I I I I I I I I: 79 : .1 130 WYPWMKKVHVNSANPNYTGG EPKRSRTAYTRQQVLELEKEFHFNRYLTRRRRI 183

.6 193 EIAHALCLSERQIKIWFQNRRMKWKKDHKILiPNTKIRSGGTAGAAGGPPGRPNGGPPAL: 251 INI III llllllllllllllllllll III: II .1 184 EIAHTLCLPERQIKIWFQNRRMKWKKDHKLPNTKGRSSSSSSCSSSAAPGQHLQPMAKDHHTDLT 2 48

.6 251 251

.1 249 250

Figure 3.5 - Comparison between the related murine homeodomain protein sequences, Hox 2.6 and Hox 5.1. The Hox 2.6 sequence from Graham et a l., 1988 and the Hox 5.1 sequence from Featherstone et a l., 1988.

Splice 1 COOH NH2 • / ••'.v.vC'O::

Hexa Homeobox Conserved N-Term S er-P ro Rich Peptide Domain Subfam ily Domain Domain

61- -1 5 -5 0 - 70-1 5 0 6 — 4 -2 5 1 5 -5 0 AA AA AA AA AA AA

Figure 3.6 - Generalised structure showing characteristic features and conserved regions of a typical vertebrate homeodomain protein. This diagram is based on several vertebrate sequences. (AA) amino acid.

37 pers. comm.). The relationship of the other two clusters to the Hox 1 and Hox 2 clusters is more obscure but again one can find related members at re la tiv e ly similar positions in each cluster. In figure 3.3 one can note the relationship between the Hox 2 homeodomains and th e ir closest murine counterparts. This comparison clearly points out a lot of significant sim ilarities between many of the mouse homeodomains, and allows one to separate many of the mouse genes into subfamilies of related sequences. These subfamilies are defined by common amino acid changes and the position o f the changes (when compared to Antp) that are shared within a family. On this basis Hox 2.5, 1.7, 3.2 and 5.2 all form a subfamily and the mouse homeobox genes f a ll into eight such subfamilies (Figure 3.3). The relatedness of genes within a subfamily is strengthened by the fact that members of a subfamily have a high degree of identity in multiple regions over their entire predicted protein sequences. Full sequences are available fo r Hox 1.4, Hox 2.6 and Hox 5.1 and comparison shows that the related areas are the N-term ini, the hexapeptide and the homeo domain and sequences flanking. This is shown for Hox 2.6 and Hox 1.4 in figure 3.2 A and for Hox 2.6 and Hox 5.1 in figure 3.5. The Hox 5.3 and Hox 2.8 genes have not been placed within a subfamily since it is possible that these two genes represent members of two other subfamilies and are not members of the other eight subfamilies. This question w ill be clarified through more cloning and sequence work. S t ill i t would appear that a ll of the murine homeobox gene clusters are related to each other. Comparisons between related genes, in the same subfamily, always shows the N-terminus, the hexapeptide and the homeodomain to be the related regions. The conservation of these regions may be due to functional constraints. It is known that the homeodomain is a DNA binding region but roles have not been suggested fo r the N-terminus and the hexa peptide. For murine homeodomain proteins the middle unique portion is often rich in prolines and serines. Thus a typical homeodomain protein can be considered as shown in figure 3.6. 3.3 S im ila ritie s between mouse Hox complexes and those o f other vertebrates I t has been shown that the human homeobox genes are organised as in the mouse (Boncinelli et a l., 1988) and that these genes show very high degrees of sequence id e n tity to th e ir mouse homologues. For example the murine and human Hox 5.1 genes show 93% amino acid id e n tity over the whole coding region (Featherstone et a l., 1988). As yet there has been no gene that has been described in one organism, either mouse or human, but not the other. More distantly related vertebrates would also seem to organise their

38 homeobox genes in a similar fashion. One can note from figure 3.2B that the murine Hox 2.3 sequence displays extensive identity over the whole protein sequence with a Xenopus X1H2 sequence (F ritz et a l., 1989). This may suggest that the Hox 2 genes are found in many vertebrate species. More convincing evidence for this suggestion comes from an analysis of sequences from the zebrafish, Brachydanio rerio. This animal has been found to possess two closely linked genes that would appear to be homologues of Hox 2.1 and Hox 2.2 (Njolstad et a l., 1989). A comparison between the murine Hox 2.1 and the zebrafish zf-21 and between the Hox 2.2 gene and the zebrafish, zf-22 is illu s tra te d in figure 3.2B. The Hox 2.1 pair o f genes share 81% sequence identity and the Hox 2.2 pair share 70%, at the amino acid level (Njolstad et al., 1988, Njolstad et al., 1989). These zebrafish genes are closer to these mouse genes than to any other murine genes so far described, suggesting that they are directly related. The genome of Brachydanio rerio also contains two linked genes that are most closely related to the murine Hox 6.1 and 6.2 genes (Njolstad et al., 1989). It would seem that the zebrafish may possess true homologues o f both the Hox 2 and Hox 3 clusters of homeobox genes. To be sure of this point, and to determine the existence, or not, of zebrafish Hox 1 and Hox 5 lo c i, more characterisation of zebrafish homeobox genes is necessary. Since the evolutionary distance between teleosts and mice is re la tiv e ly large^one would be tempted to suggest that the common features that exist between the fish and in the mouse w ill hold true for all vertebrates. 3.4 The mouse and Drosophila homeobox gene complexes have common features of organisation Many o f the Drosophila homeotic genes have been found to be clustered in two separate but adjacent complexes, Antennapedia (ANT-C) and Bithorax (BX-C) (Lewis, 1978, Kaufman et a l., 1980, Sanchez-Herrero et a l., 1985). Several homeotic lethal complementation groups have been id e n tifie d in the ANT-C. These include Antennapedia (Antp)t Sex combs reduced (Scr), Deformed (Dfd), proboscepedia (pb) and labial (lab). The BX-C has three essential domains of homeotic function which are Abdominal-B (Abd-B) , abdominal-A (abd-A) and Ultrabithorax (Ubx) . If one includes the Drosophila homeotic genes o f the BX-C and ANT-C in the homeodomain comparisons one can note that many of these sequences show s im ila rity to one subfamily. The comparisons between Abd-B and Hox 2.5, Scr and Hox 2.1, Dfd and Hox 2.6, and lab and Hox 2.9 show the highest degrees of sequence id e n tity . The Drosophila genes abd-A, Ubx and Antp are all so similar in sequence that it is d ifficu lt to assign these genes to any of the other murine subfamilies, which are represented by Hox 2.4, 2.3 and

39 HOMEOBOX I I Hox 2.6 m

Dfd n i

Figure 3.7 - Diagrammatic representation of the relationship between Hox 2.6 and the Drosophila Dfd. The shaded areas represent amino acid id e n tity , and dashed lin e s demark related areas in the two proteins.

Conservation Between Homeobox Complexes

Posterior — ------Anterior Abd-B a b d -A Ubx Antp Scr Dfd pb lab Drosophila b x - c —n i i— n —# —□ — □ — □ — d CJ- ANT-C (3)

2.5 2.4 2.3 2.2 2.1 2.0 2.7 2.8 2.9 MOUSO Hox-2 (11)

Hox-1 (6)

3.3 3.2 3.1 8.1 6.2 Hox-3 (15) 5.3 5.2 Hox-5 (2)

Figure 3.8 - Representation of the relationship between Drosophila and murine homeobox gene clusters. The sequence comparisons from figure 3.3 were used to align the clusters into an evolutionary related network, the different members of a related subfamily are represented by a vertical row of boxes. Filled boxes in the mouse clusters represent sequenced homeodomains, open boxes indicate id e n tifie d but not as yet sequenced mouse homeodomains, and hatched boxes indicate predicted genes, based on analogy to other clusters. The numbers in parenthesis represent the chromosomal location of these clusters. The numbers above each box were those that were o rig in a lly assigned as the genes were isolated. Hox 6.1 and Hox 6.2 and Hox 4.1 have been reassigned to existing clusters on chromosomes 15 and 2 respectively (Schughart et a l., 1989). Their original nomenclature has been kept until they were renamed.

40 2.2. The Drosophila gene pb would seem to f i t into both the murine subfamilies represented by Hox 2.7 and by Hox 2.8 (Cribbs and Kaufman pers. comm.). Hox 2.6 displays several regions of identity, outside of the homeodomain, when compared with the Dfd protein (Figure 3.7) (Graham et al., 1988) and the Scr protein (leMotte et al., 1989) shares identity with Hox 2.1, particularly in the amino-terminal region, where 10 of the firs t 13 amino acids are the same. Mlodzik et a l., (1988) have found extended regions o f relatedness between lab and Hox 1.6 (LaRosa and Gudas, 1988). Therefore, where protein sequence is available, mouse and Drosophila genes identified as subfamily members, based on s im ila ritie s w ithin the homeodomain, also display some identity in other domains. This is obviously also true for other subfamily members and, for example, Hox 5.1 displays the same type of relationship to Dfd as Hox 2.6 (Featherstone et a l., 1988). Again the conserved regions are at the N-terminus and the hexapeptide which would reinforce suggestions that these regions are conserved because they are fu n ctio n a lly important. 3.5 A common ancestor fo r mouse and Drosophila homeobox gene complexes One s trik in g feature of these comparisons is that i f one aligns the mouse and Drosophila subfamilies, then the physical order of the Drosophila homeobox genes along the chromosome and that of their closest murine genes is identical

(Figure 3 . 8 ) . These results suggest that the murine and Drosophila homeobox gene clusters are derived from a common ancestor. Since mice and Drosophila are representatives of the deutrostomes and protostomes respectively it is suggested that th is ancestral cluster arose before the divergence of these lineages. In the above comparisons the best overall id e n titie s between mouse and Drosophila are in those genes [lab, pb, Dfd, Antp, and Abd-B) predicted to be the most ancient by Akam et al., (1988). In addition, the identity between Scr and Hox 2.1 argues that Scr was part of the ancestral cluster. Therefore i t is suggested that there were at least six homeobox genes in the ancestral cluster. Figure 3.9 shows one possible model from which one could derive the Drosophila and the murine Hox 2 clusters from the putative ancestral clu ste r. Akam et al., (1988) have recently proposed that the earliest ancestor for the myriapod-insect lineage possessed a homeobox cluster with distinct genes related to lab, pb, Dfd, Antp and Abd-B. Subsequently by duplication and divergence the Antp gene gave rise to the Scr gene and then, more recently, to the abd-A and Ubx genes. It is interesting that the genes defined by these workers as being the most ancient are also thought to be ancient from the comparisons between Drosophila and mouse. Yet the comparisons that are described here would suggest that the cluster postulated by Akam et a l., (1988) in fact existed long before any ancestor for the myriapod-insect

41 2.5 2.4 2.3 2.2 2.1 2.6 2.7 2.8 2.9

O OOOOOO OO M o u s e

common -o O o o o o - ancestor

OOOOOQ O O Drosophila

AbdB abd-A Ubx Antp Scr Dfd 2 b lab

Figure 3.9 - A possible scheme for the evolution of the Drosophila and murine clusters from a common ancestral cluster. This diagram is based on sequence information that is currently available.

42 lineage. Also considering the relationship between Scr and Hox 2.1, outlined above, it is postulated that this gene was part of the ancestral cluster. In both models the specific abd-A, Ubx and Antp genes are thought to have been generated more recently. It is noteworthy that it is d ifficu lt to assign any o f these three genes to the murine Hox 2.4, 2.3 and 2.2 genes. This could be a result of independent duplication, in each of the major lineages, of an ancestral "Antp” like gene. So the fact that the three Drosophila genes and the three murine genes show a high degree of sequence id e n tity re fle c ts th e ir separate derivation from a common ancestral gene. In the case of pb, which shows a high degree of identity with Hox 2.7 and 2.8 (Cribbs and Kaufman pers. comm.), it is thought that there was a duplication of the ancestral pb gene in the deutrostome lineage to generate these two Hox 2 genes. Such independent duplications has been postulated for the engrailed genes (Doleki and Humphreys, 1988, Joyner and Martin, 1987). It is also important to note that the murine genome contains, not one, but four homeobox gene clusters which are a ll related to each other. Drosophila has a single homeobox cluster and i f i t is assumed that there has been no secondary loss of homeobox gene clusters from arthropods i t would seem that the four murine clusters are directly or indirectly evolved from the single ancient ancestral cluster. In each of the four clusters, the relative position of each subfamily member and the physical order and the spacing of the genes are very similar. The alignment, shown in figure 3.8, also highlights the fact that not all genes or subfamilies are represented in all the murine clusters. This may suggest that eith e r some clusters arose at different stages during the evolution of the clusters or resulted from partial duplication or there has been sufficient divergence to make some genes undetectable. Interestingly the genes that are absent from Hox 3 and Hox 5 are among those that are thought to be derived from Antp and therefore evolutionary more recent. The sequence id e n tity between the mouse and the Drosophila genes should not be thought of as representing the existence of true vertebrate homologues of Drosophila homeotic genes. Some subfamilies have four murine genes related to a given Drosophila gene. As such i t would be hard to establish a precise murine equivalent. These comparisons suggest that the Drosophila and murine homeobox clusters arose from a common ancestral cluster that existed prior to the split between the protostomes and the deutrostomes. This is supported by identity between the mouse and Drosophila proteins not only in the homeodomain but also, where information is available, in other regions outside the homeo

43 domain ( Figure 3.7, Graham et al., 1988). One d ifficu lty with this argument is that the Drosophila genes are divided in two separate clusters although both are on chromosome three. However, in a more p rim itiv e insect, the red flour beetle, Tribolium casteneum, the ANT-C and BX-C genetic equivalents are found in a single homeotic clu ste r, HOM-C (Beeman et a l., 1989). Therefore, ANT-C and BX-C may o rig in a lly have been one cluster that has been separated by more recent evolutionary events occurring in some insects.

44 CHAPTER FOUR - EXPRESSION OF HOX 2 GENES ALONG THE ROSTROCAUDAL AXIS OF THE MOUSE EMBRYO

4.1 General aspects of murine homeobox gene expression Murine homeobox genes are not expressed in a ll tissues at a ll times in embryogenesis but rather show re stricte d patterns of expression. The fa ct that they exhibit spatially and temporally restricted patterns make them all the more interesting as this would be one of the characteristics that one would expect to find in genes that were involved in controlling important developmental processes, such as regional specification or various inductive interactions. That is not to say that all genes that are important in controlling such processes w ill be restricted in their patterns of expression. Yet i f a gene were to show a general expression pattern i t would also be lik e ly that th is gene would have a house-keeping function. It is useful to consider the patterns of expression of one of these genes at a frozen time point. The time point that is often chosen is at 12.5 days of gestation since this represents the time at which expression levels peak for many of these genes. It is also a convenient stage for the analysis of the expression patterns in a number of the organ systems that are being established . A ll of these genes have been described as being expressed in ectodermal and mesodermal derivatives but not in endodermal derivatives. Their expression is also restricted within mesodermal and ectodermal tissues. This general pattern is illustrated in figure 4.1 with the Hox 2.2 gene. Within ectodermal derivatives i t is apparent from figure 4.1 that Hox 2.2 is detected in components of both the central and peripheral nervous systems. Expression is found from most posterior regions of the spinal cord and extends anteriorly all the way along the spinal cord until expression stops in the hindbrain. While there is no posterior lim it of expression within the central nervous system, there is a very clear anterior lim it - indicated with an A- This anterior lim it is interesting because at 12.5 days of gestation it does not correspond to any obvious morphological boundary. The gene is not expressed in the midbrain or forebrain. The section in figure 4.1 A demonstrates the expression of this gene in the dorsal root ganglia. These are peripheral nervous system components, paired ganglia that are arranged in a repeating pattern lateral to the spinal cord and are derived from the neural crest. Hox 2.2 can be seen to be detected in the most posterior ganglia up to the most anterior ganglia. Hox 2.2 can be found to be expressed in many mesodermal structures. Expression in prevertebrae extends from most posterior regions to the 7th

45 A

B

Figure 4.1 - In situ hybridisation of the Hox 2.2 gene in the 12.5 day mouse embryo. Both bright field (left) and dark ground (right) are shown. A number of structures are labelled: HB- hind brain, SC- spinal cord, PV- prevertebra, H- heart, L- lung, Li- liver, S- stomach, K- kidney, DR6- dorsal root ganglia The anterior limit of expression in the central nervous system is marked (A) as is that in the prevertebrae .

46 * li

Figure 4.2- Dark ground photograph of an in situ hybridisation to a 12.5 day embryo saggital section with a sense probe. One can note that the liver (li) and the blood cells (*) appear to be positive. There would also appear to be some signal around the edge of the section .

CNS Md Mx Lb

Scr

Figure 4.3 - Schematic representation of the fate map of a Drosophila blastoderm stage embryo. Anterior is to the left and dorsal to the top. The black bars represent the domains of expression of the homeotic genes. The segments are indicated at the top (Md, mandibulary; Mx,maxi11ary; Lb, labial segment; T1-T3, thoracic segments; A1-A10, abdominal segments), the primordia of the central nervous system is also indicated (CNS). Dfd, Deformed; Scr, Sex combs reduced; Antp,Antennapedi a; Ubx, Ultrabithorax; iab-2 infraabdominal-2; iab-7 , infraabdominal 7; and cad, caudal

47 prevertebrae - marked with an^in figure 4.1 B. This is analogous to the situ a tio n that is observed in the CNS, where one observes expression from posterior regions up to an anterior lim it. One also detects transcripts in the viscera. I t is important to note that in organs of composite o rig in , such as the lung, th is gene, and others, are only expressed in the mesodermal components. In figure 4.1 A one can observe the expression o f Hox 2.2 in the mesenchyme of the lung, a mesodermal component, and not in the inner epithelium which has an endodermal origin. Expression of Hox 2.2 can also be seen in the mesenchyme o f the stomach which although weak has been confirmed by northern analysis (Figure 4.1 A, N.Papalopulu pers. comm.). Expression is not restricted to mesenchyme and in the metanephric kidney Hox 2.2 transcripts are expressed abundantly in the e p ith e lia l ce lls (Figure 4.1 A, Figure 4.10). Noticeably there are other mesodermal organs which do not express the gene. This is demonstrated in figure 4.1 with the heart and expression of Hox 2.2 has not been detected in the developing limbs (see later). In figure 4.1 one can note that the liver appears to be highlighted with the Hox 2.2 probe. This in fact reflects an in situ hybridisation artefact. It is clear from figure 4.2, where a sim ila r section has been probed with a sense probe, which should not detect any mRNA, that the liver is also highlighted. In figure 4.2 it is clear that blood cells also appear as if they were positive even when one uses a sense probe. Since the liver and the blood cells are both highlighted even when one uses a probe that does not detect mRNA one can rule out these signals as being due to artefacts related to this technique. In the case of the liver it has been shown, by northern blotting that there is no expression of Hox 2.2 (N.Papalopulu pers. comm.). The 12.5 day mouse embryo liv e r is a major s ite of haemopoesis and i t would seem that the apparent positive signals related to the blood cells and the liver is due to the fact that blood cells non-specifically stick the riboprobes. In cases where expression in an organ such as the liver is not clear from in situ hybridisation the results are backed up by the more sensitive method of northern blot analysis. All of the northern blot analysis was performed by Nancy Papalopulu. One should also note that areas around the edge of the signal also appear to be positive but again since these regions are also highlighted with the sense probe it would seem that this is due to the non­ specific adherence of the riboprobes.

4.2 Relationship between the clustering of homeoboxes and their expression The clustering o f homeobox genes is a general phenomenon having been found in every case so far analysed from mouse to zebrafish to Drosophila. In

48 Drosophila one important feature of the clusters is that the physical order of the homeotic genes is identical to the order in which these genes are expressed along the anteroposterior axis of the embryo during development (Harding et a l., 1985, Akam, 1987). The domains of expression along the axis also correspond directly to the segments that are affected by mutations in these genes (Harding et a l., 1985). The expression o f the homeotic genes in the blastoderm embryo is shown diagrammatically in figure 4.3. Note that Abd- B is infraabdominal 7 and abd-A is infraabdominal 2 . The telomeric most gene of BX-C, Abd-B, is expressed caudally while the genes o f the ANT-C which lie towards the centromere, such as Antp, Scr and Dfd, are more rostrally expressed. This pattern is maintained with each more centromeric gene exhibiting a more rostral domain of expression i.e. Dfd is expressed more ro s tra lly than Scr. While many of the homeotic genes have patterns of expression that are linked to metamerism th is is not true o f a ll the homeotic genes. For example labial is expressed in non-segmented regions of the Drosophila embryo (Hoey et a l., 1986). These studies and others have lead to the suggestion that the homeotic genes are responsible fo r the specification of position along the anteroposterior axis (Slack, 1984). A given parasegment, or group of c e lls , would perceive its position along th is axis through expression of homeotic genes. As has been discussed there is a group of genes which are homologous to the Drosophila homeotic genes, as defined by genetic means, in the red flo u r beetle, Tribolium casteneum, HOM-C (Beeman et al., 1989). In this insect it is also clear that there is also a correlation between the physical order of the genes and the order, along the anteroposterior axis, of the segments affected by mutations in these genes. Thus in these two organisms, at least, the clustering of homeotic genes would appear to be important and to re fle c t aspects of there function. Consideration of the mutant phenotypes would suggest that these genes are functioning in the specification of anteroposterior position. In the previous chapter i t was suggested that the murine and Drosophila homeobox clusters are both derived from a common ancestral cluster. It is important to determine if this common evolutionary origin is also reflected in the expression patterns of the members of a murine homeobox cluster. If the mouse homeobox gene cluster also displays a correlation between the physical order of the genes and their expression along the rostrocaudal axis then one would expect such a correlation to be universal within the protostomes and the deutrostomes. It is also interesting that the murine Hox 2 cluster is so small and has nine genes within only 150 kb of DNA and that this stretch of the

49 mammalian genome is devoid of repetitive elements. Therefore in situ hybridisation has been used to determine the patterns of expression of the members of the Hox 2 clu ste r in embryogenesis. The 12.5 day mouse embryo has been extensively used because this represents the stage at which the members of Hox 2 show the highest levels of expression.

4.3 Hox 2 probes used in in situ hybridisation Portions of seven o f the members o f Hox 2 were used in the following in situ hybridisation experiments. A ll probes were subcloned into riboprobe vectors, either the pGEM 1 vector (Promega Biotech) or into the Bluescript vector (Stratagene). The multiple cloning sites of these vectors are flanked by prokaryotic promoters that allow in vitro transcription using the appropriate RNA polymerase, T7 or T3. The Hox 2.1 probe that was used is an 800 bp SacI-EcoRI fragment whose 5' SacI end is at the N-terminal coding region and whose 3' EcoRI sites lies in the 5' half of the homeo domain. This fragment was subcloned into the pGEM 1 vector (Krumlauf et a l., 1987). All other fragments fo r each of the genes were subcloned into the Bluescript vector. In a ll cases the DNA fragment of interest that was to be subcloned was isolated from an agarose gel a fte r electrophoresis by e le c tro e llu tio n . These isolated fragments were ligated into vectors that had been digested with the appropriate restriction enzyme. The ligation products were transformed into JM 109 cells and plated on L-agar containing the appropriate selectable marker. The pKS vector contains the lacZ a-peptide coding region in its poly-linker and as such after transformation into the JM 109 ce lls which carry the M15 mutation in the TacZ gene on can screen fo r recombinant plasmids by screening for b-galactosidase activity. The screening for the b-galactosidase activity involves use o f the chromogenic substrate X-gal. Recombinant colonies appear white while non-recombinant colonies are blue. Positive clones were identified either by the above blue-white selection followed by restriction digests of samples of colonies after the plasmid DNA was mini-prepped or by colony hybridisation. The genomic structure of these genes has been determined by people in this laboratory and by many other workers in this field. The Hox 2.5 probe was a 400 bp B g lll-D ra l fragment that spanned the homeobox. This fragment was derived from a lkb SacI subclone from the cosmid pCos5.4 (see Figure 3.1). The Hox 2.4 subclone that was used to generate the Hox 2.4 probe was produced by N.Eager in the laboratory and consisted of a lkb SacI genomic fragment that spanned the homeobox and the regions 3 '. A 5kb Hox 2.3 fragment was isolated from the cosmid pCos5.4 (Figure 3.1) and from th is a smaller region was subcloned to be used as a probe. This was a 800 bp EcoRI-

50 BamHI fragment that started in the intron ju s t upstream of the second exon, EcoRI site, and terminated in the 3' untranslated region (See Meijlink et al., 1987, fo r f u ll map). The Hox 2.2 probe was a 1.2 kb EcoRI-SacI fragment that spanned the homeobox and was subcloned d ire c tly from pCosH15 (Figure 3.1). This fragment was identified by southern blotting and probing with a 220 bp EcoRI-XhoI fragment containing the Antp homeobox (McGinnis et a l., 1984). The EcoRI site lies in the intron and the SacI site in the 3' untranslated region as is shown in the map of Schughart et a l. (1988). The Hox 2.6 probe was derived from a cDNA clone, cDNA 7, and is an 800 bp fragment that starts at a Sail site close to the beginning of the long open reading frame and extends to a B g lll s ite in the homeobox. The Hox 2.7 probe originated from a genomic clone and has a 5' Bglll site in the homeobox and a 3' site in the untranslated region.

4.4 Ordered expression of Hox 2 genes in the central nervous system (CNS) Using all the Hox 2 probes, overlapping but distinct domains of expression have been observed fo r each gene in the CNS. In view of the absence of a clear caudal boundary of expression, th is work has focused on examining the different rostral boundaries of expression for each member of the Hox 2 complex. The same rostral boundary of expression was consistently observed in a ll 12.5 day mouse embryos. A ll seven genes have been d ire c tly compared on serial sections, using relative distances from morphological boundaries to map the expression boundaries. Starting with Hox 2.5 one finds that as one progresses through the Hox 2 complex in a 5' to 3' direction, the lim it of expression of each gene in the CNS becomes progressively more rostral. Figure 4.4 shows that for the 5' most gene, Hox 2.5, the lim it of expression maps within the spinal cord at the level of the third/fourth cervical prevertebra. The boundary of the adjacent Hox 2.4 gene is w ithin the posterior myelencephalon, as are those of the more 3' genes Hox 2.3 (not shown) and Hox 2.2 (Figure 4.4). The difference in the rostral boundary between Hox 2.5 and Hox 2.4 is large by comparison with the difference between Hox 2.2 and Hox 2.4. However each gene does have a successively more ro stra l lim it (Figure 4.4 ). Figure 4.5 illustrates, in serial sections of an embryo, that this general trend continues through the complex. Domains of expression fo r members 3' of Hox 2.2 extend more ro s tra lly w ithin the myelencephalon, and there is a relatively large difference between the rostral boundaries of Hox 2.2 and Hox 2.1. The successively more rostral expression in the hindbrain for Hox 2.1-2.7 is shown in figures 4.5 and 4.6 .A summary of these in situ results is

51 2.5 2.4 2.2 Figure 4.4 - Comparison of the patterns of expression for Hox 2.5, Hox 2.4 and Hox 2.2 in the 12.5 day mouse embryo. The top row displays in situ hybridisation on near adjacent sections showing bright fields of the entire embryo. Below are higher powered views of these sections focusing on the anterior limits of expression. These are represented in both bright field and dark ground pictures.

52 Figure 4.5 - Anterior boundaries of expression in the central nervous system for six members of the Hox 2 cluster as revealed by in situ hybridisation. The areas (A and B) of near adjacent sections photographed in high power are illustrated in the bright field picture of the whole 12.5 day embryo at the bottom. Area A was used for Hox 2.7, Hox 2.6 and Hox 2.1 while the more posterior area B was was used for Hox 2.2, Hox 2.4 and hox 2.5. Dark ground photographs are shown for each gene as indicated at the top and bottom of the pictures. Bright field pictures of A and B are shown at the left to aid identification of boundaries.

53 2.7

2.6

2.1

54 Figure 4.6 - Patterns of expression of the Hox 2 genes that show the most anterior limits of expression in the central nervous system. A bright field photograph of a 12.5 day mouse embryo is shown at the top to indicate the area pictured in higher power. The dark ground photographs show the anterior limits of expression in the hind brain for Hox 2.7, Hox 2.6 and hox 2.1. Probes for each section are indicated at the right.

Figure 4.7 - Diagrammatic representation of the anterior boundaries of expression in the central nervous system for the Hox 2 cluster, and their correlation with gene position in the cluster. A bright field photograph of a 12.5 day mouse embryo is shown below the cluster. Arrows indicate the anterior boundaries in expression detected in near adjacent sections from this embryo. The limits were established using three or four sections for each gene and positioned by comparison of relative distances from morphological structures. P- posterior, A- anterior.

OQOOOQO — hox2 2 5 2.4 2.3 2.2 2.1 2.6 2.7

55 illustrated in figure 4.7. These findings would suggest that the position of a gene within the Hox 2 cluster reflects its relative domain of expression along the rostrocaudal axis of the animal in the CNS.

4.5 Correlation between gene order and expression is also evident in other ectodermal der i vat i ves Other ectodermal derivatives, such as neural crest and ectodermal placode structures, display expression of members of the cluster. The results presented in th is section are from 12.5 day old mouse embryos. The expression of Hox 2 genes is p a rtic u la rly noticeable in the peripheral nervous system. The dorsal root (spinal) ganglia, which are neural crest derived, exhibit expression of every member of Hox 2, from Hox 2.5 to 2.7, for example see figure 4.1 A for Hox 2.2. As has been described fo r the central nervous system, more anterior components of the peripheral nervous system, such as the vagal (Xth)/glossopharyngeal (IXth) complex, also show d iffe re n tia l expression of members of Hox 2. The components of th is complex are derived from the neural crest and from ectodermal placodes (Verwoerd and van Oostrom, 1979, D'Amico- Martel and Noden, 1983). Figure 4.8 shows that the 5' most member of the Hox 2 clu ste r, Hox 2.5, is not expressed in these cranial nerves while the more 3' members are. One can note the expression of Hox 2.2 and 2.1 in the inferior ganglion of the vagal (Xth) nerve, the nodose ganglion, but not in the petrosal ganglion, which is the inferior ganglion of the glossopharyngeal (IXth) nerve. This is demonstrated with the Hox 2.1 gene in figure 4.8 B. Clearly there is hybridisation to the nodose but not the petrosal ganglion. An analysis of the position of these two ganglia in adjacent hybridised sections may also illustrate the expression of Hox 2.7 in the more rostral inferior ganglion of the glossopharyngeal nerve, the petrosal ganglion . Expression of any of these four genes cannot be detected in superior ganglia of either the vagus or glossopharyngeal nerve are formed from more ro stra l regions than the inferior ganglia (Figure 4.8 A and for Hox 2.7 data not shown) (Altman and Bayer, 1982). The trigem inal (Vth) nerve which lie s more ro s tra lly s t i l l than these nerves does not express any member o f Hox 2. The ro stra l boundary of Hox 2.7 expression in the hind brain region is also apparent in figure 4.8 A. Cranial neural crest not only contributes to nervous tissue but is also involved in producing skeletal and connective tissue (Le Douarin, 1982) . Figure 4.9 A shows the pharyngeal region that has been probed with Hox 2.5 and Hox 2.7 on adjacent sections. The mesenchyme of the thyroid has a neural

56 B

Figure 4.8 - Differential expression of members of Hox 2 in the vagal/glossopharyngeal nerve complex. A- Adjacent sections probed with four members of the Hox 2 cluster, Hox 2.5, Hox 2.2, Hox 2.1 and Hox 2.7 are shown in dark ground. Two Bright field views of this region are shown to allow identification of structures. The probe used for each section is indicated at the top. B- High powered photograph showing Hox 2.1 expression in the nodose but not in the petrosal ganglion. X- Xth (nodose) ganglion, IX- IXth (petrosal) ganglion, 0- otocyst, S- superior ganglia of the vagal/glossopharyngeal complex, pg- petrosal ganglion. n9~ nodose ganglion

57 Figure 4.9 - Expression of Hox 2 genes in some neural crest derivatives. A- I situ hybridisation of Hox 2.5 and Hox 2.7 to adjacent sections reveals the expression of Hox 2.7 but not of Hox 2.5 in the thyroid gland. The expression patterns of these two genes are shown in dark ground and the bright field photograph at the left allows orientation. The probe used for each section is liver marked above. ^ox ^*1 expression in the enteric ganglia. G-gut, L-lung, Li-

58 2.1 2.7

PV10

Figure 4.10 - Anterior boundaries of expression of three members of Hox 2 in the prevertebrae. The probes used are indicated above each section, One can note the patterns of expression of Hox 2.2, Hox 2.1 and Hox 2.7 in the dark ground pictures. The bright field photograph shows the anatomical detail of this region of the 12.5 day embryo.

59 crest origi^ and it is apparent that these cells are highlighted with the Hox 2.7 probe but not with the Hox 2.5 probe (Le Douarin, 1982). One can also note the more anterior expression of Hox 2.7 in the central nervous system and in the prevertebrae. Figure 4.9 B displays the expression of the Hox 2.1 gene in the enteric ganglia, which are neural crest derived. Hox 2.1 hybridisation pattern in the gut is punctate which would suggest that th is probe is picking out the myenteric plexus of the gut. Thus in more anterior neural crest or ectodermal placode derivatives it is the more 3' members of the clusters that are expressed.

4.6 Ordered d iffe re n tia l expression of Hox 2 genes in mesodermal derivatives Hox 2 genes are also found to be expressed in a range of mesodermal derivatives and one can note differential expression of members of Hox 2 in the same mesodermal cells. This is apparent in the prevertebrae of the 12.5 day mouse embryo. In figure 4.10 one can note the anterior boundaries of the expression of three members of Hox 2 in the prevertebrae. Hox 2.2 is expressed from the most posterior prevertebrae to the eighth prevertebra, Hox 2.1 , the adjacent 3' gene, to the seventh prevertebra and Hox 2.7,the most 3' gene analysed, is expressed from posterior regions to the f ir s t prevertebra (Figure 4.10). Thus again, as in ectodermal derivatives, there is a correlation between the order of the genes and their expression along the rostrocaudal axis. This sort of differential expression is also apparent in visceral mesoderm. In the lung, which is a relatively anteriorly derived organ, one can observe expression of the more 3' genes but not of the 5' genes. Figure 4.11 shows adjacent 12.5 day parasaggital sections probed with Hox 2.5, 2.2, 2.1 and 2.7. I t is clear that while Hox 2.5 is not expressed in lung mesenchyme the more 3' genes, Hox 2.2, 2.1, and 2.7 are. Again one can see a clear re s tric tio n of expression in the lung to the mesodermal components and no expression in the endodermally derived epithelium. In figure 4.11 one can also see apparent signal over the liver yet this is due to in situ artefact and these genes have not been found to be expressed in liv e r by Northern blo t analysis (N.Papalopulu pers. comm.). This mesoderm versus endoderm restriction can also be observed in the stomach and the in te stin e . Figure 4.12 demonstrates in a 14.5 days transverse section that transcripts for Hox 2.5 cannot be detected in either the gut or the stomach yet those for Hox 2.1, 2.6 and 2.7 can be. In figure 4.12 the expression of Hox 2.6 is not that strong but this result has been confirmed in other embryos (see Graham et a l., 1988) and by northern b lo t analysis. I t has

60 Figure 4.11 - Differential expression of members of Hox 2 in the lung of the 12.5 day mouse embryo. A bright field photograph of an entire embryo is shown at the bottom to indicate the positions of the various anatomical structures. The dark ground pictures illustrating the expression patterns of each gene are displayed and are labelled with the probe that was used. PV- prevertebrae, H- heart, L- lung, Li- liver, S- stomach, DRG- dorsal root ganglia, K- kidney.

Figure 4.12 - Expression patterns of members of the Hox 2 cluster in the stomach and gut of a 14.5 day mouse embryo. The bright field photograph shows a transverse section through a 14.5 day mouse embryo that contains various visceral organs which are labelled. The dark ground photographs demonstrate the expression of the more 3' members of the cluster in the stomach and in the intestine. Each photograph is labelled with the probe that was used. Li- liver, S-stomach, E- epithelium, G- gut

61 2.4 2.2 2.6

Figure 4.13 - Mesonephric tubules of the 10.5 day embryo displaying expression of the Hox 2.4, Hox 2.2 and Hox 2.6 genes. The dark ground photographs clearly show strong expression of these genes in the mesonephric tubules and the probe used on each section is indicated, the bright field photograph reveals the anatomical structures. S-somites, M- mesonephric tubules.

62 2.4

2.6

Figure 4.14 - Expression of Hox 2 genes in the metanephric kidney, developing gonads and in the adrenal gland of a 14.5 day embryo, the expression of the genes in these organs is shown in the dark ground photographs and each gene probe is indicated. The bright field photograph of the transverse section of this 14.5 day embryo displays the positions of organs of interest. AG- adrenal gland, M- degenerating mesonephros, K- metanephric kidney, G- developing gonad.

63 also been shown that Hox 2.2 can be detected in the stomach (Figure 4.1 A). In both the stomach and the gut these transcripts are not detected in the inner endodermal layers but in the outer mesodermal layers. The expression of Hox 2.1 in the gut is generally more punctate and as has been discussed above this is thought to represent expression primarily in the enteric ganglia which are derived from neural crest (see Figure 4.9 above). Again in fig u re 4.12 the liver is also highlighted. Figures 4.13 and 4.14 show expression of some members of the cluster in the mesonephric and metanephric kidneys. Expression of Hox 2.4, 2.2 and 2.6 are shown in the mesonephric tubules, at the hind limb le ve l, at 10.5 days in figure 4.13. There is very strong hybridisation of these probes to the mesonephric tubules and also lower hybridisation to the other regions that are evident in this section. These other regions are the neural tube and the somites which are in the process of breaking down to yie ld sclerotome and dermamyotome. A more complete analysis of the expression of Hox 2 members in the metanephric kidney is shown at 14.5 days in figure 4.14. In this figure one can note the expression of Hox 2.5, 2.4, 2.2, 2.1, 2.6 and 2.7 in the metanephric kidney. The sections probed with Hox 2.1, 2.6 and 2.7 also reveal the expression of these genes in the adrenal gland, which has a neural crest component. Further analysis is required to determine if all of the Hox 2 genes are expressed in the adrenal gland. Thus a ll members of Hox 2 are expressed in the kidney. In figure 4.14 the developing gonad is apparent and one can observe the d iffe re n tia l expression of Hox 2 genes. Hox 2.5, 2.4 and 2.2 are not expressed while the remaining 2.1, 2.6 and 2.7 show expression in the gonad. Expression of Hox 2 genes has not been detected in the heart or in the limbs, either fore or hind. Again the liv e r artefact is seen in some sections.

The expression patterns that have been described for the murine Hox 2 genes are analogous to those of the Drosophila BX-C and ANT-C homeobox gene clusters. In both cases it is evident that the physical order of the genes along the chromosome is related to the expression of the homeotic genes along the anteroposterior axis. Besides being true fo r the blastoderm stage th is relationship is also observed in the embryonic CNS (Harding et a l., 1985, Martinez-Arias et a l., 1987, Mlodzik et a l., 1988). The telomeric most member of the BX-C Abd-B shows expression to the sixth abdominal ganglion, while the adjacent gene, abd-A, in the centromeric d ire ctio n , expresses to the f ir s t abdominal ganglion and the next centromeric gene Ubx reaches the metathoracic ganglion. ANT-C lie s towards the centromere and genes w ithin th is complex

64 maintain this trend. Antp expresses in the prothoracic ganglion, Scr to the second subesophagal ganglion and Dfd to the f ir s t subesophagal ganglion. The centromeric most member of ANT-C lab has been reported to be expressed more a n te rio rly than Dfd (Mlodzik et a l., 1988).

4.7 Discussion: Position of genes in a cluster reflect domains of expression along the rostral-caudal axis for both mouse and Drosophila

4.7.1 Ordered expression in ectoderm I t has been shown that a ll members o f Hox 2 are expressed in s lig h tly different, but overlapping, domains in the CNS with respect to the anteroposterior axis (Figures 4.4, 4.5, 4.6). Posterior lim its of expression have not been detected fo r any of these genes in the nervous system. However, as one moves from the 5' most member of the cluster (Hox 2.5) in a 3' d ire ctio n , each successive gene displays a more anterior boundary of expression (summarised in Figure 4.7). The cut-off for Hox 2.5 in the spinal cord maps opposite the third cervical prevertebra, and the other six genes have anterior boundaries in the myelencephalon. These findings cle a rly show that the position of a gene within the Hox 2 cluster is correlated with its relative domain of expression along the anteroposterior axis of the embryo, suggesting that these genes may be playing a role in establishing positional information. This is analogous to the pattern observed fo r the Drosophila BX-C and ANT-C clusters. The physical order of the genes in these clusters reflects the anteroposterior order of the segments each gene affects and their relative domains o f expression. The domains of expression of Hox 2 genes in other ectodermal derivatives mirror their domains in the CNS. In the peripheral nervous system posterior regions express all Hox 2 genes while anterior components only express more 3' members. Thus the dorsal root ganglia, which are derived from trunk neural crest and lie on either side of the spinal cord, express all members of Hox 2 (LeDouarin, 1982). Yet in more rostral regions differential expression of these genes is observed. Hox 2.5 is not expressed in the Xth or IXth cranial nerves, while Hox 2.2 and 2.1 are expressed in the in fe rio r ganglia of the Xth, the nodose ganglion, but not in the inferior ganglion of the IXth nerve (Figure 4.8). Another more 3' gene,Hox 2.7, may be expressed in the in fe rio r ganglia of both the Xth and IXth nerves (Figure 4.8). The inferior ganglia of both the Xth and IXth cranial nerves have been described as originating at a distance la te ra lly from the rhombencephalon and as such more ro stra l than the sites of Hox 2.5 expression in the central nervous system (Altman and Bayer,

65 1982). The inferior ganglia of the Xth and IXth nerves have been shown , in the avian and possibly also in the mammalian embryo, to have contributions from both neural crest and ectodermal placodes (Verwoerd and van Oostrom, 1979, D'Amico-Martel and Noden, 1983). The neural c e lls | are derived from the ectodermal placodes while the Schwann c e lls and s a te llite c e lls are formed from neural crest . It is impossible to say from these in situ hybridisation studies whether the expressing cells in the nodose ganglion are of neural crest or placodal origin. None of these genes have been detected in the more rostral superior ganglia of the Xth and IXth nerves which are exclusively derived from neural crest in the avian embryo (Altman and Bayer, 1982, D'Amico-Martel and Noden, 1983). Nor has expression been detected for any of these genes in the more ro stra l trigeminal (Vth) nerve (Altman and Bayer, 1982, Rugh 1968, Theiler, 1972). One can also observe differential expression of Hox 2 genes in the mesenchyme of the thyroid which has a neural crest o rig in (Le Douarin, 1982). This relatively anterior group of cells do not express the most 5' member of Hox 2, Hox 2.5, but do show expression of the Hox 2.7 which lies at the other end of the cluster (Figure 4.9A). These mesenchymal cells are thought to arise from the hindbrain region, in both avian and mammalian embryos (LeDouarin, 1982). Since Hox 2.5 expression does not extend into the hindbrain while Hox 2.7 does, it is not surprising that these thyroid cells show differential expression of these two genes. I t is suggested that the patterns of Hox 2 expression that are observed in the neural crest and ectodermal placode derivatives correspond with the patterns of expression that have been described in the central nervous system. As such, the described patterns of expression of these genes are consistent with suggested neural crest and ectodermal placode fate maps. 4.7.2 Hox 2 gene expression in the hindbrain and rhombomeres An important question from our results is why many of the Hox 2 genes have anterior boundaries of expression in the myelencephalon. In early stages of neural development, periodic swellings along the axis of the neural epithelium have been observed and are termed rhombomeres, in the hindbrain. Previously it had not been clear whether segmentation of the neural tube, as defined by rhombomeres, plays any role in establishing the underlying pattern of development in the hindbrain. Recently, Lumsden and Keynes (1989) have presented data that rhombomeres do play a significant role in the segmental patterns of neuronal development in the chick hindbrain. Consistent with these results, Wilkinson et a l. (1989a) have shown that rhombomeres represent domains of expression for the zinc finger gene Krox-20, providing molecular

66 support for segmentation of the CNS. It has recently been shown that the anterior boundaries of the more 3' members o f Hox 2, Hox 2.6, 2.7, and 2.8 do abut rhombomere boundaries and show a two segment periodicity (Wilkinson et al., 1989b). More curiously the Hox 2.9 gene, which is currently the most 3' member of the Hox 2 clu ste r, is expressed in the hindbrain wholly w ithin one rhombomere, rhombomere 4 i Murphy et a l., 1989, Wilkinson et a l., 1989b). Thus i t has been suggested that in the hindbrain region homeobox gene do respect segmental boundaries and are acting to confer regional identity on these segments. One problem with this model is that only Hox 2.6, 2.7, 2.8 and 2.9 respect obvious rhombomeric boundaries while Hox 2.5 has an anterior boundary in the spinal cord and Hox 2.4, 2.3, 2.2 and 2.1 have anterior lim its of expression in the unsegmented region of the hindbrain, termed rhombomere 8, r8. While chick only has seven clear rhombomeres lower vertebrates may have more. A study of the zebrafish, Brachidanyo rerio , has suggested that besides the seven that are obvious in higher vertebrates there may also be another three more caudal segments (Hanneman et a l., 1988). I t is possible that the zebrafish Hox 2 genes (Njolstad et a l., 1989) may also respect rhombomeric boundaries and that the more 5' members of the cluster may respect the more caudal segments that are apparent in th is organism. While these segments would have been obscured in subsequent vertebrate evolution i t is possible that the Hox 2 genes in higher vertebrates are respecting the boundaries that are s till morphologically evident in fish. I t is also interesting that Hox 2.5 expresses from most posterior regions to a level in the spinal cord at the third or fourth cervical prevertebrae. This boundary is the level at which the f ir s t permanent dorsal root ganglia forms and such the boundary between trunk and o ccip ita l regions. Therefore it is possible that Hox 2.5 expression marks the difference between the trunk and the occipital portions of the nerve cord or neural crest. There have been reports of differences between neural crest from these regions such as the in a b ility of o ccip ita l neural crest to form sympathetic neurons (Newgreen, 1979, Newgreen et a l., 1980). Thus i t is possible that the differential expression of Hox 2 genes acts to specify regional diversity in the neural tube. In this model Hox 2.5 would determine trunk and more anterior regions of the neural tube would be sectored through expression of the other genes. It is important to note that rhombomeres are transient structures and that the anterior lim its of the Hox 2 genes remain a fte r the rhombomeric boundaries have disappeared. Rhombomeres are most evident during the period of

67 motor neuron d iffe re n tia tio n and i t seems plausible that the Hox genes are acting to confer identity on the segments during motor neuron maturation. But at 12.5 days of gestation after the rhombomeres have disappeared there is still a lot of neurogenesis and the anterior boundaries of the Hox 2 genes are s till in the same relative positions. It would s till seem likely that at this stage and at later stages the Hox 2 genes are conferring regional cues. Indeed the more 3' genes may act as a molecular remnant of the previous rhombomeric organisation. This situ a tio n is cle a rly analogous to that of the Drosophila homeotic genes most of which do respect segmental boundaries but some of which do not. An example of a gene which does not respect segmental boundaries is that of lab which has been described as being expressed in a region specific manner independent of segmentation (Hoey et a l., 1986). The unifying theme between Drosophila genes of theANT-C and BX-C and the murine Hox genes is that they would appear to be involved in the specification of regional id e n tity along the rostrocaudal axis and that these gene respect segment boundaries when and where they encounter them. The Hox 2.9 expression pattern reported by Murphy et a l., (1989) and by Wilkinson et al., (1989b) is curious not only because it is restricted to a single rhombomere but also because while i t is currently the most 3' member of Hox 2 i t is not the most ro s tra lly expressed gene. This gene is expressed caudally of its adjacent 5' gene, Hox 2.8 (Wilkinson et a l., 1989b). Therefore i t would appear that the absolute correlation between the physical order of the genes and their expression along the rostrocaudal axis does not hold. It w ill be important to determine if Hox 2.9 is the most 3' member of Hox 2 or not and if any more 3' gene does exist if this gene extends anteriorly of the Hox 2.8 gene. 4.7.3 Hox 2 gene expression in the mesoderm The expression of all genes in posterior regions and only of the more 3' genes in anterior regions is again illustrated in mesoderm. The somitic mesoderm exhibits expression of Hox 2 genes from most posterior prevertebrae to d iffe re n t anterior prevertebrae. Hox 2.2 is expressed up to the eighth prevertebra, Hox 2.1 to the seventh prevertebra while Hox 2.7, the most 3' gene studied, is the most a n te rio rly expressed extending to the f ir s t prevertebra (Figure 4.10). As for the viscera, the metanephric kidney, which originates relatively posterior at the end of the nephrogenic cord adjacent to the cloaca, can be shown to transcribe a ll Hox 2 members (Saxen et a l., 1986) (Figure 4.14). Expression of Hox 2.4, 2.2 and 2.6 has been shown in the mesonephric kidney suggesting that 5' genes in Hox 2 are expressed in the

68 mesonephric kidney (Figure 4.13). Yet as the mesonephric tubules run fo r quite a length along the rostrocaudal axisyftit would be unfair to say that all Hox 2 genes are expressed in a ll portions of the mesonephric tubules (Torrey, 1965). Hox 2.5, fo r example, may not be expressed in more anterior portions o f the mesonephric kidney, although this point has not been properly studied. Differential expression of Hox 2 genes can be seen in the more anteriorly derived developing stomach, lung, gut and gonad (DeHaan and Ursprung, 1965, Balinsky, 1981) (Figures 4.11 & 4.12). One cannot detect Hox 2.5,or 2.4 in the stomach yet transcripts from Hox 2.2, 2.1, 2.6 and 2.7 can be found. This same situation is also true of the lung. Interestingly none of the Hox 2 genes are expressed in the heart which develops in a very anterior region (DeHaan, 1965). Yet in the gonad Hox 2.2 cannot be shown to be expressed along with 2.1, 2.6 and 2.7. While in the gut Hox 2.1 is expressed in the enteric ganglia while 2.6 and 2.7 are expressed in the musculature. Considering the sites of o rig in of the mesodermal organs i t would seem that the Hox 2 genes are again responding to rostrocaudal position and generally confirm the presumptive sites of origin of the mesodermal tissue. Yet the fa ct that Hox 2.2 is expressed in the lung but not in the gonad is confusing. I t has been suggested that the expression of fewer and more 3' genes of Hox 2 reflects a more anterior position. Therefore from the above results one would suggest that the gonad is derived more a n te rio rly than the lung mesenchyme. This is contrary to available evidence which locates the developing somatic portion of the gonad at the top of the mesonephros i.e. caudal of the developing lung (Paranko, 1987, Zamboni and Upadhyay, 1982, Yoshinga et a l., 1988). This sort of situation is also true of the developing gut which does not express Hox 2.5 but does express Hox 2.6 and 2.7 even though i t lie s in a caudal region. While the expression patterns are puzzling i t is important to note that i f a given gene is expressed then the genes 3' of i t w ill also be expressed while those that lie 5' w ill not. This sort of expression fo r members of Hox 2 is observed in a ll other instances, where there is a clearer relationship between the sites of expression and position along the rostrocaudal axis. One does not observe sporadic expression of members of Hox 2. One problem with this discussion is that it is assumed that the site where the organ is firs t evident is also the site of origin of its mesodermal c e lls . This has not been shown to be true fo r these organs in the mouse. Another factor that one must take into account is time. These results represent the situation at 12.5 and 14.5 days of gestation but at earlier times the Hox 2 genes may display different patterns of expression. It is also

69 possible that Hox 2 gene expression in the gonad, the lung and the gut are responding to different cues in separate embryonic fields. Thus although it seems that mesodermal structures generally express Hox 2 genes in relation to their position the situation that is observed is not as clear cut as that in ectodermal derivatives. While anteroposterior position is certainly a very important factor in determining Hox 2 gene expression in mesoderm it must be modified by other factors to produce the observed patterns. 4.7.4 The anterior boundaries in mesoderm and in ectoderm are not coincident In terms of actual position along the anteroposterior axis the anterior lim it of expression in mesodermal structures, as compared to ectodermal structures, is more caudal. This is best illustrated by comparing the anterior boundaries of the same gene in the CNS and in the prevertebrae (Figure 4.1). Thus the anterior boundary of expression of a given gene in ectoderm is more rostral than the anterior boundary for that gene in the mesoderm. This may indicate that these genes are under different controls in the different germ layers. This is an a ttra c tiv e idea given the complications that are observed in the patterns of expression of these genes in the visceral mesoderm. In fact th is situ a tio n has been suggested fo r the Drosophila homeotic genes (Martinez- Arias et al., 1987). Yet as with expression in ectodermal derivatives one finds that a ll members of Hox 2 are expressed in mesodermal structures that are derived from posterior locations, such as the kidney and posterior prevertebra, and that only the more 3' members of the locus are expressed in those structures that are more anteriorly derived, such as the lung and the anterior prevertebra.

I t is clear that Hox 2 genes are d iffe re n tia lly expressed along the rostrocaudal axis of the mouse embryo and that the position of a gene within the cluster is related to its extent of expression along the rostrocaudal axis. While th is is most apparent in the central nervous system i t is also evident in other ectodermal and mesodermal derivatives. 4.7.5 Correlation between gene order and expression is a general aspect of homeobox clusters The results described above are in agreement with those of Gaunt et a l. (1988) and Duboule and Dolle (1989) who have also shown a sim ila r correlation between the position of some genes within other murine clusters and their relative extent of expression along the rostrocaudal axis. In all four of the murine homeobox clusters, the genes located at the 5' end are expressed in more posterior domains than those located in the 3' part of the same cluster. It w ill be important to determine to what extent murine members of the same

70 subfamily share similar anteroposterior domains of expression. The Dfd subgroup (Hox 2.6, 1.4 , 5.1) appear to have very sim ila r patterns of expression (Gaunt et a l. 1989). However in the Abd-B subfamily the Hox 2.5 member is expressed in more anterior regions of the CNS than the Hox 5.2 member (Duboule and Dolle, 1989) and both Hox 2.5 and 2.4 are expressed more anteriorly than Hox 3.1, a member of the Hox 2.4 subfamily (Brier et a l., 1988). This suggests that genes in different clusters that are members of the same subfamily do not necessarily have identical domains of expression. In both mouse and Drosophila, anteroposterior expression of homeobox genes correlates with the organisation of the complexes. But what is even more s trik in g is that the homeobox sequence of the most p o ste rio rly expressed member of Hox 2 (Hox 2.5) is most closely related to the most posteriorly expressed member of BX-C (Abd-B). This correlation holds for the other genes in both the mouse and Drosophila clusters. For example 5cr, the Hox 2.1 related gene, is expressed posteriorly of Dfd, the Hox 2.6 related gene and from above i t is evident that Hox 2.1 is also expressed p o ste rio rly o f Hox 2.6. From this it is concluded that there is a clear relationship between the relative position, sequence identity and domains of expression along the anteroposterior axis for both the mouse and Drosophila homeobox gene complexes. By inference th is strongly argues fo r the existence of an ancestral cluster, prior to the divergence of the protostomes and deutrostomes, that also exhibited this correlation between the order of the genes and their expression along the rostrocaudal axis. From sequence comparisons i t is suggested that th is ancient cluster would have had at least six homeobox containing genes and that these genes were also involved in the interpretation of rostrocaudal position. Thus homeobox gene clusters would represent a very ancient and ubiquitous system for the interpretation of rostrocaudal positional information.

71 CHAPTER FIVE - DYNAMIC HOX 2 GENE EXPRESSION DURING DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM

5.1 Introduction Many of the Drosophila segmentation and homeotic genes are expressed in the embryonic nervous system. The segmentation genes eve, en, and ftz are expressed in a specific subset of neurons in every segment of the developing central nervous system and seem to be involved in neuronal determination (Doe et a l., 1988b, 1988c). The homeotic genes are expressed at th e ir highest levels in the embryonic nervous system (Doe and Scott, 1988a). Unlike the segmentation genes the homeotic genes are not expressed in every segment of the CNS and as such are not thought to be involved in the specification of segmentally reiterated features of the CNS. Instead these genes tend to show peak expression in given parasegments with lower levels of expression in more posterior regions and are thought to regulate correct anteroposterior differentiation in the CNS (Doe and Scott, 1988a). Mutations in these genes affect those regions that exhibit the highest levels of expression of the genes. The expression patterns of the murine homeobox genes along the length of the CNS are remarkably similar to the situation in Drosophila, with strong anterior boundaries and expression trailing back through more posterior regions. Unlike Drosophila, the role of the murine homeobox genes in the ontogeny of the central nervous system is not clear. The mammalian CNS^____ lie s dorsal ly is considerably more complex than the ventral Drosophila CNS. The mammalian CNS develops from a pseudostratified neuroepithelium and over a period generates the diverse range of cell types. The transverse organisation of the mammalian spinal cord and hindbrain is relatively similar throughout its length rostral to caudal, except for differences in timing of maturation of given classes of neurons. The different neuronal ce ll types are organised and show spatial and temporal development in the dorsoventral plane (Altman and Bayer 1984). The large motor neurons of the ventral horn are the f ir s t major group of neurons that are born and among the earliest maturing cells of the spinal cord. This is followed by the relay neurons which are located la te ra lly and la s tly by the interneurons of the dorsal horn which are among the la te s t born. While the motor neurons are the f ir s t major class of neurons to be born th e ir development is e a rlie r and more advanced in rostral regions as compared to caudal regions. This also tends to be true of the other neurons. Thus the development of the CNS occurs with rough ventrodorsal and rostrocaudal temporal gradients, such that the f ir s t

72 major class of neurons are born ve n tra lly and maturation is more advanced in rostral regions as compared to caudal regions. I f one is to approach the role of murine homeobox genes in the ontogeny of the CNS it is important to analyse their expression patterns, temporally and spatially, in the transverse plane. A number of groups have analysed the expression patterns of these genes at stages in the development o f the CNS and uniform, dorsal and ventral patterns of expression have been described (Brier et a l., 1988, Dony and Gruss, 1987, Toth et a l., 1987, Graham et a l., 1988, Duboule and Dolle, 1989, Krumlauf et a l., 1987). There has also been one study of the temporal patterns of expression of a single gene, Hox 2.5, which was shown to display different patterns at different time points (Bogarad et al., 1989). The fa ct that one observes changing patterns in the expression of one gene underlines the importance of analysing expression patterns at several time points. In this study the expression patterns of seven members of the Hox 2 cluster have been analysed during development of the CNS. Expression of all of these genes have been analysed on adjacent sections at multiple positions along the anteroposterior axis at several time points. These genes all show the same dynamic patterns of expression and only show differences along the anteroposterior axis, as has been illustrated previously (Graham et al., 1989). These rostrocaudal differences were used to suggest that these genes are involved in patterning along this axis. Yet the fact that there are no dorsoventral differences between any of these genes would suggest that they are responding to events that are occurring dorsoventral in the nervous system and are not actually involved in dorsoventral patterning. I t is suggested that these genes are expressed tra n sie n tly to confer anteroposterior position on newly born neuronal c e lls . As such one would expect these genes to show patterns of expression that mirror cell maturation in the dorsoventral axis.

5.2 Hox 2 genes are dorsally restricted at 12.5 days The is o la tio n , and use of, gene specific probes fo r 7 members of Hox 2, Hox 2.1 to 2.7, has been previously described (See chapter 4). In this study these probes were used in in situ hybridisation experiments to compare the patterns of expression of these genes in serial transverse sections of the nervous system. Embryos from d iffe re n t stages were analysed at several points along the anteroposterior axis. Since 12.5 days p.c. is the stage at which these genes show th e ir highest levels of expression this time point has often been used as a convenient stage for the analysis of the patterns of expression of Hox 2 genes, the

73 . < • '

Hox 2.5

Hox 2.4

Hox 2.2

Hox 2.7

Posterior Anterior

Mouse 2 5 2 4 2.3 2 2 2.1 2 6 2 7 2 8 2.9 Hox-2 (11)

5’ 3 ’

74 Figure 5.1 - Expression patterns of four members of Hox 2 at five different points along the rostrocaudal axis of a 12.5 day mouse embryo. Sections were taken at fiv e d iffe re n t points labelled A-E. The ro stra l to caudal order of the sections is indicated at the top of the figure. Representative bright field photographs are shown for each plane of section. The dark ground photographs display the patterns of expression of each gene at each of the five different points. The expression pattern changes from an "M" shape caudally to a sharp dorsal domain of expression in rostral regions. The probe used for each row of sections is marked on the le ft, the position of the section is marked at the top of each column. B - A bright field photograph of a 12.5 day mouse embryo is used to illu s tra te the plane of each set of sections, A-E. The organisation of the Hox 2 cluster is shown at the bottom. The number in parenthesis indicates the chromosomal location of this cluster. The genes are expressed in order from posterior to anterior as is indicated above the cluster.

75 organisation of which is again illustrated on figure 5.1 B. Figure 5.1 A shows the patterns of expression of four members of Hox 2 on adjacent transverse sections at five different points along the rostrocaudal axis, labelled A-E on figure 5.1 B. One can easily see that Hox 2.5, 2.4 2.2 and 2.7 all display similar patterns of expression at this stage . In the sections from position A, figure 5.1 A a ,f,k ,p , are caudal and cut through the metanephric kidney. One can note that all of the genes exhibit an "M” shaped pattern with strong lateral/dorsal labelling. As one moves rostrally along the axis of the animal the "M" shape is s till evident up to the level of the lungs, at B, sections b,g,l,q, in figure 5.1 A. More rostrally s till the pattern changes such that all genes exhibit a strong dorsal domain of expression with a sharp restriction. This boundary in the expression pattern is particularly interesting as it does not seem to correlate with any obvious morphological boundary. This is evident in sections from positions C and D of figure 5.1. In all cases there is generally lower or absent expression in the ventricular layer, that layer which lie s immediately adjacent to the lumen. In the hindbrain region, E in figures 5.1 A and B, there is no expression of Hox 2.5 but the other three genes, 2.2, 2.2, and 2.7 are s till expressed. The observed difference between the expression of Hox 2.5 and that o f the other three genes in the hindbrain results from the fact that there is a correlation between the physical order of genes in Hox 2 and their expression along the rostrocaudal axis (see chapter 4, Graham et al., 1989). This has also been shown to be true of the other murine Hox clusters (Gaunt et al., 1988, Duboule and Dolle, 1989). The Hox 2 cluster is illustrated in figure 5.1 B and it is clear that as one moves along the cluster in a 5' to 3' direction each successive gene has a more anterior boundary of expression. Thus Hox 2.5 is not expressed in the hindbrain, figure 5.1 A e, since its rostral extent of expression maps in the spinal cord. It is important to note that the only differences that one observes between the genes in a transverse analysis of expression in the central nervous system is that imposed by the rostrocaudal axis. Otherwise the genes show the same patterns of expression at the same positions. It is interesting that the pattern that one observes changes along the rostro-caudal axis from an "M" shape to a sharp dorsal restriction. To address how these patterns were generated the expression o f these seven Hox 2 genes were analysed at both earlier and later stages during the development o f central nervous system. Again a ll of the seven Hox 2 genes, that were analysed,show identical patterns of expression.

76 Figure 5.2 - The patterns of expression for seven members of the Hox 2 cluster in the 10. 5 day nerve cord. Transverse sections have been taken at various points along the rostrocaudal axis and have been probed with each of the seven probes and the results are shown in dark ground. Each gene is expressed across the nerve cord. The probes were as follows: a- Hox 2.5, b- Hox 2.3, c- Hox 2.4, d- Hox 2.2, e- Hox 2.1, f & h- Hox 2.6, g & i- Hox 2.7.

77 H o x 2 . 2

1 0 . 5

1 1 . 5

■ g g m

Figure 5.3 - Change in expression pattern of Hox 2.2 in the spinal cord between 10.5 and 11.5 days of gestation. Transverse sections have been taken from the spinal cord of a 10.5 and 11.5 day mouse embryo, shown in bright field on the le ft, and probed with the Hox 2.2 gene. The in situ results are displayed in dark ground on the right. DRG- Dorsal Root Ganglia, M- Motor Horn

78 5.3 Hox genes are expressed uniformly across the nerve cord at 10.5 days There is clearly a temporal aspect to the patterns of expression that one finds fo r the Hox 2 genes in the developing central nervous system since a dorsal restriction in the expression of these genes at earlier stages is not evident. Expression of all of the genes at 10.5 days is shown in figure 5.2 and one can observe that a ll genes are uniformly expressed across the neural tube at this stage. This uniform pattern of expression is observed at all points along the axis that are within the rostrocaudal domain of expression of each gene. The sections in figure 5.2 are not all adjacent but are from three different positions along the rostrocaudal axis. Sections a and b are from the hind limb level and have been probed with Hox 2.5 and 2.3 respectively while sections c and d are from the prospective lung region and have been hybridised with Hox 2.4 and 2.2 probes. Sections e, f and g are adjacent hindbrain sections that illustrate the expression of Hox 2.1, 2.6 and 2.7, respectively. The strong expression of Hox 2.6 and 2.7 across the hindbrain is also displayed in h and i. From these results it is clear that all of the Hox 2 genes analysed are expressed uniformly across the neural tube at 10.5 days of gestation. 10.5 days of embryogenesis is the period during which the dorsal root ganglia are formed (Rugh, 1968, Wentworth, 1984b). These sensory ganglia are derived from neural crest and are metamerically organised on either side of the spinal cord. In figure 2 one can observe the expression of Hox 2.5, 2.4, 2.3 and 2.2 in the forming dorsal root ganglia. Hox 2.1, 2.6 and 2.7 are also expressed in these structures although th is is not shown in fig u re 5.2 since the sections that have been probed for these genes lies more rostral than the extent of these ganglia. 5.4 Strong lateral expression of Hox 2 genes at 11.5 days This pattern changes at 11.5 days. This is shown for the Hox 2.2 gene in figure 5.3. While the expression at 10.5 days of gestation is clearly all across the nerve cord a day la te r expression is no longer spread evenly dorsal to ventral but is now abundant in la te ra l regions, down graded in motor horns and s t i l l generally expressed in other regions. One can also note the expression in the forming dorsal root ganglia (Figure 5.3). Figure 5.4 shows expression of six members of Hox 2 at various points along the anteroposterior axis of the central nervous system. Again the patterns displayed by the genes are identical at other points along this axis (data not shown). Sections a and d are from re la tiv e ly caudal regions while b,c,e, and f are from the hindbrain region. I t is clear from figure 5.4 that fo r a ll o f the genes there is stronger expression, although not exclusive, in the lateral

79 Figure 5.4 - Transverse sections of the 11.5 day mouse nerve cord probed with Hox 2 genes. The results showing strong lateral expression are displayed in dark ground. The probes used were as follows: a-Hox 2.5, b- Hox 2.4, c- Hox 2.2, d- Hox 2.1, e- Hox 2.6, f- Hox 2.7.

80 Hox-2.5 Hox-2.5 Hox-2.4 Hox-2.2

81 Figure 5.5 - Expression of three Hox 2 genes at three different points along the rostrocaudal axis of the 14.5 day mouse embryo. Each column o f sections have been probed with the same gene as is indicated at the base. Each row of sections is from the same position along the rostrocaudal axis. The results reveal a reappearance of ventral expression in more ro stra l regions and are shown in dark ground.

82 regions . At th is stage the motor horns are v is ib le and do not show as high levels of Hox 2 transcripts. This is most obvious for Hox 2.2 in figure 5.4d. 5.5 Expression of Hox 2 genes reappear ventrally at 14.5 days Analysis of la te r stage of development reveals that the pattern described fo r the ro stra l 12.5 day cord is also observed caudally at 14.5 days. This is illustrated very clearly for Hox 2.5, 2.4 and 2.2 in figure 5.5 particularly in sections c,f, and i which cut through the metanephric kidney and the more ro stra l sections b,e and h which are at the lung le v e l. Again a ll o f the Hox 2 genes are dorsally restricted and a sharp demarcation, that does not correlate with any morphological boundary, is revealed. This pattern changes as one move more anterior with expression turning on again in the ventral half of the neural tube. This is shown for Hox 2.4 and 2.2 in figure 5.5 on sections d and g. Hox 2.5 does not exhibit this pattern since this pattern is only evident in regions which are more anterior than the boundary of Hox 2.5 expression. Thus again the only difference that one observes between these genes relates to their differential expression along the anteroposterior axis. This difference in expression is not restricted to ectodermal tissues but is also found in mesodermal derivatives. One can note the expression of Hox 2.2 in the mesenchyme of the lung in figure 5.5 section h but not the expression of Hox 2.5 or 2.4 in the adjacent sections, b and e. 5.6 Hox 3.1 is expressed in stripes The sharp dorsal restriction in expression is particularly interesting. This pattern was compared with that of a homeobox gene from another cluster. The Hox 3.1 gene was chosen since th is gene had previously been described as exhibiting a ventral domain of expression in the 12.5 day nerve cord although without a sharp re s tric tio n ^Breieret a l., 1988). Adjacent sections were probed with Hox 2.5 and Hox 3.1 and the expression in the neural tube analysed. In rostral regions Hox 2.5 displayed a sharp dorsal domain of expression while Hox 3.1, as previously reported, was most abundant ventrally and did, not display a sharp demarcation in expression. Yet more in te re stin g ly in caudal regions while Hox 2.5 displayed the typical "M" shape pattern Hox 3.1 exhibited a strikingly different pattern. This pattern is shown in figure 5.6. Hox 3.1 is expressed in a strip e across each h a lf of the neural tube projecting la te ra lly from the edge of the ve n tricu la r zone. There is also expression of this gene along the sides of the ventricular layer. Expression is absent from other regions. Figure 5.6 shows the expression of Hox 2.5 on an adjacent section displaying the previously described "M" shaped pattern. Thus the expression pattern of at least two genes from different clusters are radically different at the same stage of development.

83 84 Figure 5.6 - A comparison between the expression patterns of the Hox 2.5 and the Hox 3.1 genes in the caudal 12.5 day mouse spinal cord. The bright fie ld photographs show the regions that were probed and the very different hybridisation patterns that were revealed are shown in dark ground in the lower two rows in lower and higher power. While Hox 2.5 is expressed dorsally Hox 3.1 is expressed in stripes. The probe used is indicated at the base of each column.

85 5.7 Discussion The expression patterns, in transverse sections of the nerve cord, for seven members of Hox 2, Hox 2.1 to 2.7, have been compared on serial sections. This comparison has used embryos at d iffe re n t stages and sections have been taken from numerous points along the rostrocaudal axis. The results show dynamic patterns of expression fo r these genes during the development o f the central nervous system. I t is important to note that a ll seven genes display essentially identical patterns of expression. The only differences that one can observe between any of these genes at any of the stages analysed, from 10.5 - 14.5 days of gestation, is in their expression along the rostrocaudal axis. As has been previously reported the genes of the Hox 2 cluster show differential expression along the rostrocaudal axis such that there is a correlation between the physical order of the genes and their expression along this axis. As one moves from Hox 2.5 in a 3' direction each successive gene shows a progressively more anterior lim it of expression (Graham et a l., 1989). From the earliest times analysed (9 days of gestation) these genes show expression across transverse sections of the neural tube and show expression in the closing neural folds (data not shown). At 10.5 days of gestation the expression of these genes can s till be seen to be all across transverse sections of the neural tube. A day later members of this cluster show strong la te ra l expression, n il or very low levels in the motor horns and general expression in other regions of the nerve cord. By 12.5 days the caudal regions of the spinal cord exhibit a more dorsalised pattern with expression being found in lateral dorsal regions but not in the ventricular zone or in ventral areas, giving an "M" shape. Yet in rostral portions of the 12.5 day CNS the transcripts of these genes show dorsal restriction with a sharp demarcation, that does not correspond to any obvious morphological boundary. Dorsal restriction has also been described for the Hox 1.4 gene and again at 12.5 days th is gene was found not to be expressed in the ve n tricu la r zone (Toth et a l. 1987). The ro stra l 12.5 day pattern can s t i l l be observed in caudal portions of the 14.5 day spinal cord but not in more rostral regions. In ro stra l regions of the 14.5 day CNS do not display th is sharp dorsal restriction but expression can be found across the neural tube in both ventral and dorsal regions. These results are consistent with those of earlier studies on the expression patterns of members of Hox 2 and in particular confirm the results of Bogarad et al., (1989) with the Hox 2.5 gene who describe identical spatial and temporal patterns of expression. The pattern that was previously described

86 for Hox 2.6 with expression across the cord is thought to represent an intermediate pattern between that observed at 11.5 days and that at 12.5 days (Graham et a l., 1988). One problem in using mice of the CBA stra in is the lack of synchrony in any given litte r, thus it is d ifficu lt to give accurate estimations based on timings of the stage of a given embryo. The expression of Ghox 2.1 in the CNS, the chicken homologue of Hox 2.1, has been shown to be predominantly dorsally located at stage 25, a stage that is approximately equivalent to a 12 to 13 day mouse embryo (Hamburger and Hamilton, 1951, Wedden et a l., 1989). Thus in two vertebrate species two homologous genes show homologous patterns of expression in the CNS. This may suggest that these genes are performing identical functions in both species. The Hox 3.1 pattern of expression is obviously very different from that of members of Hox 2. Not only is it ventrally abundant in the rostral portion of the nerve cord at 12.5 days of gestation, while the Hox 2 genes display a sharp dorsal domain of expression, but in caudal regions Hox 3.1 is expressed in a stripe across each side of the spinal cord extending laterally from the edge of the ventricular zone. At this level it is also expressed along the side of the ventricular zone. A study of earlier patterns of expression of Hox 3.1 has not been conducted and i t w ill be important to analyse these e a rlie r stages i f we are to understand the observed 12.5 day patterns. It is clear that the Hox 2 genes show differential rostrocaudal expression and this fact has been used to suggest that they may play a role in rostrocaudal patterning, yet these same genes show no difference in the transverse plane of the spinal cord. Since none of these genes show any differences in their transverse patterns of expression in the developing spinal cord i t is suggested that the dynamic patterns that are observed reflect events in the spinal cord. If these genes were involved in dorsoventral patterning of the spinal cord one would expect to observe difference between the genes. 5.7.1 Hox 2 gene expression correlates with the time and place of the birth of the major classes of spinal neurons An important question now emerges as to what developmental events these genes are following? I t would appear not to be something as simple as proliferation of cells since short term thymidine labelling, that would mark c e lls in S-phase, of mouse neural tubes at various stages does not produce the same patterns, at comparable stages, as are observed with the Hox 2 probes (Nornes and Carry 1978). Nor does i t seem lik e ly that these genes are involved in the basic neuronal differentiation since neurons of the same class located at different points along the rostrocaudal axis do not express the same genes.

87 The dynamic patterns of Hox 2 expression are more sim ilar, in both temporal and spatial aspects, to that observed fo r the b irth of the major classes of spinal neurons (Sims and Vaughn, 1979, Altman and Bayer, 1984, Wentworth, 1984a, 1984b). The f ir s t major class of neurons to d iffe re n tia te are the motor neurons, which arise ve n tra lly at about day 9 to 11. The next major class are the commissural relay neurons whose ce ll bodies have been described as lying in lateral regions from ventral to dorsal. The early differentiating commissural neurons originate between 10.5 and 12.5 days p.c. Association or funicular relay neurons are born ju s t behind the commissural neurons and have ce ll bodies located dorsal of the oval bundle of His and as fa r ventral as the la te ra l funiculus (Wentworth, 1984b) . Lastly the in te r neurons of the dorsal horn are born between 12 and 14 days p.c. I t is suggested that the Hox 2 genes are expressed across the early neural tube and as neurons are born they tra n sie n tly express Hox 2 genes at high levels. The transient expression of these genes is to allow the differentiating neuron to understand its position along the rostrocaudal axis, through which Hox 2 genes i t expresses. Once the ce lls have begun to be fu lly differentiated they already know their position and turn off expression of these genes. This scenario would suggest that the early pattern of expression across transverse sections of the neural tube is temporally and spatially modified as each of the major classes of neuron are born. F irs tly one would expect to see expression in the ventrally lying motor neurons and then these ventral cells would turn o ff and expression would be high in the forming commissural neurons whose c e lls bodies should lie la te ra l at about 11.5 days. Expression would then recede dorsally, but s till be lateral, as the next major class, the association neurons, are born. Finally one would expect to see general dorsal expression as the in te r neurons of the dorsal horn are being born between 12 and 14 days. Since the neural tube matures in a rostral to caudal d ire ction we would suggest that the differences between caudal and ro stra l portions at 12.5 days re fle c ts difference in development along th is axis. This suggestion is also supported by the fact that the rostral 12.5 day pattern is s t i l l observed in caudal portions of older embryos. I t would moreover explain the differences observed at 14.5 days of gestation along the rostrocaudal axis. 5.7.2 The Hox 2 genes and the Hox 3.1 gene must be responding to different signals Thus the role of the Hox 2 genes would be to allow cells to understand their rostrocaudal position and the dynamic patterns that are observed in the

88 neural tube result from the birth of the different classes of neuron. This would seem to be the most plausible association between the observed patterns and events during ontogeny of the central nervous system. Although from the Hox 3.1 pattern that has been described it would seem that this is not the case, for this gene at least. The expression pattern displayed by this gene does not f it with the above interpretation of Hox 2 expression pattern and it seems likely that this gene must be responding to different cues than those that organise the Hox 2 expression. I t has been demonstrated that genes that share sim ila r positions w ithin different Hox clusters exhibit high degrees of sequence identity and it has been suggested that these gene clusters a ll arose from a common ancestral clu ste r (see chapters 3 and 4, Duboule and Dolle, 1989, Graham et a l., 1989). Related genes within different clusters have been shown to display identical rostral lim its of expression in the CNS (Gaunt et al., 1989). If, as has been proposed, these genes are involved in the establishment of rostrocaudal positional information it would seem fruitless to utilise many Hox genes to specify the same position. Yet the difference between the expression in the CNS of Hox 3.1 and Hox 2.4, its closest Hox 2 gene, may point out that while genes have the same anterior boundary in expression they are displaying very different patterns of expression in the dorsoventral plane. Thus it is possible that during deutrostome evolution as the central nervous system became more complex the Hox gene clusters were duplicated to increase the potential to specify different positions. It w ill obviously be important to determine the expression patterns of the genes from the other Hox clusters. 5.7.3 Re-expression ventrally and the sharp dorsal demarcation in the expression patterns There are s till some unexplained aspects of the patterns of Hox 2 gene expression. Firstly if the expression of these genes does correlate with aspects of neurogenesis why are these genes re-expressed ventrally in the rostral 14.5 day spinal cord? Possibly the ventral expression reflects aspects of gliogenesis, although this is uncertain. Another major point is the sharp line that demarks the dorsal expression of these genes at 12.5 and 14.5 days of gestation. As has been said this line does not correspond with any obvious morphological marker that is evident at this stage yet the transient nature of our lin e , which has been revealed by at least seven d iffe re n t homeobox genes would suggest that i t is of significance. One morphological marker that would lie in a similar position and divide alar and basal plates is the sulcus lim itans that has been described in ra t (Altman and Bayer, 1984) and human

89 (Muller and O'Rahilly, 1988) embryos. Yet this structure has been noted at comparatively earlier stages in rat and human and would seem not to be obvious at 12.5 days in the mouse and later. A similar restriction in the murine spinal cord has also been observed with immunohistochemistry fo r the c e llu la r retinol binding protein (CRBP) which has revealed a ventral pattern with a sharp lin e (Maden et a l., 1989). This CRBP pattern is established at about 10.5 days of gestation and it is interesting that CRBP respects a line in the neural tube 2 days before the Hox 2 genes. Comparative studies between CRBP and Hox 2 probes reveals that these probes respect the same lin e ( A.Graham, R.Krumlauf and M.Maden unpublished). We have not as yet determined whether the Hox 3.1 lin e that is apparent in caudal regions of the 12.5 day nerve cord lies along the boundary of CRBP expression. The fact that a transient line that separates alar and basal plates at mid-gestation times is revealed by homeobox gene expression and the presence of retinoid binding protein would suggest that it is of biological/developmental importance. One would predict that other molecules w ill also exhibit similar types of restriction in the developing nerve cord. It is intriguing that the line should be revealed by homeobox and re tin o id probes since there would appear to be an association between the expression of homeobox genes and re tin o ic acid such that most members of Hox 2 are activated upon addition of retinoic acid to F9 teratocarcinoma cells (N.Papalopulu pers. comm.). It should also be noted that the line that is evident through CRBP staining appears prior to the Hox 3.1 line which is prior to the sharp dorsal demarcation of Hox 2 genes. This is interesting since in tissue culture the Hox 2 genes respond to the addition of retinoic acid and in the embryonic CNS the re tin o l/re tin o ic acid is acting before the homeobox genes. Again i t is of interest that the c e llu la r re tin o ic acid binding protein (CRABP) can be detected at high levels in the forming commissural and association neurons and in neural crest derivatives which also express Hox 2 genes (Holland and Hogan, 1988a, Graham et a l., 1988, Maden et a l., 1989).

While this study actually focuses on the dynamic spatial and temporal patterns of Hox 2 genes in the transverse plane of the spinal cord it serves to reinforce the importance of their differential expression along the rostrocaudal axis. Hox 2 genes in th is system would seem to be acting to confer rostrocaudal position on newly forming neurons, when they need to understand their position. The expression pattern of Hox 3.1 is clearly d iffe re n t and i t would seem to be responding to d iffe re n t developmental cues. Yet the roles o f murine homeobox genes can be seen to be s trik in g ly sim ila r to

90 th e ir Drosophila counterparts. In both organisms these genes can be seen to be involved in the specification of anteroposterior position. Whether the murine and Drosophila central nervous systems are actually homologous is in question but i t is clear that these genes which are derived from a common ancestor are fu lfillin g very similar roles i.e. conferring anteroposterior position.

91 CHAPTER SIX - GENERAL DISCUSSION

6.1 Positions of genes in a cluster reflect domains of expression in the rostrocaudal axis for both mouse and Drosophila This thesis commenced with a discussion of regional specification (Wolpert, 1971). The work presented in th is thesis is consistent w ith, and strengthens, previous suggestions that murine homeobox genes play a role in the establishment of regional specification (see Holland and Hogan, 1988b). Evidence includes both expression studies and evolutionary considerations. It has been shown that there is a correlation between the physical order of the members of Hox 2 along the chromosome and their order of expression along the rostrocaudal axis, such that each successive 3' gene is expressed more rostrally (see chapter 4). The differential expression of the Hox 2 genes along the rostrocaudal axis is apparent in both ectodermal and mesodermal derivatives. This ordered pattern of expression would be consistent with these genes having a role in regional specification. This is supported by comparison with the Drosophila homeotic genes of the ANT-C and BX-C. I t was fo r these genes that a correlation between the physical order of the genes and their expression along the anteroposterior axis was firs t described (Harding et al., 1985, Akam, 1987). As has been discussed in chapter one i t was clear that these genes are involved in the establishment of regional specification along the Drosophila rostrocaudal axis (Harding et a l., 1985, Akam, 1987). The relationship between the murine and Drosophila clusters extends fu rth e r than aspects of th e ir expression patterns. Sequence comparisons indicate that the murine Hox clusters and the Drosophila homeobox clusters are both derived from a common ancestral cluster (Chapter 3, Duboule and Dolle, 1989, Graham et a l., 1989). The clustering and the ordered expression of the homeobox genes is therefore homologous in both Drosophila and mice and i t seems very lik e ly that the murine genes, in common with the Drosophila genes, are acting to confer rostrocaudal regional specification. Since mice and Drosophila are representatives of the deutrostome and protostome lineages, respectively, i t is suggested that the ancestral Hox cluster existed prior to the divergence of these two lineages. By inference it is proposed that the correlation between the order of the genes and their expression along the rostrocaudal axis may be a general feature of all higher metazoans. I t is also suggested that the Hox clusters are involved in regional specification in all higher metazoans.

92 6.2 Evolution of homeobox gene clusters and the evolution of the body plan Sequence comparisons have been used to suggest that the ancestral cluster contained 6 homeobox genes that subsequently expanded during evolution to y ie ld the Drosophila and mouse clusters (Figure 3.9, Graham et al., 1989). If these genes are involved in regional specification one would be tempted to link an increase in the number of these genes with an increase in the complexity of the body plan. The common ancestor to protostomes and deutrostomes is thought to have been acoelornate, b ila te ra l and non-metameric, possibly o f a comparable grade of organisation to that of a Plathyhelminthe or a Nemertean ( Clark, 1964, Akam, 1989). At this stage in evolution there must already have been molecular representations of back, front and of subdivisions of the middle region of the body. Interestingly during the evolution of the relatively complex organisation of Drosophila from that of the putative ancestor it is felt that the cluster of homeobox genes only expanded by two, with the evolution of abd- A and Ubx (Figures 3.8 and 3.9, Akam, 1989). It is these genes that are responsible for distinguishing between the thoracic and abdominal trunk segments, a d is tin c tio n which is it s e lf thought to be a re la tiv e ly recent event in arthropod evolution (Akam, 1989). I t would seem that the ends of an animal had been fixed early in evolution and that the duplication of these genes is to cater for internal specialisation. It is also evident from this that one cannot link the evolution of homeotic genes to the more drastic changes in the body plan that must have occurred during the evolution of the diptera (Akam, 1989). Unlike Drosophila, during the evolution of mammals from the common ancestor of the protostomes and deutrostomes the homeobox gene cluster not only expanded but it also duplicated and diverged to give at least four separate but related clusters (Figure 3.8). It would be interesting to understand how morphological changes that occurred during the evolution of the deutrostomes could possibly be related to the increased homeobox gene/cluster diversity. It is likely not to be due to the formation of a coelom as such, since the protostomes also evolved a coelom but did not duplicate the homeobox gene clusters. However, since it is fe lt that the coelom evolved separately in the protostome and deutrostome lineages the duplication of the Hox cluster(s) could be partially due to both the evolution of, and to the type of coelom that arose in the deutrostome lineage (Clark, 1964). There have been other major changes to the body plan that could be linked to the gene evolution such as the acquisition of a notochord and repeated blocks of muscle along the rostrocaudal axis. Mesodermal segmentation in this lineage could account for

93 some o f the duplication events, as could the subsequent evolution o f the vertebral column or the increased complexity of the viscera. The actual order of events in the evolution of the deutrostomes is obscure and even less is known about the temporal sequence of homeobox clu ste r duplication and diversity, therefore\at present it would be very d ifficu lt to tie the genetic events with the morphological events. From figure 3.8 it is clear that not all of the genes or subfamilies are represented in all the murine clusters. Thus although some clusters such as Hox 1 and Hox 2 have expanded from the ancestral clu ste r, the Hox 3 and Hox 5 clusters appear to be missing some subfamily members that are represented in Hox 1 and Hox 2. The genes that are absent are internal and thought to be derived from Antp and therefore evolutionarily more recent. Considering these points it is important to note that it has been proposed by Ohno (1970) that during chordate evolution there have thought to have been two whole genome duplications. The f ir s t of these is suggested to have occurred between the tra n s itio n from urochordates to cephalochordates and the second during the evolution of mammals from fish. Since i t is suggested that the four murine homeobox clusters arose from a single cluster during deutrostome evolution this has obvious relevance to homeobox gene clusters. It should also be noted that in Brachydanio rerio a Hox 2 and a Hox 3 cluster have been described but not a Hox 5 or a Hox 1 cluster. The Hox 1 and Hox 2 clusters are very close to each other as are the Hox 3 and Hox 5 clusters and therefore a complete genome duplication between fish and mammals could have resulted in the current four homeobox clusters, if this model is true it would also imply that there was a single homeobox cluster in urochordates but due to genome duplication there would be two in cephalochordates. It would be very interesting to extend the studies presented in this thesis to a phylogenetic study of the organisation and the expression pattern of the Hox genes. Hopefully one could begin to understand when the Hox clusters were duplicated and in what sort of animal such an event may have occurred. It may then be possible to relate the evolution of the Hox genes to that of the body plan and to prove or disprove the above proposed theory o f genome evolution as i t relates to homeobox genes. For example a comparison amongst the various organisms that one finds w ithin the proto- chordates could be very inform ative. A comparison between a cephalochordate, such as Amphioxus, which has a f u ll notochord extending from its t a il to the tip of the head and has segmental muscle although not a vertebral column, with an ascidian, which does not have segmented muscle, although i t has longitudinal muscle fib re s , nor does i t have as fu ll extended notochord, could

94 be very informative (Romer, 1976).

6.3 Relationships between the expression patterns of genes from related murine Hox clusters If these genes are involved in regional specification one would expect that the expansion of these genes/clusters would increase the number of instructions or degrees of freedom available to an embryo. Aspects of th is should be revealed by comparing the expression patterns of subfamily members . An analysis of three murine genes of the same subfamily, Hox 1.4, 2.6 and 5.1, has shown these genes to have the same anterior boundaries in the CNS and in the somitic mesoderm (Gaunt et al., 1989). This study also highlighted important differences between the stage- and tissue-dependent expression of these genes. For example Hox 5.1 is re la tiv e ly abundant w ithin ectoderm and mesoderm at early stages (8.5 - 9.5 days p.c.), yet was only weakly detected in the mesodermal components of th e lun(3anc5stomachat 10.5 days and was absent at 12.5 days. In contrast Hox 1.4 and 2.6 transcripts were relatively weak at 8.5 - 9.5 days p.c. but were abundant at 12.5 days. These three genes also show differential expression in retinoic acid induced differentiation of F9 teratocarcinoma cells (N.Papalopulu pers. comm.). Other subfamilies show even more drastic differences between their members as fo r example between Hox 2.5 and Hox 5.2. While Hox 2.5 has an anterior boundary at the level of the firs t permanent dorsal root ganglia, the Hox 5.2 boundary is located very much more p o ste rio rly. Dolle and Duboule (1989) describe very strong expression of Hox 5.2 in the developing forelimb while Hox 2.5 cannot be detected in this structure, at any stage of development. Another example of drastic differences in the expression patterns of subfamily members is that described for Hox 2.4 and Hox 3.1 in the developing nervous system (see chapter 5). While Hox 2.4 exhibits an "M" shaped transverse p ro file of dorsal expression in the caudal 12.5 day spinal cord the Hox 3.1 gene is expressed in la te ra l stripes across the neural tube and along the side of the ventricular zone. The expression patterns of related genes from different subfamilies give no obvious clues as to the possible changes in the body plan that accompanied the duplication and divergence of the Hox clusters. That having been said the differences that are observed in the spinal cord in transverse sections are particularly striking and could well reflect different roles for each cluster in the generation of the complex organisation o f the mammalian central nervous system. Thus the evolution of a complex central nervous system could have been associated with the evolution of more Hox gene clusters and with the

95 complexity of those clusters. The fact that there are sim ilarities in the expression patterns between subfamily members might indicate that these genes share some overlap and redundancy in function, and the differences observed could also usefully increase the variety of instructions available to the embryo. These differences would not necessarily need to be as gross as whole tissue or stage differences in expression. Since these genes are thought to encode transcription factors, it is possible that by varying the levels of these factors one could change the developmental decisions that a cell could make.

6.4 Hox 2 gene expression and the vertebrate head It is intriguing that the expression of members of Hox 2, or of any other murine Hox genes, in the ectoderm does not extend ro stra l of the hindbrain. This may suggest that there are more 3' members o f Hox 2, as yet undiscovered, which would be expressed in these more rostral regions. Given that the murine and the Drosophila clusters both evolved from a common ancestral clu ste r that is thought to have had a lab like gene it is curious that while lab is expressed a n te rio rly of its adjacent telomeric gene pb, the Hox 2 gene that is most related to lab, Hox 2.9, is expressed caudally of the adjacent 5' gene Hox 2.8 (Pultz et al., 1988, Diedrich et al., 1989, Wilkinson et al., 1989b). Therefore i t would be tempting to suggest that there is at least one other member of Hox 2 that is also related to lab and that this gene maintains the ordered expression. While this may or may not be the case it would s till seem u n like ly, taking in to account the above evolutionary arguments, that there are enough extra members of Hox 2 to provide regional information all the way from the hindbrain to the tip of the forebrain. Contrastingly the Drosophila lab gene is expressed in very anterior regions of the embryo (Hoey et al., 1986, Mlodzik et a l., 1988, Diedrich et a l., 1989). The vertebrate head shows numerous differences from the rest of the body particularly in its embryological origin. Unlike the rest of the body, in the head the neural crest functions as mesoderm and forms connective, skeletal and tissue (Le Douarin, 1982). I t has been suggested th a t, in the transition from protochordates to vertebrates and in the switch to an active mode of predation, many features occurring only in vertebrates were concentrated in the head (Gans and Northcutt, 1983). Thus it was necessary to add this "new head" on to the protochordate body and the onus for building much of this structure fe ll to the neural crest cells (Gans and Northcutt, 1983). The ends of homeobox gene clusters, and therefore the most a n te rio rly expressing gene, would have been defined p rio r to th is m odification. Therefore

96 the fact that Hox 2 genes do not extend beyond the hindbrain may suggest that these genes s till respect the anterior end of the "old body" prior to the addition of the vertebrate head. To return to phylogenetic approaches it would obviously be of interest to know i f a lab lik e gene existed in an animal such as Amphioxus, a cephalochordate, which is fe lt to be close to the sort of animal that existed prior to the vertebrates. If such a gene did exist it would be interesting to determine its extent of expression along the rostrocaudal axis. One would also expect that there would be more recently evolved genes that are used to interpret position in more anterior regions. In light of this it is interesting that sea urchins possess one Engrailed like gene while mice have two and that the murine En-2 gene is expressed in the midbrain (Dolecki and Humphreys, 1988, Joyner and Martin, 1987, Davis et a l., 1988). Thus the two engrailed genes were duplicated in the deutrostome lineage, possibly to cope with the vertebrate head.

6.5 Hox genes are involved in the interpretation of regional cues While th is work points towards homeobox genes having a role in the establishment of rostrocaudal regional specification in the mouse this role is probably in the interpretation of information rather than being the primary source of the information. This is clearly homologous to the role played by the Drosophila homeobox gene clusters (see introduction, Akam, 1987, Ingham, 1988). It is also important to note that the homeodomain proteins are nuclear proteins (Harvey et a l., 1986, Kessel et a l., 1987, Odenwald, et a l., 1987). Unlike Drosophila where regional specification is achieved in a scyncitial blastoderm this process in most other animals must deal with cellular structures. Therefore it is d ifficu lt to imagine how the products of the Hox genes which are nuclear proteins could actually specify the positional information in cellular embryos. This last point is important since there are many aspects o f murine/mammalian embryogenesis that would preclude the use of some o f the mechanisms that have been elucidated in Drosophila.

6.6 Differences between Drosophila and mouse development th a t could preclude the use of identical genetic mechanisms In Drosophila the establishment of anteroposterior axis and of the regional specification of points along that axis is achieved through the interaction of a number of transcription factors in the scyncitial blastoderm (see introduction, Akam, 1987, Ingham, 1988). The rostrocaudal axis of the mouse embryo is not established prior to fertilisation but is generated in the

97 cellular embryo during development, and is firs t made apparent at gastrulation. As has been discussed above it is possible that this axis is a consequence of the orientation of the implanting embryo with respect to its uterine environment (Smith, 1985). Therefore one would not expect to fin d the anteroposterior polarity in the mammal generated by bicoid or oskar lik e molecules. The signal(s) for rostrocaudal patterning in cellular embryos is more likely to be due to molecules that can communicate inter-cellularly. One would also not expect to find homologous pair rule or segment polarity genes in the mouse since with the possible exception of rhombomeres there do not appear to be any segments of developmental significance in the mouse embryo. That is not to say that genes which are structurally related w ill not be found but rather that if these genes are found then they w ill not have a homologous function. For example murine genes with homeodomains related to that of the Drosophila pair rule gene paired have been described although their expression patterns would seem to be very different than that of the Drosophila gene (Deutsch et a l., 1988). This is also true fo r the engrailed like genes ( Joyner and Martin, 1987, Davis et al., 1988, Davis and Joyner, 1988, Davidson et a l., 1988). A recent study o f the expression patterns o f the engrailed related proteins in a number of arthropods, annelids and chordates suggested that while these proteins probably have related biochemical functions in these different species they w ill not have homologous developmental functions in all the species analysed (Patel et al., 1989). Although the expression patterns within the arthropods, including Drosophilay are consistent with a common role in this phylum, in no other phyla were engrailed related proteins expressed in developing metameres (Patel et a l., 1989). While it seems that the functions of structurally related proteins in the mouse and Drosophila may not be fu n ctio n ally conserved i t might be possible that the molecular interactions between some of the proteins w ill be the same in both organisms. This might explain why regions of the Dfd, en, and caudal proteins outside of the homeodomain are conserved. They may help maintain a useful molecular pathway that could be activated in both Drosophila and mice but for different reasons. Another important difference between Drosophila and murine embryogenesis is that the body plan of the Drosophila embryo is established very rapidly. In contrast the body plan of the mouse embryo is generated over a larger time period (see Introduction). With regards to th is temporal problem i t would be of interest to examine the events and the molecules involved in the development o f a short germ insect. Short germ band insects, unlike long germ

98 band insects such as Drosophila, do not have a ll the segments of the embryo represented on the blastoderm but rather the early embryo only consists of very anterior regions and the more posterior portions are generated over time. In these embryos the regional signals have to cope with a cellular embryo and there is a strong temporal aspect to the regional specification. Therefore in these regards the mechanisms that could be elucidated in these embryos could be more applicable to the mammalian embryo.

6.7 Molecules that could organise Hox gene expression in vertebrates It is interesting to consider the nature of the hierarchical interactions that occur, and of the molecules that may be involved, in mammalian development and how these signals could re sult in the observed patterns of Hox gene expression. There are as yet no such candidate molecules in mammalian development but progress has been made in other vertebrate systems, most noticeably in Xenopus. Although Xenopus d iffe rs from the amniotes in a number of ways, such as in the induction and organisation of the body plan where it employs more determinative processes and in achieving a ll the primary body plan specification before growth begins, it is the best understood vertebrate with regards to these processes (Cooke, 1989a). I t is also f e lt that since the body plan is conserved across a ll early vertebrate development the features described in Xenopus w ill help c la rify the fundamental mechanisms.

6.7.1 Growth factors and regional specification in Xenopus embryogenesis It has been postulated that there are three signals that are involved in organising the mesoderm of the Xenopus embryo (Dale and Slack, 1987). There are two signals that emanate from the vegetal region, o f which one is the ventrovegetal signal which induces the overlying marginal region to become ventrally specified mesoderm and the other is the dorsovegetal signal which induces its overlying dorsolateral marginal zone to become the organiser, which represents dorsal anterior mesoderm. The th ird signal is that which is thought to be released from the organiser to dorsalize the ventrally specified mesoderm in a graded fashion such that the mesoderm that lies closest to the organiser is most dorsal and that furthest away most ventral. A number of molecules have been id e n tifie d which can mimic some of these e ffe cts. Ventral mesoderm can be induced with fib ro b la st growth fa cto r, while a TGF-b lik e factor derived from the XTC cell line, XTC-MIF, can induce dorsal type mesoderm and this tissue can act as an organiser (Smith, 1987, Slack et al., 1987, Cooke, 1989b). The factor fo r the dorsalisation of the ventral mesoderm may be of a similar biochemical nature to heparin (Dale and Slack, 1987).

99 Therefore perhaps molecules such as these may activate homeobox gene expression in vertebrate embryos. There have been two recent reports that would indicate that these are the sort of molecules that one should be looking fo r. F irs tly a novel frog homeobox gene, not of the Antp class, was found to be induced in competent ectoderm by mesoderm inducing facto r secreted by the XTC c e ll lin e (Rosa, 1989). This factor is probably XTC-MIF. This homeobox gene, M ix .l, is not expressed in ectoderm or mesoderm in the embryo but is re s tric te d to endoderm (Rosa, 1989). Secondly there is another Xenopus homeobox gene, Xhox3, which is again not of the Antp class but is most closely related to the Drosophila eve homeodomain, which is predominantly located in posterior regions of the embryo (Ruiz I Altaba and Melton, 1989a, Ruiz I Altaba and Melton, 1989b). This gene has been shown to be down regulated upon XTC-MIF treatment but up-regulated by FGF treatment (Ruiz I Altaba and Melton, 1989b). Thus XTC-MIF which can induce anterior dorsal mesoderm down regulates th is gene which is predominantly expressed in posterior regions while FGF which induces more ventral mesoderm acts to increase the level of Xhox3. These growth factor molecules can clearly regulate the expression of homeobox containing genes, although since these genes are not of the Antp class it is d ifficu lt to know how these results relate to the Hox genes. It is possible that genes such as Xhox3 which display broad domains of expression are activated by these growth factor molecules and they in turn activate the genes o f the Hox clusters which are d iffe re n tia lly expressed along the rostrocaudal axis and generate smaller domains. If this were the situation then th is would be very reminiscent of that in Drosophila where the embryo is in itia lly subdivided into large units and then in turn split into even smaller units (see introduction, Akam, 1987, Ingham, 1988).

6.7.2 Retinoic acid and rostrocaudal regional specification There are other molecules besides growth factors that could be involved in the early stages of regional specification in the vertebrate embryo. I t has been recently demonstrated that a l l -trans retinoic acid has an effect upon the body plan of the developing Xenopus embryo. This molecule has been shown to cause a transformation in the developing central nervous system of Xenopus (Durston et a l., 1989). The transformation observed is that of anterior neural tissue to a more posterior specification. One implication from this work is that there exists a posterior source of retinoic acid in the embryo. It is therefore interesting that the chick ta il bud mesoderm has ZPA activity when grafted to the appropriate region of the limb bud (Saunders and Gasseling, 1983). This may suggest that there is a localised posterior source

100 of retinoic acid in the ta il bud of the chick and possibly also in mammals.

6.7.3 Retinoic acid and homeobox gene expression Retinoic acid may be particularly interesting with regards to homeobox genes since many of these genes respond very fast to retinoic acid in teratocarcinoma cells (Colberg-Poley et al., 1985, Hauser et al., 1985, Deschamps et a l., 1987, Baron et a l., 1987, Murphy et a l., 1988). Results from this laboratory (N.Papalopulu pers. comm.) have demonstrated that the Hox 2 genes are very quickly responsive to re tin o ic acid and that the response is not the same fo r each member o f Hox 2. The speed o f the response of these genes to retinoic acid is exceptionally fast, in the order of 20-30 minutes, and therefore these genes may be primary targets for the action of retinoic acid. This fa s t response is thought to be mediated p o st-tra n scrip tio n a lly (N.Papalopulu and R.Krumlauf pers. comm.). If there is a posterior source of retinoic acid in the embryo and the genes are found to be differentially responsive across the cluster one could imagine a scheme that would establish progressively more rostral boundaries of expression for each successive 3' member of Hox 2. In such a situation one would be predicting that the most caudally expressed gene, such as Hox 2.5, would need the highest concentration of re tin o ic acid to respond while the more ro s tra lly expressed genes would be able to respond to lower concentrations of retinoic acid and therefore their domains of expression could extend more rostrally away from the source of retinoic acid. Another a ttra c tiv e aspect of th is model is that since the homeobox genes encode proteins that are transcriptional regulators these genes once turned on could maintain their own expression through autoactivation. Whilethere is no good evidence in vertebrates for autoactivation of homeobox genes, there is in Drosophila, e.g. Dfd (Kuziora and McGinnis, 1988). Therefore the action of retinoic acid to order the expression of these genes need only be transient since the genes once activated could maintain their own expression. With regards to this it had been noted that when the Xenopus embryos were treated with retinoic acid, and showed a posteriorisation of the front of the nerve cord, the expression of some homeobox genes was upregulated (Durston et a l., 1989 and N.Papalopulu pers. comm.). The relationship between re tin o l binding proteins and homeobox gene expression is also s trik in g ly demonstrated in the central nervous system. I t has been shown that the cytoplasmic retinol binding protein (CRBP), which is expressed in a reciprocal domain to the Hox 2 genes. CRBP is found in the ventral portion of the spinal cord while Hox 2 genes are expressed in a dorsal

101 domain. These two complementary domains of expression abut. The lin e which marks the boundary between these two domains does not correspond to any morphological marker. Thus again one can note a relationship between homeobox genes and re tin o ls .

These examples from Xenopus serve to point out the sort of molecules that may act as the regional cues in mammalian regional specification. As was discussed in the introduction one approach to the study o f development was to utilise molecules that were found to be important in other biological processes, such as cellular transformation, and to analyse their possible developmental roles. Thus i t is encouraging that a number of molecules that have been found to be important in regional specification of the Xenopus embryo have growth factor properties or are steroid hormones.

6.8 Other roles fo r homeobox genes While a role for Hox genes in anteroposterior specification may be that which has been evolutionary conserved and therefore ancient there may also be other roles for these genes in the mouse. The differences in the anterior boundaries in expression between the ectoderm and the mesoderm would suggest that the genes are responding to different signals in the different germ layers. This is substantiated by the fact the the Hox 2.1 gene exhibits a different 5' start site in the lung versus the spinal cord and therefore must be responding to different factors in these different situations (N.Papalopulu pers. comm.). Thus possibly the Hox 2.1 gene, and the other Hox genes that are expressed in the lung, may be performing a function that is related to lung organogenesis. Since these genes are expressed in the mesenchyme, which induces growth and branching on the developing endodermal bud, i t is possible that these genes are participating in these functions (Sorokin, 1965). This may also be true in the other organ systems of the mouse. Another example that may be similar is that of the expression patterns in the transverse plane of the developing nerve cord (Chapter 5). I t would seem, that although the rostral boundaries of expression do not change, the transverse pattern of expression is s p a tia lly modified as development proceeds. Therefore while there are signals that establish, or have established, the rostral boundary of expression, superimposed upon these re s tric tio n s are other signals which cause the observed patterns. These signals may be those that are involved in generating the ordered array of diverse c e ll types in the nerve cord. I f th is were so i t would be expected that other molecules would display similar restrictions in their expression

102 patterns and indeed at least in the case o f cytoplasmic retinol binding protein (CRBP) th is would appear to be the case (Maden et a l., 1989). I t has also been noted that a number o f the Hox genes have been found to be expressed in haemopoetic c e lls (Kongsuwan et a l., 1988, B la tt et a l., 1988b). The patterns of expression of some of the Hox genes varied in different cell lines (Kongsuwan et a l., 1988). Some authors have pointed out altered aspects of homeobox gene organisation or regulation that are found in leukaemic c e lls . There has been described a deletion on mouse chromosome 2 that is is associated with myeloid leukaemia which spans at least part of the Hox 5 clu ste r (B la tt and Sachs, 1988a). There has also been reported a genomic rearrangement in a myeloid leukaemic cell line that involves the insertion of an intracisternal A particle upstream of the Hox 2.4 gene (Blatt et a l. 1988b, Kongsuwan et al., 1989). This rearrangement results in the constitutive expression of the Hox 2.4 gene in this leukaemic cell line (Blatt et al., 1988b, Kongsuwan et a l., 1989). These observations may indicate that the Hox genes are involved in the d iffe re n tia tio n process of haemopoetic c e lls . Since the Hox gene fam ily represents a large collection of transcriptional regulators that could help define a wide range of diverse ce ll types i t would not be too surprising i f during the evolution of the haemopoetic system these genes were recruited.

6.9 Homeobox target genes It is important to note that while the homeobox genes may be able to self regulate themselves they may also be able to cross regulate each other and modify th e ir patterns of expression. For example i f homeobox genes do autoactivate themselves it is possible that other subfamily members, with very sim ila r homeodomains, could compete fo r the same sites and therefore affect transcription. This could be synergistic or antagonistic. It is clear that, unlike the situation in Drosophila, the posterior murine genes do not repress the expression of the more anterior genes. Thus instead of observing anteriorly and posteriorly restricted domains of expression one observes overlapping domains of expression with different anterior boundaries. There has been l i t t l e consideration of what genes homeodomain proteins may activate. Since it is fe lt that these genes act to interpret positional cues i t would seem lik e ly that they would activate molecules that would determine, directly or indirectly, the differences appropriate to a given position. For example, considering the prevertebrae one would not expect the homeobox genes to be implicated in aspects of cellular differentiation that are common to all prevertebrae since different combinations of homeobox genes

103 are expressed in d iffe re n t prevertebrae. I t would seem more lik e ly that the homeobox genes w ill e ffe ct genes which w ill be involved in the morphogenesis of the tissue. In the introduction an example of a graft from the hind limb bud to the fore limb bud, in the chick, was cited. In this experiment the grafted material remembered that it came from the hind limb and induced a claw in its new location in the fore limb. I t was suggested that th is piece o f material carried with it a record of its history and that this record could be thought of in terms of a gene network where genes are in an "on" or "off" state and that this record was established after the in itia l positional signal had been imparted. This is particularly interesting in the light of the observed patterns of homeobox gene expression, not only that of Hox 2 genes but also of the genes of the other clusters. One could think of the Hox genes as being part of the network of genes that can be "on" or "off" which are remembered after the in itia l positional information has been imparted and are thus part of the record of any tissue. It is certain that the expression of Hox genes in the mouse persists until late stages and even into the adult (e.g. Graham et a l., 1988).

6.10 Future experimental approaches to homeobox gene function While the studies presented in this work support the idea that the murine genes are involved in regional specification they are descriptive and it w ill be important to couple work of this kind with more embryological and genetic experiments. Hopefully from performing these types of experiments more information could be gained on the roles of Hox genes in murine embryogenesis.

6.10.1 Tissue grafts and Hox gene expression If these genes are involved in the interpretation of rostrocaudal regional information embryological manipulations could be performed that would confirm this hypothesis. For example, material from the hindbrain region could be transplanted to more caudal portions and the pattern of Hox 2 gene expression examined. This would more s p e c ific a lly mean comparing the expression of Hox 2.5, the caudally expressed gene and that of a more 3' gene which is more rostrally expressed, such as Hox 2.8. Since embryonic tissues become gradually re stricte d in th e ir developmental potency with time, there should be an early period when the transplanted material w ill respond to its new environment and differentiate accordingly and a later period when it will differentiate as if it were in its original location. These two different situations could be determined by the

104 use of histological or tissue-specific markers. If Hox 2 genes are involved in the interpretation of rostrocaudal regional specification one would predict that if the graft was done early enough then the piece of hindbrain that was grafted should express Hox 2.8 but not Hox 2.5 but when transplanted to its new location if it responded to this environment this piece of tissue should now switch on Hox 2.5 as a marker of its new posterior location. In the second situ a tio n when the transplanted material would not respond to its new environment one would predict that the material after transplantation would not switch on Hox 2.5. In this latter case one would be predicting that the Hox genes are acting as part of the mechanisms that allowed this piece of tissue to remember its previous history. Given the available methods fo r mouse embryo culture one could carry out such experiments on the mouse although it would be technically more convenient to work with chick embryos. Since the Hox genes are so strongly conserved (see chapter 3) one would be confident that the results from experiments on the chick embryo would be d ire c tly relevant to the mouse embryo.

None o f the mouse Hox gene clusters map to any known genetic lo ci therefore if one is to embark upon a genetic analysis of these genes the mutations must be created. Technology now exists in the mouse to create a range of mutations using DNA micro-injection into one cell embryos or through manipulation of embryonic stem (ES) c e lls followed by reintroduction into the animal (Rossant and Joyner, 1989).

6.10.2 Generation of gain of function mutants Micro-injection of Hox gene constructs containing a variety of regulatory sequences into one c e ll embryos could be used to inappropriately express these genes. In e ffe ct a gain of function mutant is being created and one would then screen fo r a mutant phenotype. This approach has been u tilis e d fo r two murine homeobox genes Hox 1.4 and Hox 1.1 (Wolgemuth et a l., 1989, B alling et a l., 1989). The Hox 1.4 gene was introduced into mice and expressed from a construct that contained lOkb of coding and 5' flanking DNA and about 2kb of SV40 sequences. This construct caused the Hox 1.4 gene to be overexpressed and resulted in the development of an abnormal gut although i t did not a ffe ct other tissue where i t was normally expressed (Wolgemuth et a l., 1989). The Hox 1.1 gene was put into transgenic mice behind the chicken b-actin promoter (Balling et al., 1989). This ectopic expression of Hox 1.1 resulted in craniofacial abnormalities which were interpreted as being due to the ectopic

105 expression of the Hox 1.1 gene in the head. The effect of the Hox 1.1 gene is fe lt to be in the respecification of the positional information received by the cephalic neural crest and therefore results in craniofacial abnormalities since the affected structures are derived from this cell population (Balling et a l., 1989). These results are very encouraging, in particular those with the Hox 1.1 gene, as they strengthen suggestions that the Hox genes play important roles in mammalian development. It w ill obviously be important to attempt such experiments with the genes of the Hox 2 cluster. One caveat that must be taken into account is that there are^uggestions that ubiquitous high level expression of these genes causes embryonic le th a lity . B alling et a l., (1989) have suggested that the mice carry the Hox 1.1 gene behind the chicken b- actin promoter survived to birth because this promoter is so weak. One possible reason for this supposed lethality is that since the homeobox genes have the capabilities to regulate themselves and to regulate each other the overexpression of any given Hox gene could perturb the whole system by interfering with the normal regulatory network. Therefore overexpression of one Hox gene could result in the repression or activation of a range of other Hox genes and therefore of their target genes. If these genes are involved in the establishment of regional specification then a large scale perturbation of this large gene family and its normal regulation could result in the death of the embryo. I t would be interesting to express any member of Hox 2 throughout the neural tube at a ll times in development and then to analyse any resultant phenotype. If one could direct expression to a specific region of the embryo, instead of generally overexpressing the gene everywhere, one may avoid lethality or at least delay it until reasonably late periods. Another attraction of this approach is that instead of having to deal with effects of overexpression of a gene in the whole animal, which can be exceptionally d ifficu lt to interpret, the overexpression could be restricted to a single organ or tissue where one would hopefully have a better chance of understanding any phenotype that was generated. This approach could possibly be applied to an analysis of the role to the Hox genes in the development of the central nervous system. This could be achieved by driving expression of any of these genes from any of the neural filam ent promoters which are active from 9 days o f gestation s p e c ific a lly in the neural tube (Cochard and Paul in, 1984). Expression from these regulatory regions should result in general expression of this gene at all places in the developing neural tube and therefore the gene w ill not exhibit the normal

106 dynamic patterns of expression in the neural tube. This experiment while expressing the gene at all points across the transverse plane of the neural tube would also result in inappropriate expression of the gene in more rostral regions. Thus one could analyse the effect of inappropriate expression across the transverse plane at positions along the rostrocaudal axis where the gene should normally be expressed and the effects of expressing a gene more rostrally than it normally is. The roles of homeobox genes in haemopoetic c e lls could be tested by specifically manipulating cells of this type. One could irradiate an animal to k ill its haemopoetic cel Is land then repopulate th is animal with bone marrow cells from another animal that had been infected with a retroviral construct that overexpressed one of the Hox genes. If these genes do have a role in haemopoetic lineages then possibly one could perturb th is process. The e ffe ct j of overexpression of the gene on the differentiation process would be assessed by analysing the cell types and the proportions of each cell type in the blood and tissues of the animal. Another aspect of these experiments is that one should analyse any transgenic overexpressing a given Hox gene for inappropriate expression of other Hox genes. One could also attempt to direct the expression of one Hox gene by the regulatory sequences of another. There has as yet been no identification of such regulatory sequences and the regulatory sequences fo r any gene may be spread throughout the whole cluster (Graham et a l., 1989). Experiments in transgenic mice using the b-galactosidase gene as a reporter gene behind regions from the 5' portion of the Hox 1.3 gene have only shown partial reconstruction of the patterns of expression (Zakany et al., 1988). This would support the above suggestion that the control elements for these genes are spread throughout the cluster. If this is true, to express one homeobox gene in the domain of another the genes would have to be swapped. That is , to express the Hox 2.5 gene in the Hox 2.8 domain then the Hox 2.5 gene would have to be placed in the Hox 2.8 position in the cluster. One way of achieving this end is to make use of the YAC clone that contains eight members of Hox 2 (see Figure 3.1). By using the recombination systems o f yeast, which are very e ffic ie n t in comparisons to mice, the Hox 2.8 gene, fo r example, on the YAC could be replaced with the Hox 2.5 gene by performing this recombination in the yeast cells. This recombinant YAC would then be isolated and introduced into mice the phenotype o f the resulting embryos analysed. Hopefully this would result in the expression of Hox 2.5 in the normal Hox 2.8 spatial and temporal pattern. Inappropriate expression

107 could well result in the posteriorisation of regions that normally do not express Hox 2.5. This may result in the alteration of structures in the hindbrain and peripheral nervous system and in the mesodermal organs.

6.10.3 Generation of loss of function mutants Gain o f function mutants can in themselves be confusing and i t is important to couple this approach with those that involve the generation of other types of mutation. These experiments hinge upon the use of ES cells. As has been stated in the introduction these cells are derived from the inner cells mass of the embryo, they can be maintained in culture, manipulated and then reintroduced back into the animal and they w ill contribute to the germ lin e (Rossant and Joyner, 1989). These c e lls have been used to create mutations in a number of genes including the Hox 1.1 gene and in the En-2 gene (Zimmer and Gruss, 1989, Joyner et a l., 1989). These mutations have been created by transfecting the ES cells with modified versions of the gene of interest and then screening for homologous integration events that have resulted in the replacement of an endogenous copy with the mutated copy. In the case of the Hox 1.1 gene a premature stop codon was introduced into the homeobox and this should result in the production of a truncated protein (Zimmer and Gruss, 1989). Joyner et a l., (1989) homologously integrated a neomycin resistance cassette into the En-2 gene and this integration event resulted in the removal of the homeobox. Thus using ES c e lls one could generate a vast range of different mutations in any gene of interest. In this laboratory and in collaboration with Dr. A.Bradley this technology has been used to disrupt the Hox 2.6 gene (A.Bradley and R.Krumlauf, pers. comm.). I t w ill be interesting to analyse the embryonic phenotypes once the mutant genes have been passed through the germ lin e and the mice bred to homozygosity. Mutations that severely disrupt a gene may cause very extreme phenotypes such as early embryonic death and therefore i t w ill be important to create mutations that do not result in such drastic changes in the protein product. Using this system one could create individual point mutations in any gene and generate mice carrying such a mutation. Such animals may have less severe phenotypes that could be very inform ative. Having proposed that severe gene disruptions may cause embryonic lethality mice carrying such mutations may well survive until relatively late times. This could happen because the early expression patterns of related subfamily members which share very sim ila r N-term ini, hexapeptides and homeodomains, such as Hox 1.4, Hox 2.6 and Hox 5.1, are very sim ila r during gastrulation stages (Gaunt et al., 1989). Thus if one of these genes were

108 deleted it is possible that the other subfamily members could take over the early functions of th is gene, while they are expressed in the same domains. The actual effect of the mutation would then only be fe lt when the specific patterns o f expression of the mutated gene arose. There is precedence fo r th is sort of situ a tio n in Drosophila. The Ubx and abd-A genes o f Drosophila are the most closely related genes of the homeotic genes in th e ir homeodomains (Akam et a l, 1988). These two genes e xh ib it some redundancy where th e ir expression patterns overlap, such as in the nervous system, and in these cases the a c tiv ity o f eith e r gene w ill su ffice (Ghysen and Lewis, 1986). There has also been described a mutation in the BX-C which results in a deletion of part of the Ubx and abd-A genes (Rowe and Akam, 1988, Casanova et a l., 1988). This mutation actually causes a fusion protein to be produced which consists of the amino terminal part o f abd-A and the carboxy terminal of Ubx. Embryos of th is type, which lack abd-A and Ubx genes but carry the fusion abd-A/Ubx gene can develop relatively normally with this fusion protein being able to rescue many o f the functions of e ith e r of these two proteins. Thus proteins with similar but not identical homeodomains can compensate fo r other homeodomain proteins that are absent.

These embryological and genetic approaches should begin to test many of the ideas presented in this thesis. Besides providing information on homeobox gene they w ill also be invaluable to any studies on mammalian embryology. These experiments and those by other labs, on other interesting molecules, w ill hopefully be able to begin to address the hierarchy of interactions that are involved in regional specification in mammalian development.

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134 The Murine and Drosophila Homeobox Gene Complexes Have Common Features of Organization and Expression

Anthony Graham, Nancy Papalopulu, of positional values along the anteroposterior axis of the and Robb Krumlauf animal. Thus, it would seem that homeoboxes are in­ Division of Eucaryotic Molecular Genetics volved not only in the process of segmentation but also in National Institute for Medical Research specifying positional information. The Ridgeway, Mill Hill Support for a role of homeobox genes in mouse devel­ London NW7 1AA opment is derived from their patterns of expression ob­ England served in the embryo. Homeobox genes are expressed in derivatives of the ectoderm (both central nervous system [CNS] and peripheral nervous system [PNS]) and meso­ Summary derm (lung, somites, gut, and kidney), but generally not in endoderm (Feinberg et al., 1987; Krumlauf et al., 1987; In situ hybridization analysis of mouse embryos shows Dressier and Gruss, 1988; Graham et al., 1988). The the seven members of the Hox-2 complex to be genes also display similar but not identical overlapping differentially expressed in the central and peripheral domains of expression within the same tissue (Gaunt et nervous system and in mesodermal derivatives (so­ al., 1986,1988; Graham et al., 1988; Holland and Hogan, mites and lung). Beginning at the 5' end of the cluster, 1988), which suggests that they may have a role in the es­ each successive gene displays a more anterior bound­ tablishment of positional information. ary of expression in the central nervous system. A Many of the Drosophila homeobox-containing genes gene’s position in the Hox-2 cluster therefore reflects have been found to be clustered in two separate but adja­ its relative domain of expression along the antero­ cent complexes, Antennapedia (ANT-C) and Bithorax (BX- posterior axis of the embryo, a feature observed with C), which are located on the right arm of chromosome Drosophila homeotic genes. Sequence comparisons three (Lewis 1978; Kaufman et al., 1980; S6nchez-Herrero of the Hox-2 cluster with other mouse and Drosophila et al., 1985). One important feature of these complexes is homeobox genes have defined subgroups of related that the physical order of the homeotic genes in the genes; in the mouse there are four clusters related by clusters is identical to the order in which these genes are duplication and divergence. Alignment shows a clear expressed along the anteroposterior axis of the embryo relationship among genes in the mouse and Drosoph­ during development (Harding et al., 1985; Akam, 1987). ila complexes, based on relative position, sequence The domains of expression along this axis also cor­ identity, and domains of expression along the ros­ respond directly to the segments that are affected by mu­ tral-caudal axis. Our results argue that these com­ tations in these genes (Harding et al., 1985). Therefore, plexes arose from a common ancestor, present before there is a correlation in Drosophila between the relative the divergence of lineages that gave rise to arthropods position of a gene within a cluster, its domain of expres­ and vertebrates. sion, and its effects on the specification of structures along the anteroposterior axis of the embryo (Harding et Introduction al., 1985; Akam, 1987). The organization of homeobox genes in clusters is a Drosophila genes implicated in specifying segmental pat­ general phenomenon that has also been described in tern and segment identity, such as engrailed and Anten­ mouse (Duboule et al., 1986; Hart et al., 1987b; Graham napedia (Antp), have been found to contain a common ele­ et al., 1988; Breier et al., 1988; Sharpe et al., 1988), hu­ ment, the homeobox (reviewed in Gehring, 1987). An man (Boncinelli et al., 1988), Xenopus (Harvey et al., initial study of the phylogenetic distribution of Anfp-related 1986), and zebra fish (Njolstad et al., 1988). The murine homeoboxes suggested that the presence of this se­ genome contains more Anfp-related homeoboxes than quence in the genome of an animal may correlate with a Drosophila, with at least 25 members identified to date in segmented body plan (McGinnis, 1985). Yet it was subse­ this large gene family. These genes are organized into quently shown that homeoboxes are evolutionarily more four clusters, termed Hox-1, Hox-2, Hox-3, and Hox-5, lo­ widespread, with copies of this element being found in the cated on chromosomes 6,11,15, and 2, respectively (Bu- genomes of animals from widely separate phyla including can et al., 1986; Hart et al., 1985; Breier et al., 1988; nonsegmented invertebrates and mammals (Holland and Featherstone et al., 1988). Analysis of the sequence and Hogan, 1986). Of the genes in which the homeobox was organization of genes from these clusters revealed that first discovered, only fushi tarazu is a segmentation gene, some members had substantial identity in several regions while the others are homeotic genes believed to be re­ of their predicted proteins in addition to the homeodomain sponsible for anteroposterior specification (Slack, 1984). (Regulski et al., 1987; Featherstone et al., 1988; Graham Later studies uncovered Drosophila homeobox-contain­ et al., 1988). These highly related members form a sub­ ing genes that exhibit patterns of expression that suggest group, and based on their identity and alignment within roles in positional information independent of the process the clusters, it has been suggested that the mouse of segmentation (Hoey et al., 1986). In the case of Antp- clusters are related to each other by the duplication and related homeoboxes, this role may be in the specification divergence of an ancestral cluster (Hart et al., 1987b; EMBRYO

S. CORD

LUNG

Figure 1. Expression of the Hox-2 Complex in Embryonic Tissues The Hox-2 cluster and the relative positions of its seven members are shown at the top. Hox-2.5 represents the most 5' gene and Hox-2.1 the most 3' gene. The expression in poly(A)+ RNA extracted from whole 12.5 day embryos, and spinal cord and lung from 14.5 day embryos, was analyzed by RNA blotting. Probes were 32P-labeled single­ stranded RNA, and each lane contains 2 ng of poly(A)+ RNA. The sizes of the major transcript of each gene are as follows: Hox-2.5, 2.4 kb; Hox-2.4, 1.4, 2.2, and 7 kb; Hox-2.3, 1.3 kb; Hox-2.2, 1.4 and 9 kb; Hox-2.1, 2.1, 4, 8, and 10 kb; Hox-2.6, 2.1 and 4.1 kb; Hox-2.7, 3.6 and Figure 2. In Situ Hybridization of the Hox-2.2 Gene in a Sagittal Sec­ 6.8 kb. tion of a 12.5 Day Mouse Embryo Both bright field (top) and dark ground (bottom) are shown. The bound­ aries of expression in the CNS (sc) and in the somitic mesoderm, at the seventh prevertebra (pv), are indicated.

Duboule et al., 1989; Graham et al., 1988; Papalopulu et al., 1989). The high degree of conservation in both sequence and organization of the mouse homeobox gene clusters is in­ genomic fragments for each gene and to isolate cDNAs, triguing, and may by analogy to the Drosophila clusters re­ which have allowed us to generate gene-specific probes flect aspects of their regulation or function. Thus, it is im­ for analyzing the structure, sequence, and expression pat­ portant to examine the existance of such relationships in terns of the Hox-2 complex. Figure 1 shows the ordering the same and different organisms. Toward this end, we of the genes in the Hox-2 complex (the numbering reflects have analyzed members of the Hox-2 complex with re­ the order in which the genes were discovered). All seven spect to correlations between their sequence, organiza­ genes have the same 5'-to-3' orientation with respect to tion, and patterns of expression in the mouse embryo. transcription, as revealed by sequence and expression Such a correlation has been found to exist and has re­ data, with Hox-2.5 as the most 5' member. vealed important similarities between the mouse and Dro­ A comparison of the expression in embryonic tissue for sophila homeobox gene complexes. These observations adjacent genes in the Hox-2 cluster is shown in Figure 1. shed light on the possible evolutionary history of homeo­ Because of the high degree of identity between many box gene clusters. We suggest that both the mouse and mouse homeobox genes, we used probes for each gene Drosophila homeobox clusters have arisen from a com­ that would minimize cross-hybridization. Clearly, every mon, ancient ancestral complex. This ancient cluster of member of the cluster is expressed during embryogene- genes is related to both its murine and Drosophila coun­ sis, and in RNA from 12.5 day embryos many of the genes terparts by the sequences and patterns of expression of display complex patterns of transcription as evidenced by the genes along the anteroposterior axis of the animal. multiple transcripts that differ in both size and intensity. There are tissue-specific differences in the transcript dis­ Results tribution of some genes, as seen for Hox-2.6 in Figure 1. The two major transcripts, of 2.4 and 4.1 kb, are observed Organization and Expression of Hox-2 in the RNA from whole embryos; however, the 4.1 kb band We have recently reported the isolation of cosmid clones is reduced in relative intensity in the spinal cord and is spanning the Hox-2 complex on chromosome 11, which barely detectable in lung. We do not know to what extent contains seven homeobox genes (Graham et al., 1988). the multiple bands represent differential splicing or multi­ These clones have now been used to prepare subcloned ple start sites, and it is possible that these transcripts Figure 3. Comparison of the Patterns of Ex­ pression for Hox-2.5, Hox-2.4, and Hox-2.2 in the 12.5 Day Mouse Embryo The top row represents in situ hybridization to near-adjacent sections showing bright fields of the entire embryo. Below are high-power mi­ crographs of these sections focusing on the regions in which anterior limits of expression are observed. These are represented in both bright field and dark ground with the probe used for each indicated at the bottom of each column of pictures.

could encode more than one protein. However, in the case correlates with genes located in the 3' part of the Hox-2 of Hox-2.1 we have found that there are multiple initiation cluster. sites that differ between tissues (Garbern et al., sub­ mitted). In Situ Analysis of Hox-2 Gene Expression Most mouse homeobox genes are expressed in ec­ along the Anteroposterior Axis todermal derivatives (CNS and PNS), and all members of The Hox-2 complex is expressed (as shown above) in the Hox-2 show high levels of expression in RNA from 14.5 day CNS, which is an ectodermal derivative that lies along the embryonic spinal cord (Figure 1). The expression in the length of the anteroposterior axis of the animal. We have spinal cord of all Hox-2 genes persists throughout devel­ used in situ hybridization to examine the spatial restric­ opment up to birth, and, in the one case we have exam­ tions in the patterns of expression of every member of ined, expression in the spinal cord continues in adult Hox-2 within the CNS along the anteroposterior axis. Fig­ stages (Krumlauf et al., 1987). Expression of the complex ure 2 shows an example of these results with the Hox-2.2 in a mesodermal derivative, lung, shows a different pat­ gene in a sagittal section of a 12.5 day mouse embryo. tern. The three most 5' members of the cluster, Hox- There is an anterior boundary in the domain of expression 2.3-2.5, are not expressed in this tissue, while the four in the CNS that maps to the hindbrain (sc). However, in genes at the 3' end of the complex display high levels of this section and other serial sections there is no clear expression in lung. The lung is generated from anteriorly posterior limit to this expression. A similar type of spatial derived mesoderm, and these results support the idea restriction is also observed in the somitic mesoderm, (see Discussion) that expression in anterior structures where expression extends from the most posterior prever- 2.7 2.6 2.1 Figure 4. Anterior Boundaries of Expression in the CNS for Six Members of the Hox-2 C o m ­ plex, Analyzed by In Situ Hybridization

The areas (A and B) of the near-adjacent sec­ tions photographed in high power are illus­ trated in the bright-field picture of the whole embryo (12.5 day) at the bottom. Area (A) was used for Hox-2.7, Hox-2.6, and Hox-2.1. The more posterior boxed area (B) was used for Hox-2.2, Hox-2.4, and Hox-2.5. Dark-ground photographs are shown for each gene as indi­ cated at the top and bottom of the pictures. Bright-field pictures of areas (A) and (B) are shown at the left to aid in identification of boundaries.

2.2 2.4 2.5

tebrae to an anterior boundary corresponding to the sev­ shown) and Hox-2.2 (Figure 3). The differences in the an­ enth prevertebra (Figure 2). The anterior cutoff of expres­ terior boundary between Hox-2.5 and Hox-2.4 are large by sion for Hox-2.2 within the somitic mesoderm is more comparison with the differences between Hox-2.2 and posterior than in the CNS, and these overlapping but off­ Hox-2.4\ however, each of the genes does have a succes­ set domains of expression in ectoderm versus mesoderm sively more anterior limit (see Figure 6). appear to be a common feature of homeobox expression Figure 4 illustrates, in serial sections of an embryo, that in the mouse (Gaunt, 1988; Gaunt et al., 1988; Dony and this general trend continues throughout the complex. Do­ Gruss, 1987). mains of expression for members 3' of Hox-2.2 extend Using all the Hox-2 probes, we have observed overlap­ more rostrally within the myelencephalon, and there is a ping but distinct domains of expression for each gene in relatively large difference between the anterior bound­ the CNS. In view of the absence of a clear posterior bound­ aries of Hox-2.2 and Hox-2.1. The successively more ros­ ary of expression, we have focused on examining the dif­ tral expression in the hindbrain for Hox-2.1-2.7 is shown ferent anterior boundaries of expression for each member in Figures 4 and 5. A summary of these in situ results is of the Hox-2 complex. We consistently observe the same illustrated in Figure 6. Based on these findings we con­ relative anterior boundaries of expression in all 12.5 day clude that the position of a gene in the Hox-2 cluster mouse embryos. All seven genes have been directly com­ reflects its relative domain of expression along the an­ pared in serial sections, using relative distances from mor­ teroposterior axis of the embryo in the CNS. In similar in phological structures to map the boundaries. Starting with situ hybridization experiments, this pattern appears to be Hox-2.5, we find that as one progresses through the Hox-2 already established in earlier embryos (9.5 day). This complex in a 5' to 3' direction, the limit of expression of correlation between position and expression in Hox-2 also each gene in the CNS becomes progressively more an­ appears to apply to patterns of expression in somitic terior. Figure 3 shows that for the most 5' gene, Hox-2.5, mesoderm (prevertebrae) and is consistent with expres­ the limit of expression maps within the spinal cord at the sion in lung (Figure 1). level of the third prevertebra. The boundary of the adja­ This correlation in limits of expression is analogous to cent Hox-2.4 gene is within the posterior myelencephalon the Drosophila BX-C and ANT-C homeobox gene clusters, (hindbrain), as are those of the more 3' genes Hox-2.3 (not where the physical order of the genes along the chromo- — 0 O O O O O O—hox2 2 5 2 4 2 3 2.2 2.1 2.6 2.7

2.7

Figure 6. Diagrammatic Representation of the Anterior Boundaries of Expression in the CNS for the Hox-2 Complex, and Their Correlation 2.6 with Gene Position in the Cluster A bright-field photograph of a 12.5 day embryo used in the serial- section analysis is shown below the cluster. Arrows indicate the actual anterior limits of expression detected in the near-adjacent sections from this embryo (Figures 2, 3, and 5). The limits were established using results from three or four sections for each gene and positioned by comparison of relative distances from morphological structures ob­ served in bright field. P, posterior; A, anterior.

2.1 pression to the prothoracic ganglion, and Sex combs reduced (Scr) to the second subesophageal ganglion. Deformed (Dfd) transcripts can be detected in the first r subesophageal ganglion (Harding et al., 1985), and labial Figure 5. Patterns of Expression for Hox-2 Genes That Show the Most (lab) has been reported to be expressed more anteriorly Anterior Limits of Expression in the CNS than Dfd (Mlodzik et al., 1988). A bright-field photograph of of a 12.5 day embryo is shown at the top to indicate the area pictured in high power. The dark-ground photo­ Homeodomain Sequence Comparison between graphs show the anterior limits of expression in the hindbrain for Hox- 2.7, Hox-2.6, and Hox-2.7. Probes for each gene are indicated to the Murine and Drosophila Clusters right of the pictures. Recently we reported (Graham et al., 1988) that the Hox- 2.6 gene shares substantial sequence identity in multiple regions of the protein with other mouse (Featherstone et al., 1988) and vertebrate homeobox genes (Harvey et al., some is related to the anteroposterior order of the seg­ 1986). Furthermore, Hox-2.6 formed part of a group of ment that recessive mutations of each gene affect (Hard­ mouse genes that displayed significant identity with the ing et al., 1985; Akam, 1987). A similar genetic correlation Drosophila Dfd gene (Regulski et al., 1987). Based on has also been described for the homeobox gene cluster these identities and the similarities in the expression pat­ HOM-C of Tribolium castaneum (Beeman, 1987). In situ tern described above, we wanted to examine whether hybridization analysis of the expression of the Drosophila other genes in the Hox-2 cluster might also show related­ homeotic genes in BX-C and ANT-C also reveals a rela­ ness to Drosophila homeobox genes (Krumlauf et al., tionship between the transcript distribution of each gene 1987; Graham et al., 1988). Sequences of the Hox-2.2 and in the CNS and its relative chromosomal position (Harding Hox-2.3 genes have been published (Hart et al., 1987b; et al., 1985; Martinez-Arias et al., 1987; Mlodzik et al., Meijlink et al., 1987; Schughart et al., 1988), and we have 1988). These studies have shown that the anterior bound­ now isolated and sequenced all the homeoboxes and ary of Abdominal-B (Abd-B) expression maps to the sixth most of the predicted proteins of the remaining members abdominal ganglion, while Abd-A transcripts extend to the of the Hox-2 complex (Krumlauf et al., 1987; Graham et al., first abdominal ganglion and Ultrabithorax (Ubx) expres­ 1988; Figure 7; unpublished data). sion reaches the metathoracic ganglion. The homeotic In Figure 7 we compare the sequences of all the Hox-2 genes of ANT-C maintain this trend with Antp showing ex­ homeodomains and their closest murine and Drosophila RKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHAICLTERQIKIWFQNRRMKWKKENK quences show similarity to one subfamily. The compari­ Hox2.5 SRKK-CP-K...... L--M— -D--H-V-RL-N-S---V...... M--M-- sons between Abd-B and Hox-2.5, Scr and Hox-2.1, Dfd Hoxl.7 TRKK-CP-KH...... L-M— -D-Y-V-RL-N... V...... H O X 3.2 TRKK-CP-K...... L-M— -D--Y-V-RV-N... V...... M--M-- and Hox-2.6, and lab and Hox-1.6 show the highest degree Hox5.2 TRKK-CP-K...... L--M D--Y-V-RI-N... V...... M--MS- Abd-B VRKK-KP-SKF------L-A-VSKQK-W-L-RN-Q--- V-...... N--NSQ of sequence identity. The Hox-2.7 subfamily shows iden­

Hox2.4 -R--- S- ...... l - P — K— -VS— G... V—-...... N tity with the zerknullt (zen) homeodomains, but zen is not Hox3.1 -RS--- S...... L--P K VS— G--- V-...... N a homeotic gene; this group may be more related to Hox2.3 ...... Y...... T...... Hoxl.l ...... — H- proboscepedia (pb; Pultz et al., 1988), which has a Hox2.2 GR...... -.... Y...... S- homeodomain very similar to that of zen (Cribbs and Kauf­ Hoxl.2 GR...... - ...... N...... man, personal communication). The remaining Drosoph­ Hox6.1 -R I-S...... -N ...... SN ila genes including Abd-A, Ubx, and Antp are all so similar Abd-A -R..... F...... H------L---LR Ubx -R...... T-H------M...... L— IQ in sequence that it is difficult to assign these genes to any Antp ...... of the remaining murine subfamilies, which are repre­ Hox2.1 G - A - T A...... S...... D ~ Hoxl.3 G--A-TA...... -...... S...... D-- sented by Hox-2.4, -2.3, and -2.2. Hox-2.6 displays several Scr T--Q-TS...... L--H- regions of identity, outside the homeodomain, when com­ HOX2.6 P--S-TA— Q-V...... Y...... V..... S..... DH- Hoxl.4 P— S-TA— Q-V...... T— S— V-...... DH- pared with the Dfd protein (Graham et al., 1988), and Hox5.1 P— S-TA— Q-V...... T--P ...... DH- Mlodzik et al. (1988) have found extended regions of ho­ Dfd P--Q-TA— H-I...... Y—-...... -T-V-S...... D-- mology between lab and Hox-1.6 (Duboule et al., 1986; Hox2.7 S--A-TA-SA-L V...... C-P--V-M-NL-N-S...... Y--DQ- Hoxl.5 S-— TA— P-LV...... M.p..V-M-NL-N...... Y--DQ- LaRosa and Gudas, 1988). The Scr protein (LeMotte et Hox4.1 S— A-TA--SA-LV...... FV-P-VQM-NL-N-S...... Y--DQ- ZenZl L-S-TAF-SV-LV— N - K S - M - Y - T QR-S-C--V...... F-DIQ al., 1989) shares identity with Hox-2.1, particularly in the ZenZ2 S-S-TAFSSL-LI— R— L-K-A-T... SQR-A V...... L-STN Pb ? amino-terminal region, where 10 of the first 13 amino acids

Hoxl.6 PNAV-TNS-TK-LT------K------AA-V—AS-Q-N-T-V...... Q--RE- are the same. Therefore, where protein sequence is avail­ Labial NNS-TNF-NK-LT A NT-Q-N-T-V------Q--RV- able, mouse and Drosophila genes identified as subfamily members, based on similarities within the homeodomain, y y y y 2 j 2j6 2 7 2j also display some identity in other domains. OOOOOOOO One striking feature of these comparisons is that if we align the mouse and Drosophila subfamilies, then the

com m on physical order of the Drosophila homeobox genes along -0 o 0 0 0 0 -a nce stor the chromosome and that of their closest related murine genes are identical (Figure 7). These results suggest that the murine and Drosophila homeobox gene clusters are OOOOOOOO D rosophila derived from a common ancestor that arose before the AbdB AbdA Ubx Antp Scr Dfd pb lab divergence of the lineages that gave rise to arthropods Figure 7. Amino Acid Sequence Comparisons of Mouse and Drosoph­ and vertebrates. Akam et al. (1988) have recently pro­ ila Homeodomains, Showing the Existence of Related Subgroups posed that the earliest ancestor for the myriapod-insect The Antp sequence at the top was used as the basis for all compari­ lineage possessed a homeobox cluster with distinct sons. Dashes represent identical amino acids. The separation into genes related to lab, pb, Dfd, Antp, and Abd-B. Subse­ subgroups is based on the sequence changes shared by the various quently, by duplication and divergence the Antp gene members. The relative order of the Drosophila genes on the chromo­ some is indicated at the left. References for the sequences used in gave rise to the Scr gene and then, more recently, to the these comparisons are detailed in Experimental Procedures. At the Ubx and the Abd-A genes. In our comparisons the best bottom of the diagram is a possible scheme for the evolution of murine overall identities between mouse and Drosophila are in and Drosophila clusters from a common ancestral cluster. This dia­ those genes (lab, Dfd, Antp, and Abd-B) predicted to be gram is based on sequence data currently available. the most ancient by Akam et al. (1988). In addition, the identity between Scr and Hox-2.1 argues that Scr was a part of the ancestral cluster. It appears that both flies and mice have four genes related to Antp. These could be de­ (ANT-C, BX-C) counterparts against Antp, the prototypi­ rived, wholly or in part, from duplication events that oc­ cal Drosophila homeodomain. This comparison clearly curred prior to the separation of the lineages that gave rise shows that there are significant similarities between many to arthropods and vertebrates, and have been part of the of the mouse homeodomains, and allows one to separate ancestral cluster. They may also have arisen by indepen­ the murine genes into subfamilies of related sequences. dent events in each of the major lineages, as has been These subfamilies are defined by the common amino acid postulated for the engrailed genes (Dolecki and Hum­ changes and positions of the changes (when compared phreys, 1988; Joyner and Martin, 1987). Even if the Antp with Antp) that are shared within a family. On this basis group of genes arose independently, our results argue for Hox-2.5, -1.7, -3.2, and -5.2 all form a subfamily, and the a common evolutionary origin between the mouse and mouse homeobox genes can be divided into eight such Drosophila clusters. subfamilies (Figure 7). The relatedness of genes within a mouse subfamily is strengthened by the fact that mem­ Discussion bers of a subfamily have a high degree of identity in multi­ ple domains over their entire predicted protein sequences. The Homeobox Gene Complex If we extend this comparison to include the Drosophila In this report we have compared aspects of the structure homeodomains, it is also clear that many of these se­ and expression of the Hox-2 complex with other mouse Posterior Anterior

Abd-B Abd-A Ubx Antp Scr Dfd Pb lab Drosophila

BX-C - / h h ANT-C (3)

i r i r Mouse 2.5 2.4 2.3 2.2 2.1 2.6 2.7 2.8 H O X -2 (11)

1.7 1.1 1.2 1.3 1.4 1.5 1.6 Hox-1 (6)

3.3 3.2 3.1 6.1 6.2 Hox-3 (15)

5.3 5.2 5.1 4.1 - ■ ------■ ---- ■ ----- Hox-5 (2)

Figure 8. Diagrammatic Representation of the Relationships between Drosophila and Murine Homeobox Gene Clusters The sequence comparisons shown in Figure 7 were used to align the clusters into an evolutionarily related network. The different members of a related subfamily are represented by a vertical row of boxes. Both the murine and Drosophila clusters show a correlation between their organization and patterns of expression along the anteroposterior axis of the embryo (see text for details). Filled boxes in the mouse clusters represent sequenced homeodomains, open boxes indicate identified but not yet sequenced mouse homeodomains, and hatched boxes indicate predicted genes, based on analogy to other clusters. The numbers in parentheses represent the chromosomal location of these clusters. The numbers above each box were those originally assigned as the genes were isolated (see Experimental Procedures for references); however, Hox-6.1 and -6.2 and Hox-4.1 have been reassigned to existing clusters on chromosomes 15 (Schughart et al., 1989) and 2, respectively, and we have kept their original nomenclature they are until renamed.

and Drosophila homeobox gene clusters. Based on iden­ make some genes undetectable. Using this alignment we tity shared within homeobox sequences and their pre­ might also predict the potential position and existance of dicted proteins, it is possible to divide the murine genes other genes in the parts of the clusters that have not been into subfamilies, also related by specific common differ­ cloned. Based on the extensive similarity between the ences they share when compared with the Antp homeodo­ Hox-2 and Hox-1 clusters, we isolated clones 3' of Hox-2.7 main (Figure 7). We and others have used this type of while looking for a Hox-1.6-related gene, and identified a comparison, in part, to support the idea that the mouse new gene (as yet unsequenced) that we term Hox-2.8. In Hox-2 and Hox-1 clusters are related by duplication events the same manner we have looked for genes 5' of Hox-2.5 (Fibi et al., 1988; Graham et al., 1988; Hart et al., 1987a; that would be related to Hox-5.3. We have walked 30 kb 5' Papalopulu et al., 1989). of Hox-2.5, but have no evidence for additional genes. In light of the newly defined Hox-5 cluster (Duboule and Dolle, 1989) and the assignment of Hox-6.1 and -6.2 to the Hox-3 cluster on chromosome 15 (Schughart et al.f 1989), A Common Ancestor for Mouse and Drosophila it appears that duplication and divergence of homeobox Homeobox Complexes genes have lead to at least four evolutionarily related com­ A remarkable feature of our comparisons with mouse and plexes in the mouse. In each of the four clusters, the rela­ Drosophila homeodomains is the substantial similarity be­ tive position of each subfamily member (Figure 7) and the tween many of the mouse subfamilies and genes in the physical order and spacing of the genes are very similar. ANT-C and BX-C complexes (Figure 7). The identity is This allows us to align the Hox clusters into an evolutionar­ based on common conserved changes within the homeo­ ily related complex, as shown in Figure 8. The different domains; however, in several cases where sequence data members of a related subfamily are represented by the are available these comparisons reveal that mouse and vertical rows of solid boxes. For example, Hox-2.5, -1.7, Drosophila members in the same subfamily (Dfd, Hox-2.6; -3.2, and -5.2 form one such family related to the Drosoph­ Scr, Hox-2.1; lab, Hox-1.6) also have conserved regions in ila Abd-B gene. The aligment also reveals that not all other domains of the predicted proteins (Graham et al., genes or subfamilies are represented in each mouse clus­ 1988; Featherstone et al., 1988; Mlodzik et al., 1988; ter, and suggests that either some clusters arose by partial LeMotte et al., 1989). There is a strong correlation be­ duplication or there has been sufficient divergence to tween both the position of genes and their relatedness within the mouse and Drosophila homeobox clusters (Fig­ Positions of Genes in a Cluster Reflect Domains ure 7). In Figure 8 we have positioned the Drosophila of Expression in the Rostral-Caudal Axis homeobox genes above the four mouse clusters and indi­ for Both Mouse and Drosophila cate the most closely related subfamilies to illustrate this In this paper we have demonstrated that all of the mem­ point. It is difficult to make an absolute one-to-one correla­ bers of the Hox-2 complex are expressed in slightly differ­ tion between the mouse and Drosophila genes, but sub­ ent overlapping domains in the spinal cord, with respect groups of Abd-B, Scr, Dfd, pb, and lab are observed. How­ to the anteroposterior axis (Figures 2-6). We have not de­ ever, because the three Drosophila genes (Antp, Ubx, and tected any posterior limits for expression of these genes Abd-A) are all highly similar to each other in the homeodo­ in the CNS. However, as one moves from the most 5' mem­ main, direct alignment of these with the mouse genes ber of the cluster (Hox-2.5) in a 3' direction, each succes­ Hox-2.2, -2.3, and -2.4 is uncertain. sive gene displays a more anterior boundary of expres­ It is important to note that Akam et al. (1988) have pro­ sion (summarized in Figure 6). The cutoff for Hox-2.5 in the posed that the ancestor of the Drosophila homeobox clus­ CNS maps opposite the third prevertebra, and the other ter possessed a set of four or five distinct genes of the lab, six genes have anterior boundaries in the myelencepha­ pb, Dfd, Antp, and Abd-B type, and that the Ubx and Abd-A lon. Our findings clearly show that the position of a gene genes arose by more recent duplication from the Antp in the Hox-2 cluster is correlated with its relative domain gene. The Scr homeobox is also very similar to Antp, and of expression in the anteroposterior axis of the embryo, it is not clear if Scr resulted from an early duplication sepa­ suggesting that these homeobox genes may be playing a rate from events leading to Abd-A and Ubx or was part of role in the establishment of positional information. This is the primordial cluster. The best sequence identities be­ analogous to the pattern observed for the Drosophila BX- tween the mouse and Drosophila genes in Figures 7 and C and ANT-C clusters. The physical order of the genes in 8 are observed for the set of genes predicted to be mem­ these clusters reflects the anteroposterior order of the bers of the ancestral cluster of Akam et al. (1988). In the segments each gene affects and their relative domains of mouse there also appear to be four genes related to Antp expression. (Hox-2.1-2.4). This may suggest that these genes arose by Our results are in agreement with the recent findings of independent duplication of the ancestral Antp-type gene Gaunt et al. (1988) and Duboule and Dolle (1989) showing also in the lineage that yielded chordates or that they are a similar correlation between the position of some genes derived from a similar, earlier duplication event in a com­ within other murine clusters and their relative extent of ex­ mon ancestor. This earlier event would have produced pression along the rostral-caudal axis. In all four of the more than one Antp-type gene in the ancestral cluster murine homeobox clusters, the genes located at the 5' end prior to the divergence between arthropods and ver­ are expressed in more posterior domains than genes lo­ tebrates. We favor the latter possibility at least in the case cated in the 3' part of the same cluster. It will be important of Hox-2.1, which also shows identity with Scr and sup­ to examine to what extent murine members of the same ports the idea that Scr was a part of this common ances­ subfamily all share similar anteroposterior domains of ex­ tral cluster. Interestingly, this group of Anfp-related genes pression. The Dfd subgroup (Hox-2.6, -1.4, -5.1) appears to in the mouse also appears to be the most variable part of have very similar patterns of expression (Gaunt, Duboule, some clusters, as illustrated in the Hox-5 cluster where and Krumlauf, unpublished data). However, in the Abd-B there are no members of the Abd-A, Ubx, Antp, or Scr sub­ familyHox-2.5 is expressed in more anterior regions of the family (Duboule and Dolle, 1989). CNS than Hox-5.2 (Duboule and Dolle, 1989), and both The sequence identity between the mouse and Dro­ Hox-2.5 and -2.4 are expressed more anteriorly than Hox- sophila genes does not necessarily imply that these 3.1 (Breier et al., 1988). This suggests that genes in the mouse genes are true vertebrate homologs of the Dro­ different clusters that are members of the same subfamily sophila homeotic genes. Some subfamilies have four do not necessarily have identical domains of expression. members related to a given Drosophila gene. As such, a An important question from our results concerns why precise murine equivalent would be hard to establish. many of the Hox-2 genes have anterior boundaries of These comparisons suggest that the murine and Dro­ expression in the myelencephalon. At 12.5 days these sophila gene clusters arose from an ancestral cluster that boundaries do not appear to correspond to any morpho­ existed prior to the divergence of the lineages that eventu­ logical structures within the hindbrain. However, in early ally gave rise to mice and flies. This is supported by the stages of neural development, periodic swellings along observation that some regions of the proteins besides the the axis of the neural epithelium have been observed, and homeodomain have been conserved (Regulski et al., these structures are termed neuromeres (rhombomeres 1987; Graham et al., 1988). One difficulty with this argu­ in the hindbrain). Previously, it had not been clear whether ment is that the Drosophila genes are divided into two sep­ segmentation of the neural tube, as defined by the rhom­ arate clusters although both are on chromosome three. bomeres, plays any role in establishing the underlying pat­ However, in a more primitive insect, the red flour beetle tern of development in the hindbrain. Recently, Lumsden Tribolium castaneum, the ANT-C and BX-C equivalents and Keynes (1989) have presented data suggesting that are found in a single homeotic cluster, HOM-C (Beeman, rhombomeres do play a significant role in the segmental 1987). Therefore, ANT-C and BX-C may originally have patterns of neuronal development in the chick hindbrain. been one cluster separated by a more recent evolutionary Consistent with these results, Wilkinson et al. (1989) have event occurring in certain insects. shown that rhombomeres represent domains of expres­ sion for the zinc finger gene Krox-20, providing molecular tire protein with Hox-2.1 and is expressed in anterior parts support for segementation of the CNS. of the CNS (Njolstad et al., 1988; personal communica­ The repeated array of spinal motor and sensory roots tion). It has been shown that related subfamilies are also and sympathetic ganglia in the PNS is a consequence of present in many vertebrates, including Xenopus (Regulski segmentation in the somitic mesoderm and is not an in­ et al., 1987; Graham et al., 1988; Harvey et al., 1986). Fritz trinsic property of the spinal cord or the PNS (Keynes and et al. (1989) have linked three Xenopus genes (Xlhbox 6, Stern, 1988). In the hindbrain the alternating pattern of 7, and 2) and suggest that they represent the Xenopus cranial nerve nuclei and their association with particular homologs of three mouse Hox-2 genes (Hox-2.5, -2.4, and rhombomeres (Lumsden and Keynes, 1989) is established -2.3). The Xlhbox 2 and mouse Hox-2.3 proteins have 75% in the absence of input from somitic mesoderm, implying homology over the entire protein. Therefore we suggest that segmentation in the hindbrain proceeds by a different that homeobox complexes in all vertebrates may have a mechanism. Therefore a mechanism involving inherent structural and functional organization analogous to that segmentation of the neural epithelium may be significant, we have described for the mouse. particularly in the hindbrain which serves as a complex How can we account for the strong correlation between coordination center, and help to establish the regional genetic linkage and domains of expression in the mouse differences between the hindbrain and the spinal cord. and Drosophila homeobox clusters? The conservation in The expression of Krox-20 and many of the homeobox expression and organization we believe to be a conse­ genes may be involved in achieving this regional specifi­ quence of c/s-acting regulatory elements distributed along cation. The different boundaries of Hox-2 genes could rep­ a cluster. If the clusters are broken apart, they would then resent limits of expression that correspond to different lose their appropriate coordinated expression. We sug­ rhombomeres in the hindbrain, suggesting that they may gest that each gene may be controlled by multiple ele­ play a role in establishing the rhombomeric pattern. It will ments over a distance, and the normal pattern of gene be important to make direct comparisons at early stages expression results from the additive effects of these ele­ of mouse development with Krox-20 to determine whether ments. Consistent with this idea is the fact that complex these anterior limits represent rhombomere boundaries. transcription patterns involving multiple transcripts and initiation sites are observed for many of the Hox-2 genes, Similarities of Mouse, Other Vertebrate, and in the human Hox-3 cluster differential splicing of at and Drosophila Complexes least three genes is observed (Simeone et al., 1988). In both mouse and Drosophila, anteroposterior expres­ Zakany et al. (1988) have also found that transgenic mice sion of homeobox genes in the CNS correlates with the or­ carrying Hox-1.3 constructs are only able to generate part ganization of the complexes. But what is even more strik­ of the normal spatial pattern of regulation in the nervous ing is that the homeobox sequence of the most posteriorly system and do not express in mesodermal tissues. These expressed member of Hox-2 (Hox-2.5) is most closely constructs contained large amounts of flanking DNA, im­ related to the most posteriorly expressed member of BX-C plying that other sequences in the cluster are required for (Abd-B). This correlation holds for the other genes in both normal regulation. Therfore, we feel that the maintenance the mouse and Drosophila clusters. From this we con­ of homeobox gene clustering in vertebrates is a result of clude that there is a clear relationship between relative po­ selection based on regulatory mechanisms. In this way sition, sequence identity, and domains of expression each of the mouse clusters may have a slightly different along the anteroposterior axis for both the mouse and Dro­ type of regulation, and closely related proteins with poten­ sophila homeobox gene networks, which strongly argues tially similar functions may be independently regulated in for a common ancestral origin of these complexes. some tissues. This may represent a mechanism, different These similarities with Drosophila are not unique for the from the evolution of new genes, whereby existing genes mouse. Homeobox genes have been found to be orga­ are recruited for new purposes by altering control pro­ nized in clusters in all vertebrates so far analyzed (see In­ cesses. troduction), and their overall structure is very similar to Experimental Procedures that of the mouse clusters. Boncinelli et al. (1988) have shown that the human homeobox complexes are orga­ Hybridization Probes nized as in mouse and display identity to Drosophila. The All probes from the Hox-2 cluster were subcloned into the Bluescript chicken genome has Hox-2 and Hox-1 complexes that are vector for double-strand sequencing using Sequenase (United States Biochemical Corp.) according to the suppliers and for in vitro transcrip­ highly related to those of the mouse in both structure and tion with either T7 or T3 RNA polymerase. For in situ hybridization the expression (Fainsod and Kuroiwa, personal communica­ probes were made with 35S-UTP and for blotting analysis with tion; Wedden et al., 1989). The high degree of identity be­ [32P]UTP supplied by New England Nuclear (Du Pont). Probes were tween the chick and mouse Hox-2.1 and Hox-2.6 genes al­ as follows: Hox-2.5 (a 3'400 bp Bglll-Dral fragment), Hox-2.4 (1 kb Sacl lowed us to examine their expression by Northern blot fragment), Hox-2.3 (800 bp EcoRI-BamHI fragment), Hox-2.2 (1.2 kb EcoRI-SacI fragment), Hox-2.1 (800 bp Sacl-EcoRI fragment), Hox-2.6 analysis (Papalopulu et al., 1989) and in situ hybridization (800 bp Sail—Bglll fragment), and Hox-2.7 (1.4 kb Sacl-BamHI frag­ in chick embryos (A. G., unpublished data). Our prelimi­ ment). The Hox-2.1 and Hox-2.6 probes were those reported by Krum­ nary results suggest that the patterns of expression in lauf et al. (1987) and Graham et al. (1988). chick embryos are nearly identical. In zebra fish a Hox-2 RNA Isolation and Northern Hybridization cluster has also been isolated. One member, ZF-21, dis­ Poly(A)+ mRNA was isolated from mouse tissues essentially as de­ plays 85% homology at the amino acid level over the en­ scribed in Krumlauf et al. (1987). In brief, the samples were homo­ genized in 3 M LiCI-6 M urea on ice for 2 min, and stored overnight Acknowledgments at 4°C to precipitate the RNA. The RNA was harvested by centrifuga­ tion, redissolved in 10 mM Tris-HCI (pH 7.6), 1 mM EDTA, 0.5% SDS, We thank Paul Hunt and Jo Lorimer for homeodomain sequences. We and extracted several times with phenol-chloroform. Following ethanol also thank Denis Duboule for the generous sharing of ideas and data precipitation the poly(A)+ mRNA was isolated by oligo(dT)-cellulose prior to publication, and Steve Gaunt, Peter Gruss, Dado Boncinelli, chromatography. The RNA samples were electrophoresed in denatur­ Paul Sharpe, and Abraham Fainsod for valuable discussions. We are ing formaldehyde-agarose gels, transferred to GeneScreen (Du Pont) grateful to Leanne Wiedemann, David Wilkinson, and Andrew Lums- in 20x SSC, and coupled to the membrane by ultraviolet cross-linking den for discussions on data and critical reading and comments on the and baking as described in Krumlauf et al. (1987). The filters were hy­ manuscript. N. P. was supported by the Greek State Scholarship Foun­ bridized using 32P-labeled single-stranded antisense RNA probes dation, and A. G. by an MRC studentship. produced by in vitro transcription of subcloned gene-specific frag­ The costs of publication of this article were defrayed in part by the ments with T7 or T3 RNA polymerase. Hybridization was in 60% forma- payment of page charges. This article must therefore be hereby mide, 5x SSC, 0.1% bovine serum albumin, 0.1% Ficoll 400, 0.1% marked “advertisement” in accordance with 18 U.S.C. Section 1734 polyvinylpyrrolidone, 20 mM sodium phosphate (pH 6.8), 1% SDS, 7% solely to indicate this fact. dextran sulfate, 100 ng/ml tRNA, 10 |ig/ml poly(A) at 65°C for 16 hr. The filters were washed in 0.1 x SSC, 0.5% SDS at 80°C for 2 hr, and in the Received November 15, 1988; revised February 7, 1989. cases where high-stringency conditions were required to ensure that the signal was not derived from related genes, the filter was further References treated with RNAase A. The filters were incubated in 2x SSC, 0.1% SDS at 52°C for 1 hr. Akam, M. E. (1987). The molecular basis for metameric pattern in the Drosophila embryo. Development 101, 1-22. In Situ Hybridization Akam, M. E., Dawson, I., and Tear, G. (1988). Homeotic genes and the The protocol used was basically that of Wilkinson et al. (1987) with control of segment diversity. Development 104 (Suppl.), 123-133. some modifications, and is essentially as follows. Mouse embryos Baron, A., Featherstone, M. S., Hill, R. E., Hall, A., Galliot, B., and were fixed in 4% paraformaldehyde in PBS overnight at 4°C, and then Duboule, D. (1987). Hox-1.6\ a mouse homeo-box-containing gene embedded in paraffin wax. Sections (6 urn) were cut and dried onto member of the Hox-1 complex. EMBO J. 6, 2977-2986. gelatin-subbed slides. The section were dewaxed in xylene, treated Beeman, R. W. (1987). A homeotic gene cluster in the red flour beetle. with 0.2 M HCI, refixed in paraformaldehyde, subjected to proteinase Nature 327, 247-249. K treatment, and then treated with acetic anhydride. After dehydration, probes were redissolved at a final activity of 1-2 x 105 dpm/kb/nl in Boncinelli, E., Somma, R., Acampora, D., Pannese, M., D’Esposito, M., hybridization buffer (50% formamide, 0.3 M NaCI, 20 mM Tris, 5 mM and Simeone, A. (1988). Organization of human homeobox genes. EDTA, 10 mM sodium phosphate, 10% dextran, 1x Denhardt’s solu­ Hum. Reprod. 3, 880-886. tion, 0.5 mg/ml tRNA). Hybridization was overnight at 55°C. Sections Breier, G., Dressier, G. R,, and Gruss, P. (1988). Primary structure and were washed at 65°C in 50% formamide, 2x SSC, 100 mM DTT for developmental expression of Hox 3.1, a member of the murine Hox 3 40 min followed by incubation with RNAase A at 20 ugAnl in 0.5 M homeobox cluster. EMBO J. 7, 1329-1336. NaCI, 10 mM Tris, 5 mM EDTA for 30 min. Sections were again washed Bucan, M., Yang-Feng, T., Colberg-Poley, A. M., Wolgemuth, D. J., Ge­ in 50% formamide, 2x SSC, 100 mM DTT at 65°C for 30 min followed net, J., Franke, U., and Lehrach, H. (1986). Genetic and cytogenetic lo­ by two 15 min washes in 2x SSC and 0.1 x SSC. Sections were de­ calization of the homeobox containing genes on mouse chromosome hydrated through alcohol solutions containing 0.3 M ammonium ace­ 6 and human chromosome 7. EMBO J. 5, 2899-2905. tate. Slides were dipped in a mix of Ilford K5 nuclear emulsion and Dolecki, G. J., and Humphreys, T. (1988). An engrailed class homeo­ glycerol-water (6 ml emulsion in 8.82 ml H20 and 0.18 ml glycerol) box gene in sea urchins. Gene 64, 21-31. and kept at 4°C until developed. Sections were stained in 0.02% tolu- Dony, C., and Gruss, P. (1987). Specific expression of the Hox 1.3 idine blue for 1 min and then mounted in Permount. homeobox gene in murine embryonic structures originating from or in­ duced by the mesoderm. EMBO J. 6, 2965-2975. Sequences and Organizational Data Used for Comparisons Dressier, G. R., and Gruss, P. (1988). Do multigene families regulate The sequences used for the homeodomain comparisons in Figure 7 vertebrate development? Trends Genet. 4, 214-219. are from the sources indicated in parentheses: Hox-2.5 and Hox-2.4 Duboule, D., and Dolle, P. (1989). The murine Hox gene network: its (this work); Hox-2.6 and Hox-2.7 (Graham et al., 1988); Hox-2.1 (Krum­ structural and functional organization resembles that of Drosophila lauf et al., 1987); Hox-2.2 (Hart et al., 1987b); Hox-2.3 (Meijlink et al., homeotic genes. EMBO J., in press. 1987); Hox-1.7 (Rubin et al., 1987); Hox-1.1, -1.2, -1.4, and -1.5 (Duboule et al., 1986); Hox-1.3 (Odenwald et al., 1987); Hox-1.6 (Baron et al., Duboule, D., Baron, A., Mahl, P., and Galliot, B. (1986). A new homeo­ 1987); Hox-3.1 and -3.2 (Breier et al., 1988); Hox-6.1 (Sharpe et al., box is present in overlapping cosmid clones which define the mouse 1988); Hox-4.1 (Lonai et al., 1987); Hox-5.1 (Featherstone et al., 1988); HOX-1 locus. EMBO J. 5, 1973-1980. Hox-5.2 (Duboule and Dolle, 1989); Abd-B (Regulski et al., 1985); Abd- Duboule, D., Galliot, B., Baron, A., and Featherstone, M. S. (1989). Mu­ A (Akam et al., 1988); Ubx and Antp (Scott and Weiner, 1984); Scr rine homeobox genes: some aspects of their organization and struc­ (Kuroiwa et al., 1985); Dfd (Regulski et al., 1987); zen z1 and z2 (Rush- ture. In Cell to Cell Signals in Mammalian Development, NATO ASI Se­ low et al., 1988); lab(F90-2) (Hoey et al., 1986; Mlodzik et al., 1988). ries, Vol. 26, S. W. deLaat, ed. (Berlin: Springer-Verlag), pp. 97-108. The references for the four mouse clusters and the Drosophila Featherstone, M. S., Baron, A., Gaunt, S. J., Mattei, M.-G., and clusters used to compile the network diagram in Figure 8 are as indi­ Duboule,

Anthony Graham, Nancy Papalopulu, Joanne Lorimer, John H. McVey, 1 Edward G.D. Tuddenham , 1 and Robb Krumlauf Laboratory of Molecular Embryology, National Institute for Medical Research, London NW7 1AA, UK

GENES & DEVELOPMENT 2:1424-1438 © 1988 by Cold Spring Harbor Laboratory ISSN 0890-9369/88 $1.00 Characterization of a murine homeo box gene, Hox-2.6, related to theDrosophila Deformed gene

Anthony Graham, Nancy Papalopulu, Joanne Lorimer, John H. McVey, 1 Edward G.D. Tuddenham , 1 and Robb Krumlauf Laboratory of Molecular Embryology, National Institute for Medical Research, London NW7 1AA, UK

The Hox-2 locus on chromosome 11 represents one of the major clusters of homeo-box-containing genes in the mouse. We have identified two new members (Hox-2.6 and Hox-2.7), which form part of this cluster of seven linked genes, and it appears that the Hox-2 locus is related by duplication and divergence to at least one other mouse homeo box cluster,Hox-1. The Hox-2.6 gene encodes a predicted protein of 250 amino acids, which displays extensive similarity in multiple regions to certain mouse, human,Xenopus , and zebra fish homeo domain proteins. The Drosophila Deformed (Dfd) gene also shares these same regions of similarity, and based on this sequence conservation, we suggest thatHox-2.6 forms part of a vertebrate 4Dfd-like' family. Hox-2.6 is expressed in fetal and adult tissues and is modulated during the differentiation of F9 teratocarcinoma stem cells. In situ hybridization analysis of mouse embryos shows that theHox-2.6 is expressed in ectodermal derivatives: spinal cord, hindbrain, dorsal root ganglia, and the Xth cranial ganglia. In the central nervous system, expression is observed in the most posterior parts of the spinal cord, with the anterior limit residing in a region of the hindbrain and no expression in the mid- or forebrain. In mesodermal structures, Hox-2.6 is expressed in the kidney, the mesenchyme of the stomach and lung, and the longitudinal muscle layer of the gut. Expression has not been observed in derivatives of embryonic endoderm. The patterns of Hox-2.6 expression in both mesoderm and ectoderm are spatially restricted and may reflect a role for the gene in the response to or establishment of positional cues in the embryo. [Key Words: Hox-2.6; homeo box; mouse embryo;Drosophila Deformed; in situ hybridization; teratocarcinoma cells] Received June 3, 1988; revised version accepted August 25, 1988.

The homeo box was first discovered in Drosophila in rate phylogenetic groups, including nonsegmented in­ genes that were implicated in controlling pattern forma­ vertebrates and mammals (McGinnis 1985; Holland and tion by specifying segmental pattern and segment iden­ Hogan 1986). Homeo box genes are organized in clusters tity (for review, see Gehring 1987). This process involves in the Drosophila and mammalian genomes. Murine the determination of positional values along the antero­ homeo-box-containing genes represent a large gene posterior body axis. More recently, it has been show that family with at least 21 members, which are organized in several Drosophila homeo-box-containing genes are ex­ several (at least four) gene clusters. The two major pressed in pattern that suggest roles in positional specifi­ mouse homeo box clusters, Hox-1 and Hox-2, are lo­ cation, independent of the process of segmentation cated on chromosomes 6 and 11, respectively (Hart et al. (Doyle et al. 1986; Hoey et al. 1986; Akam 1987; 1985; Bucan et al. 1986). To date, theHox-1 cluster has Gehring 1987). at least seven members (Duboule et al. 1986; Rubin et al. The homeo box can encode a 61-amino-acid protein 1987), and theHox-2 cluster has five (Hart et al. 1987); domain that has a putative helix-tum-helix motif. This however, the size and organization of the clusters has motif is implicated in DNA-binding activity (Desplan et not been completely established. al. 1985; Fainsod et al. 1986; Cho et al. 1988), and it is In general, very little overall similarity has been ob­ possible that the biochemical function of the homeo do­ served between the proteins of Drosophila homeo-box- main is to direct DNA binding and potentially regulate containing genes and their vertebrate counterparts. The transcription. Homeo-box-containing genes have been homeo box itself and a short hexapeptide region (Krum­ identified and cloned from the genomes of widely sepa­ lauf et al. 1987) are the major related elements among many of these genes from different species. Recently, 1Present address: Haemostasis Research Group, Clinical Research Regulski et al. (1987) presented data showing that the; Centre, Middlesex HA1 3UJ UK. Drosophila Deformed [Dfd] gene exhibits similarity

1424 GENES & DEVELOPMENT 2:1424-1438 © 1988 by Cold Spring Harbor Laboratory ISSN 0890-9369/88 $1.00 Hox-2.6 structure and expression

with two vertebrate genes not only in the homeo do­ probe to isolate clones that span part of the locus. From main but also in other regions of the protein sequence. these clones we were able to characterize the structure, Information of this type suggests that vertebrate analogs sequence, and patterns of expression for one member of of previously characterizedDrosophila genes may exist. the locus, Hox-2.1 (Krumlauf et al. 1987). Subsequent ge­ Therefore, it is important to determine and compare the nomic walking yielded more cosmid clones extending 5' coding information of more vertebrate homeo-box-con­ and 3' of this region. Restriction mapping, sequencing, taining genes to allow definition of closely related and hybridization analysis, using mouse andDrosophila homeo box gene subfamilies, not based solely on the homeo box probes, allowed construction of a map for the homeo box. Hox-2 locus that positions seven homeo boxes over 80 Whether conservation of structure reflects homolo­ kb (Fig. 1). This map is in agreement with previous re­ gous functions for homeo box genes in the development sults assigning five genes,Hox-2.1 to Hox-2.5, to the of different species has yet to be shown. However, in locus (Hart et al. 1985,1987b) and, in addition, identifies view of the difficulty in defining mammalian genes re­ two new members of the locus, which we term Hox-2.6 sponsible for regulating important developmental pro­ and Hox-2.7. The Hox-2.6 and Hox-2.7 genes are, at cesses, they provide an opportunity to study molecules present, the 3'-most members of the locus and lie ~13 that are potentially involved in control during embryo­ kb and 31 kb downstream of Hox-2.1. genesis. The patterns of expression of mammalian A 600-bp Hox-2.6 probe containing part of the homeo homeo box genes during development are consistent box and 3'-flanking sequences was generated from a sub­ with such a role (for review, see Feinberg et al. 1987). A cloned 4.4-kb BamHL fragment {Bglll-BamHb, probe 1, number of groups have analyzed the expression of mu­ Fig. 1). This probe was used to isolate a number of rine homeo box genes and found them to be temporally Hox-2.6 cDNA clones from a \gtl0 library, prepared and spatially restricted in their patterns of expression from 8.5-day p.c. mouse embryo RNA (Fahrner et al. (Gaunt et al. 1986; Dony and Gruss 1987). The murine 1987). The two longest cDNA clones (2 and 7, Figs. 1 and homeo box genes appear to be expressed in similar but 2) and genomic clones from the Hox-2.6 region were se­ not identical overlapping domains, which often display quenced. Sequence analysis reveals that the cDNAs con­ differences in their anterior-posterior, dorsal-ventral, tain a long open reading frame encoded by two exons, and medial-lateral levels of expression (Feinberg et al. which in the genomic DNA are separated by a 790-bp 1987; Krumlauf et al. 1988; Gaunt 1988; Holland andintron, and contain consensus splice donor-acceptor Hogan 1988). The complex and overlapping patterns of sites. Hox-2.6 is transcribed in the same direction as transcription suggest that these genes may be involved Hox-2.1, and it appears that all seven genes in the locus in several different processes during embryogenesis, in­ have the same 5 '-» 3 ' orientation (Hart et al. 1987b). cluding the setup of positional cues. Figure 2 shows complete sequence of the two cDNAs The fact that vertebrate homeo box genes are found in and some additional 3' sequence from a genomic sub­ similar types of clusters in all organisms so far analyzed clone. The size of the major Hox-2.6 transcript, esti­ is intriguing and may be a consequence of their modes of mated by Northern RNA analysis (see Fig. 4), is 2.4 kb, regulation or function. Thus, it is important to extend and the cDNA clones account for only 1300 bp. The 3' our understanding of the organization and expression of ends of these clones have no adjacent polyadenylation genes within these mouse clusters, to examine evolu­ signal and appear to have arisen by specific internal tionary or functional relationships between members of priming from an A-rich region between position 998 and the homeo box gene family. This paper presents a fur­ 1104 (Fig. 2). There is a potential polyadenylation signal ther characterization of the Hox-2 locus, identifying two (AATAAA) at position 890 in the 3'-untranslated region; new genes (Hox-2.6 and Hox-2.7) and shows that there however, RNase protection experiments have indicated are at least seven linked members of this cluster. Anal­ that this site is not used (data not shown). Genomic ysis of the structure and expression of one of the new probes 3' of the cDNA clones detect mRNA, suggesting members, Hox-2.6, is detailed. The Hox-2.6 gene is ex­ that most of the sequence missing from our clones is pressed in a temporally and spatially restricted pattern derived from the 3'-untranslated end of the mRNA. Also during embryogenesis, and its predicted protein displays contained within the 3'-untranslated region of Hox-2.6 extensive identity to other homeo domain proteins. is an AUUUA motif implicated in modulating the level Based on multiple regions of similarity to the Droso­ of mRNA stability (Shaw and Kamen 1986). phila Dfd gene, Hox-2.6 can be seen to be a member of a Conceptual translation of the sequence in Figure 2 re­ 'D/d-like' family of vertebrate genes. veals that both cDNAs contain a long open reading frame. The first AUG, 36 bp after an in-frame stop codon, has been denoted the methionine-initiator codon. Results The predicted protein contains 250 amino acids, corre­ sponding to a predicted molecular weight of 27,500, with an in-frame hexapeptide (Krumlauf et al. 1987) and Hox-2.6gene structure and protein sequence homeo domain in the carboxy-terminal portion of the A number of overlapping genomic clones, which span protein. Like Hox-2.1 and many other homeo-domain- the Hox-2 locus on chromosome 11, have been isolated containing proteins, Hox-2.6 is rich in prolines (18%), from a mouse cosmid DNA library. Initially, a 220-bp including a run of 14 consecutive prolines in the amino- fragment containing the Antp homeo box was used as a terminal region.

GENES & DEVELOPMENT 1425 Graham et al.

HOX 2 C luster - Chromosome 11

______pcos 5.4 .pcosH15 pcosH9 ------pcos3.1

HOX HOX HOX HOX HOX HOX HOX 2.5 2.4 2.3 2.2 2.1 2.6 2.7 TT T !~ TTT TT“ r i i "TT TT ~i—r RB BR RB R BB R RR BBR BR BB B R 2kb

) V) i i n / 1 I 1

Hexa ' 1 ' ' HOX 2.6 100 bp Peptide, Homeobox AUG COOH

cDNA 2 CDNA7 PROBES

Figure 1. Map of the Hox-2 locus, which positions seven homeo boxes, and a detailed map of the Hox-2.6 gene structure. The overlapping cosmid clones that span this cluster are illustrated. The predicted Hox-2.6-coding regions are shown, as are the two cDNA clones that have been used for sequence analysis. Probes 1 and 2 are the probes used in all later hybridization experiments. (R) £coRI; (B) BamHl.

Evolutionary relationships to the Hox-2.6protein—a quences in addition to the homeo domain are highly Dfd fam ily conserved in these proteins (Fig. 3A). The three proteins Sequence comparisons of the Hox-2.6 homeo domain are different in size (232-255 amino acids); but if posi­ with other homeo domain sequences are summarized in tioned for maximal alignment, one can see substantial Table 1. The Hox-2.6 homeo domain shows 83% amino conservation in the amino terminal region. This is more acid identity to that of Antp (McGinnis et al. 1984) and extensive in the case of Xhox 1A than in the case of is therefore a member of the ANT-C/BX-C class of HHO.cl3; yet in both instances, the first 30-35 amino homeo domain. Considering murine homeo domains, acids show a high degree of sequence similarity. Within the Hox-2.6 homeo domains show the closest similarity these amino acids, many of the differences represent to those of Hox-1.4 and Hox-5.1, both with 93% amino conservative changes, and there is a block of 12 amino acid identity (Duboule et al. 1986; Featherstone et al. acids that are identical in all three proteins. The other 1988). In addition, sequences immediately adjacent to region of notable similarity in these proteins begins the Hox-1.4 and Hox-5.1 homeo domains, plus the hexa- around the hexapeptide and extends through the homeo peptides and amino-terminal regions, show extended re­ domain and the first five to six amino acids on its car- gion of identity with Hox-2.6 (Featherstone et al. 1988; boxy-terminal side. The remaining regions of these pro­ B. Galliot, pers. comm.); these genes appear to form a teins are highly divergent. subfamily of related genes. It was noted previously that HHO.cl3 and Xhox 1A Significant similarities to Hox-2.6 are also found in shared related regions with the Drosophila homeo-do­ other vertebrate homeo-domain-containing proteins. main-containing protein Dfd (Regulski et al. 1987). We These are the human HHO.cl3 (Mavillo et al. 1986), the therefore compared Hox-2.6 with Dfd, and Figure 3B Xenopus Xhox 1A (Harvey et al. 1986), and the zebra fish diagrams blocks of sequence similarity identified with ZF 13 (Njolstad et al. 1988) genes. Both HHO.cl3 and an optimal alignment between the Hox-2.6 and Dfd pro­ Xhox 1A show 95% amino acid identity within the teins. The Dfd protein is much larger thanHox-2.6 (586 homeo domain, whereas ZF 13 shows 98% amino acid vs. 250 amino acids). This difference appears to be the identity. All three proteins exhibit a high degree of iden­ consequence of several regions rich or repetitive in tity in sequences immediately flanking the homeo do­ single amino acids, such as Gly, His, Gin, or Asn, which main, with those of ZF 13 being closest to Hox-2.6 are not present in the mouse gene. The Hox-2.6 homeo (Njolstad et al. 1988). domain has 89% amino acid identity with the Dfd An alignment comparison between Hox-2.6, Xhox 1A, homeo domain. The overall amino acid identity to Dfd and HHO.cl3 over the entire protein shows that se­ is 40% (102/250 amino acids) and is more extensive

1426 GENES & DEVELOPMENT Hox-2.6 structure and expression

TTTCGGAAACAGGAAAACCGAGTCAGGGGTCGGAATAAATTTTAGTATATTTTGTGGGCAATTCCCAGAAATTA genes also share many of the same amino acid sequence

( 7 ) ( 2 ) differences in the homeo domain when compared to the 1 30 60 ATG GCT ATG AGT TCC TTT TTG ATC AAC TCA AAC TAT GTC GAC CCC AAG TTC CCT CCG TGC Antp homeo domain. Therefore, based on the multiple MET A la MET Ser Ser Phe Leu tie Asn Sec Asn T y r V a l Asp P ro Lys Phe P ro P ro Cys regions of homology it appears that Hox-2.6, HHO.cl3, 90 120 GAG GAG TAT TCA CAG AGC GAT TAC CTA CCC AGC GAC CAC TCG CCC GGG TAC TAC GCC GGC Xhox 1A, Hox-5.1, Hox-1.4, and ZF 13 are all members G lu G lu T y r Ser G in Ser Asp T y r Leu P ro Ser Asp H is Ser Pro Gly Tyr Tyr A la G ly of a vertebrate Dfd-like family related by a common pro­ 150 1B0 GGC CAG AGG CGA GAG AGC GGC TTC CAG CCG GAG GCG GCC TTT GGG CGC CGG GCG CCG TGC genitor that evolved before the divergence between the G ly G in A rg Arg Glu Ser Gly Phe Gin P ro G lu A la A la Phe G ly A rg A rg A la P ro Cys lineages that gave rise to vertebrates and arthropods. 210 240 ACT GTG CAG CGC TAC GCG GCC TGC CGA GAC CCC GGG CCC CCG CCA CCT CCG CCG CCC CCG T h r V a l G in A rg T y r A la A la Cys A rg Asp P ro G ly P ro P ro P ro P ro P ro P ro P ro P ro

270 300 CCG CCC CCG CCA CCG CCC GGG CTG TCC CCT CGG GCT CCA GTG CAG CCA ACA GCC GGG GCC The Hox-2 and Hox-1 clusters are related by P ro Pro P ro P ro Pro Pro Gly Leu Ser P ro A rg A ia P ro v a l G in P ro T h r A la G ly A la duplication 330 360 CTC CTC CCG GAG CCC GGG CAG CGC AGC GAG GCG GTC AGC AGC AGC CCC CCG CCG CCT CCC Leu Leu P ro Glu Pro Gly Gin A rg S er G lu A la V a l Sec Ser Ser Pro P ro P ro P ro P ro To extend our analysis of theHox-2 locus further, we

390 420 have isolated the Hox-2.7 gene and sequenced the TGC GCC CAG AAC CCC CTG CAT CCC AGC CCG TCC CAC TCC GCG TGC AAA GAG CCC GTCI[g tc Cys Ala Gin Asn Pro Leu His Pro Ser P ro Ser H is S er A la Cys Lys G lu P ro V a l [ v a l homeo domain (Fig. 4). It shares only 68% amino acid identity (42/61) with the Antp homeo domain and, 450 V 480 TAC CCC TGG ATG CGC AAA GTT CAC GTG AGO ACG'GTA AAC CCC AAT TAC GCC GGC GGG GAG among other Drosophila homeo domains, is most Tyr Pro Trp MET Arg Lys Val His Val Ser Thr Val Asn Pro Asn Tyr Ala Gly Gly Glu closely related to S60Z1 (Doyle et al. 1986). The pre­ CCC AAG CGC TCT CGG ACG GCC TAC ACT CGC CAG CAG GTC CTG GAG TTG GAG AAG GAG TTT dicted sequence in the homeo domain varies in only two Pro Lys A rg Ser A rg T h r A la T y r T h r A rg G in G in V a l Leu G lu Leu G lu Lys G lu Phe positions with mhl9, also assigned to chromosome 11 570 600 CAC TAC AAC CGC TAC CTG ACG CGC CGC CGG AGG GTG GAG ATC GCC CAC GCG CTC TGC CTG (Lonai et al. 1987), and may represent the same gene. His Tyr Asn Arg T y r Leu T h r A rg A rg A rg A rg V a l Glu tie Ala His Ala Leu Cys Leu When compared with mouse homeo domains, Hox-2.7 630 660 TCC GAG CGC CAG ATC AAG ATC TGG TTC CAG AAT CGG CGC ATG AAG TGG AAA AAA GAC CAC displays the strongest identity with the Hox-1.5 domain Ser Glu A rg G in l i e Lys l i e T rp Phe G in Asn Arg Arg MET Lys Trp Lys Lys Asp H is and flanking regions (Fig. 4). This represents another ex­ 690 720 ample (like Hox-2.6) of a gene in the Hox-2 cluster, ap­ AAG TTG CCC AAC ACC AAG ATC CGC TCG GGT GGC ACC GCG GGC GCA GCC GGA GGC CCC CCT LySj Leu P ro Asn T h r Lys l i e A rg Ser Gly Gly Thr A la G ly A la A la G ly G ly P ro P ro parently more highly related to a gene in the Hox-1

750 787 cluster than to other Hox-2 members. A comparison of GGC CGG CCC AAC GGA GGC CCC CCT GCG CTC TAG TGCCCCCCAAGCAGGAGTTCGAACATGGGGGGGT Gly Arg Pro Asn Gly G ly P ro P ro A la Leu the sequence and organization of the seven genes in the

826 866 Hox-2 cluster with the Hox-1 cluster sheds some light GGGGGGAACAGCGAGCACCGAAGGGGGTGCGGGGTATGGGAGGGTCCCCGGGCTTGAGCCCAGAAAAAATCTATCTACC on the possible genealogy of these murine homeo box 905 945 CTACCCTCACTTTATCTATATGGAATAAACACAGAGAAGGGGGGTAGGGAAGCCTTATTTATAGAAAGGACAATAAGGG genes. The genes in both clusters are transcribed in the same 5' -» 3' orientation, and based on intergenic dis­ 984 1024 AGCCGGGTAAAGTCTTCGGAGACAAGATTCGAGTCTCTTGCTTTCTTCCTTTAAAAAAAAAAAGAAAGAAAGAAGAGAA tances and sequence comparisons between members of

1064 (2> 1104 the Hox-1 (Duboule et al. 1986) andHox-2 clusters, it is GTAAAGAAAGAGAGAGAGAGAAAAAGAAGAAAGGAAGGAAGCAAGAAAAGGAGGAAGAAAGGAAAAGACAGAAGAGAAA (7) possible to align some of the genes in these loci as 1143 1183 TGGAGGAGTCTGCTGCGCCTGGTTTTCAGCTTTGGTGAAGATGGATCCAGGCTTCATCTTTAATCACGCCAGGCCCGGG shown in Figure 5.

1222 CCATCTGTCTCTGTTTACTTGCCGAGGAGAAGGACGGGC Figure 2. Nucleic acid and protein sequence ofHox-2.6 from cDNA and genomic clones. The longest open reading frame and Table 1. Homeo domain homologies to Hox-2.6 its conceptual translation are shown, and numbering starts on the first AUG 36 bp downstream of an in-frame stop codon. The Antp 82% (50/61) Drosophila McGinnis et al. intron is indicated by the large arrowhead. The beginning and (1984) end of the two cDNA clones (2 and 7), shown in Fig. 1, are Dfd 89% (54/61) Drosophila Regulski et al. marked by small arrowheads and their respective numbers. The (1987) open boxes outline the hexapeptide and the homeo domain. ZF 13 98% (60/61) zebra fish P.R. Njostad et al. (pers. comm.) Xhox 1A 95% (58/61) Xenopus Harvey et al. (1986) HHO.cl3 93% (57/61) human Mavillo et al. (1986) (52%) when conservative changes are taken into ac­ Hox-1.4 93% (57/61) mouse Duboule et al. count. (1986) Hox-2.6 shares almost exactly the same pattern of Hox-5.1 93% (57/61) mouse Featherstone et al. similarity with Dfd as it does with Xhox 1A and (1988) HHO.cl3. Several domains, including the highly con­ Hox-2.1 89% (54/61) mouse Krumlauf et al. served amino-terminal sequences inHox-2.6, which are (1987) shared with the Xenopus and human genes, are also Hox-2.3 82% (50/61) mouse Meijlink et al. present in the Dfd protein. However the N-terminal (1988) identities in Dfd are partially disrupted by some of the Hox-2.7 75% (46/61) mouse Fig. 4 (this paper) repetitive amino acid regions (see Fig. 3B). All of these

GENES & DEVELOPMENT 1427 Graham et al.

2.6 1 MAMSSFLINSNYVDPKFPPCEEYSQSDYLPSDHSPGYYAGGQRRESGFQPEAAFGRRAPCTVQRYA 66 C13 1 -V---YMV—K------LEGG— GEQGAD — G— AQGAD PGLYP-PDFGEQPFGG 64 X1A 1 -R------S------H S-K------S- -HG- 46

2.6 67 ACRDPGPPPPPPPPPPPPP PGLSPRAPVQPTAGALLPEPGQRSEAVSSSPPPPPCAQNPLHPSPS 131 Cl3 65 SGPG— SAL-ARGHGQE-GG— GHYA— GE-CPAPPA-P-APLPG-RAY-QS D-KQ-PSG 124 X1A 47 PYPRSVSSNSSSSASYSSC Q-SVRQSARL-HSSGLVPGEKAHL-PHMPTSS— S-SLKSSEHKHP 111

2.6 132 HSACKEPWYPWMRKVHVSTVNPNYAGGE PKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHA 197 C13 125 TALKQPA------K ANS T--- X1A 112 D-PEQD------K-A-I-KATST-SD A------T 177

2.6 198 LCLSERQIKIWFQNRRMKWKKDHK LPNTKIRSGGTAGAAGGPPGRPNGGPPAL 250 C13 191 ------G— SSSSSSSSCSSSVAPSQHLQPMAKDHHTDLTTL 255 X1A 178 -R------K-NPSVNLQIAGGSPNQNKGNHVCQ 232

B HOMEOBOX I I

Hox 2.6 m ii iiiiii

Dfd rx ME II III

Splice

COOH

Conserved N-Term Ser-Pro Rich Hexa Homeobox Subfamily Domain Domain Peptide Domain

1 5 -5 0 - 70-150■ 6 - -4 -2 5 • 61- -1 5 -5 0 AA AA AA AA AA AA 1 Figure 3. Evolutionary relationships betweenHox-2.6 and homeo domain proteins of other species. (A) Comparison of a related subclass of vertebrate homeo domain proteins, including mouse[Hox-2.6), human (HHO.cl4; Mavillo et al. 1986), andXenopus (Xhox 1A; Harvey et al. 1986). The boxed region represents the homeo domain, and dashes indicate amino acid identity. B( ) Diagrammatic representation of the relationship between Hox-2.6 and Drosophila Dfd. The shaded areas represent amino acid identity, and dashed lines demark related areas in the two proteins. (C) Generalized structure showing characteristic features and conserved regions based on comparison of several vertebrate homeo domain proteins. (AA) amino acid.

In cases where only the homeo domain and flanking in the previous section), and several other regions of the sequences have been determined, comparisons reveal two proteins are also related (D. Duboule, pers. comm.). that the pairs Hox-2.7-Hox-1.5 and Hox-2.2-Hox-1.2 Comparisons of other members of these clusters over (Colberg-Poley et al. 1985b; Hart et al. 1987a) display the the entire predicted protein sequences show that highest levels of sequence identity with each other, Hox-2.1-Hox-1.3 (Krumlauf et al. 1987; Odenwald et al. sharing in common specific amino acid changes when 1987; Fibi et al. 1988) andHox-2.3-Hox-l.l (Kessel et al. compared with the Antp homeo domain. The Hox-2.6 1987; Meijink et al. 1987) are closely related to each gene shows the strongest similarity with the Hox-1.4 other. The related region spans the hexapeptide region, homeo domain and surrounding sequences (as described the homeo domain plus surrounding sequence, and a

1428 GENES & DEVELOPMENT Hox-2.6 structure and expression

-1 GGG AAC AAG AGC CCC CCG GGG TCG GCC GCG Hox-2.6 gene. The carboxy-terminal region of these re­ Hox 2.7 Gly Asn Lys Ser Pro Pro Gly Ser Ala Ala lated proteins and the 3'-untranslated regions are highly Hox 1.5 G l y ...... Gin --- Ser diverged; therefore, the probe used was a single-stranded 15 TCC AAG CGG GCG CGC ACG GCG TAC ACA AGC GCG CAG CTG GTG GAG antisense RNA from this region of Hox-2.6 (Fig. 1, probe Hox 2.7 Ser Lys Arg Ala Arg Thr Ala Tyr Thr Ser Ala Gin Leu Val Glu Hox 1.5 — . ... — _ Gly — ...... Arg Pro — ...... 1). Although it does contain the last 15 amino acids of 30 the homeo domain, using high-stringency washing con­ CTG GAG AAG GAG TTC CAC TTC AAC CGT TAT TTG TGC CGG CCG CGC Hox 2.7 Leu Glu Lys Glu Phe His Phe Asn Arg Tyr Leu Cys Arg Pro Arg ditions with RNase A treatment (see Methods; Krum­ Hox 1.5 ...... M e t ...... lauf et al. 1987), this probe does not detect transcripts or 45 cloned DNA from the related genes {Hox-1.4 and CGG GTC GAGATG GCC AAT CTG CTG AAC CTC AGC GAG CGC CAG ATC Hox 2.7 Arg Val Glu MET Ala Asn Leu Leu Asn Leu Ser Glu Arg Gin lie Hox-5.1) in control experiments and is specific for Hox 1.5 ------Thr -...... - Hox-2.6. 60 AAG ATC TGG TTC CAG AAC CGT CGC ATG AAG TAC AAG AAA GAC CAG In tissues expressingHox-2.6, the probe detects at Hox 2.7 Lys lie Trp Phe Gin Asn Arg Arg MET Lys Tyr Lys Lys Asp Gin Hox 1.5 least six transcripts ranging from 2.4 to 10 kb, including 75 two major species at 2.4 and 4 kb (Fig. 6A). The complex AAG GCC AAG GGG CTG GCC TCG TCC TCT GGG GGT CCC TCT CCG GCC pattern of transcription reflects multiple transcripts Hox 2.7 Lys Ala Lys Gly Leu Ala Ser Ser Ser Gly Gly Pro Ser Pro Ala Hox 1.5 G l y Met Leu Tyr from the Hox-2.6 gene and is analogous to results that we obtained with another member of the locus, Hox-2.1 Figure 4. Sequence of the Hox-2.7 homeo domain. The (Krumlauf et al. 1987). The direct comparison of these Hox-2.7 homeo domain and the flanking region with the pre­ adjacent genes in the Hox-2 cluster (Fig. 6) shows that dicted amino acid sequence are shown aligned with the Hox-1.5 homeo domain. Dashes represent identical amino acids, and they both have multiple transcripts that differ in size the homeo domain is in brackets. and intensity from each other. The bands do not appear to be unprocessed precursors, because intron probes do not hybridize to the multiple transcripts. It is possible large region (30-50 amino acids) at the amino-terminal that they represent RNAs capable of encoding multiple end of the proteins (Fibi et al. 1988; Papalopulu et al. proteins from the same gene, as has been observed for a 1988; J. Garbem et al., unpubl.). Taken together, the data Xenopus homeo box gene (Cho et al. 1988). demonstrate that at least five genes in Hox-2 are related The major tissues that expressHox-2.6 are the lung, to five genes in Hox-1 via duplication and divergence of spinal cord, and kidney, and a selected time course of an ancestral cluster (Fig. 5). No mouse counterparts have expression in these tissues is shown in Figure 6A. The been found as yet for the Hox-1.6 gene in the Hox-2 expression in lung is observed in all fetal, neonatal, and cluster, or for the Hox-2.4-Hox-2.5 genes in the Hox-1 adult stages, and we have also detected transcripts by cluster. However, it seems likely that further character­ Northern analysis as early as 14.5 days p.c. The levels or ization of the overlapping regions of these clusters will complexity of expression in the lung do not appear to reveal more related members and define the limits of the change with time, but the 5-kb transcript is reduced in duplication. abundance when compared with other tissues. In the spinal cord, Hox-2.6 is expressed at the same levels in 14.5-day and 16.5-day embryos and is also present in the 18.5-day embryo, but we have not examined expression Developmental expression of Hox-2.6 in the adult spinal cord. In contrast to the constant rela­ To begin to define the pattern of Hox-2.6 expression in tive levels ofHox-2.6 mRNA in the lung and spinal cord the embryo and adult, we analyzed poly(A)+ RNA ex­ during development, expression in the kidney undergoes tracted from teratocarcinoma cell lines and tissues at a 10-fold drop between neonatal (1 week) and adult (6 various stages by Northern hybridization. In view of the weeks) stages. This same pattern of kidney expression is extensive sequence identity betweenHox-2.6 and other observed with the Hox-2.1 gene (Krumlauf et al. 1987). subgroup members, such asHox-1.4 and Hox-5.1, it was Hox-2.1 is expressed in the adult kidney (Jackson et al. important to select probes that were specific for the 1985) (although not observable on the exposure shown in Fig. 6B) at greatly reduced levels when compared with fetal and neonatal stages. Hox-2.6 is also expressed in adult testes and fetal liver, stomach, and gut but not in HOX1 Ch6 adult heart, liver, or spleen (data not shown). In general, it seems that Hox-2.6 is expressed at a higher level in HOX 2 CH11 most tissues and developmental stages examined rela­ tive to Hox-2.1, with the exception of spinal cord. Both Figure 5. A schematic alignment of the Hox-1 and Hox-2 Hox-2.6 and Hox-2.1 show a reduction at the level of homeo box gene clusters. Dotted lines indicate sequence simi­ high molecular transcripts in the lung, suggesting larities between genes in the two clusters that are potentially related by duplication. Hox-1 is located on mouse chromosome tissue-specific differences in transcription or processing 6 (Ch6), andHox-2 on mouse chromosome 11 (Chi 1). All genes of these complex RNA species. in both clusters have the same orientation with respect to tran­ Based on the isolation of cDNA clones from an 8.5-day scription and are drawn with the 5' -> 3' direction (left to right). p.c. library, Hox-2.6 is expressed in 8.5-day p.c. embryos. See text for references and details. It is difficult to obtain sufficient material to examine

GENES & DEVELOPMENT 1429 Graham et al.

Tissues F9 Ceils > o o > > -5 0 Q) lO to > > io CD 0) > > > -o cd C O CO CD CD oo CD CD CD < □ co cb Q Kb

10 8 6.5

5.0 Hox 2.6 4.0

2.4

B Kb

12 f t i M m i m m 10

F t -6.0 Hox 2.1 -3.8

2.2 m i

Figure 6. Northern analysis of Hox-2.6 and Hox-2.1 in F9 teratocarcinoma cells and mouse tissues from different developmental stages. The tissue of origin and stage of poly(A)+ RNA sample are indicated above each lane. In all tissue samples, 2 p,g of RNA per lane was loaded, and the F9 cell samples contained 2.5 |xg per lane. (A) Same filter as B reprobed with Hox-2.6; [B] filter probed with Hox-2.1. (RA) Cells treated with retinoic acid (5 x 10-8); (CHX) cells treated with cycloheximide (75 (xg/ml) for 6 hr.

earlier stages by Northern blots, but there have been a undetectable in F9 stem cells and accumulates to high number of reports describing patterns of expression of levels after 1 day of induction (Fig. 6B). This accumula­ murine homeo box genes in the F9 embryonal carci­ tion is transitory, and levels continually fall during fur­ noma cell line (Colberg-Poley et al. 1985a,b; Breier et al. ther differentiation stages to very low levels by day 5. 1986; Baron et al. 1987; Kessel et al. 1987; Rubin et al. Treatment of F9 stem cells with the protein synthesis 1987). The F9 cells are able to differentiate in vitro in inhibitor cycloheximide also inducedHox-2.6 and response to chemical inducers to form derivatives of the Hox-2.1 RNA levels (Fig. 6). In the case ofHox-2.1, this extraembryonic endoderm, and we have used them to induction is not mediated by transcription but reflects determine whether Hox-2.6 might be active in early mRNA instability involving the AUUUA motif (Shaw stages of embryonic differentiation. Expression of and Kamen 1986) in the 3 '-untranslated region of the Hox-2.6 was analyzed, by Northern blots, in stem cells Hox-2.1 mRNA (R. Krumlauf et al., in prep.). Similar se­ and at 1,3, and 5 days after retinoic-acid-induced differ­ quences reside in the 3' end of the Hox-2.6 mRNA (Fig. entiation (Fig. 6A).Hox-2.6 transcripts are present in un­ 2), and cycloheximide induction suggests thatHox-2.6 differentiated F9 stem cells at low levels. Transcript transcripts are also unstable in F9 stem cells. Thus, both levels are increased after 1 day of differentiation to near Hox-2.1 and Hox-2.6 show an increase in expression fol­ maximal amounts and maintained at this high level lowing cycloheximide treatment and initial differentia­ throughout the remainder of the differentiation time tion of F9 stem cells. However, the kinetics or temporal course. This is in direct contrast toHox-2.1, which is pattern of their response differs and depends on the de­

1430 GENES & DEVELOPMENT Hox-2.6 structure and expression gree of differentiation of the F9 cells. In summary, the tissue. This is obvious in the lung at 12.5 and 14.5 days, Northern analysis shows that Hox-2.6 and Hox-2.1 dis­ where the inner endodermal epithelial layer of cells is play similar overlapping patterns that only differ in the not expressing and the outer mesenchymal layer of cells timing or level of expression in cell lines and a number is expressingHox-2.6 (Fig. 8A,B). This mesodermal-en­ of mouse tissues. dodermal restriction can also be seen in the stomach (Fig. 8C), whereas we can observeHox-2.6 transcripts in the muscle layer of the gut in Figure 8D. The restriction of expression to the longitudinal muscle and not the cir­ Hox-2.6—in situ hybridization cular muscle is more obvious at 14.5 days (Fig. 8D). Ex­ pression of Hox-2.6 has not been observed in other me­ In situ hybridization has been used to extend and com­ sodermal organs such as the heart, the limbs, or those plement the analysis of expression by Northern blots derived from somites. Those tissues that do express that described the general patterns of Hox-2.6 expres­ sion. Antisense probes (probes 1 and 2, Fig. 1) and a sense Hox-2.6 are derived from intermediate and lateral plate probe were hybridized to sections of 12.5- and 14.5-day mesoderm. This overall pattern of expression is very similar to mouse embryos. We chose these stages, as they repre­ the one observed for Hox-2.1 (Holland and Hogan 1988). sent the stages of maximal expression ofHox-2.6. The sense probe acts as a control for nonspecific probe 'stick­ One exception is the extent of expression that each gene iness' to the sections. This probe did not highlight any shows along the rostral caudal axis. Most homeo box features of the 12.5-day mouse embryo except red blood genes examined by in situ hybridization are expressed in cells. the CNS,- however the domains are overlapping but not identical and often display differences in the anterior Of the three germ layers of the embryo,Hox-2.6 is ex­ limits of expression (Gaunt et al. 1988). Figure 7 shows pressed in derivatives of mesoderm and ectoderm but near adjacent sections of the hindbrain region of a 12.5- not in derivatives of endoderm. More specifically, day mouse embryo that have been probed for either Hox-2.6 is expressed in the following ectodermal deriva­ tives: spinal cord, hindbrain, and some ganglia of the pe­ Hox-2.6 or Hox-2.1 (the Hox-2.1 probe is probe 1 of Krumlauf et al. 1987). One can clearly see that the ex­ ripheral nervous system (Fig. 7).Hox-2.6 is not detected pression of Hox-2.6 extends more anteriorly than that of in the forebrain or midbrain; hence, expression within Hox-2.1. The limits of expression of either gene do not the central nervous system is restricted to more poste­ seem to correspond to any clear morphological or biolog­ rior regions. Transcripts are detected all along the spinal ical cues at this embryonic stage. Thus,Hox-2.6 shows cord, from posterior portions (Fig. 7A) into the hindbrain (Fig. 7B,D). The anterior limit of expression maps to the similar but subtly different patterns of expression to those of Hox-2.1 and, in common with all other homeo hindbrain and does not seem to correlate with any ob­ box genes, is spatially restricted in its patterns of expres­ vious morphological change. This is clearly observed at sion during development. In view of the structural simi­ 14.5 days, where one can see expression of Hox-2.6 ex­ larity between Hox-2.6 and other members of the Dfd- tending along the spinal cord past the cervical flexure like subgroup,Hox-1.4 and Hox-5.1, it will be very inter­ into the hindbrain (Fig. 7B). There appears to be no pos­ esting to determine whether their patterns of expression terior limit of expression, and Hox-2.6 can be detected in are also highly related. the most posterior portions of the CNS. In contrast to Hox-2.1 and other homeo box genes that have been ana­ Discussion lyzed by in situ hybridization,Hox-2.6 is expressed uni­ formly in transverse sections of the spinal cord (Awgule­ In this paper we present data relating to the characteriza­ witsch et al. 1986; Toth et al. 1987; Holland and Hogan tion of a new murine homeo box gene, Hox-2.6. This 1988) (Fig. 7C). It shows no dorsoventral or mediolateral gene and Hox-2.1, another new gene we have identified, spatial restriction at 12.5 days. Hox-2.6 is expressed in form part of a group of seven tightly linked homeo box the peripheral nervous system in dorsal root ganglia and genes in the Hox-2 cluster located on mouse chromo­ at very high levels in a ganglion that is probably the no­ some 11 (Fig. 1). To date, nearly 25 murine homeo box- dose ganglion of the Xth cranial nerve (Graham et al. containing genes have been identified. Therefore, the 1988). Figure 7D shows expression of the gene in the Hox-2 locus represents part of a large gene family related hindbrain, in a dorsal root ganglion, and in the nodose by at least one common element, the homeo box. Se­ ganglion. Expression of Hox-2.6 has not been observed in quence analysis reveals a structure forHox-2.6 that is other cranial ganglia. The CNS is derived from the similar to that of many other murine and vertebrate neural tube, and the peripheral nervous system has a homeo box genes. Typically,Hox-2.6 is comprised of neural crest origin. Thus,Hox-2.6 is expressed in both two exons separated by an intron of about 1 kb (790 bp). neural tube and neural crest derivatives. The protein-coding information (250 amino acids) is lo­ Expression of Hox-2.6 in mesodermal structures is cated in the 5' portion of the mRNA, which has a long also spatially restricted. Hox-2.6 is expressed in the mes­ 3'-untranslated region. A conserved hexapeptide is lo­ enchyme of the stomach and the lung, in the kidney, and cated close to the homeo box but on a separate exon, in the longitudinal muscle layer of the gut. Figure 8 with the splice site located 15-40 bp upstream of the shows clearly the restriction of the expression of this homeo box. The predicted proteins are very rich in pro­ gene to mesodermal tissues and not to endodermal lines and serines, and the distribution of these prolines

GENES & DEVELOPMENT 1431 Graham et al.

A RBC B PV

s c

HB

SC

"V

NG HB

DR G

Figure 7. Expression of Hox-2.6 in ectodermal derivatives, as shown by in situ hybridization to embryo sections. (A)Hox-2.6 expres­ sion in posterior spinal cord of a 12.5-day p.c. embryo. [B] Expression of Hox-2.6 at the junction of the hindbrain and spinal cord of a 14.5-day p.c. embryo. (C) Transverse section of thoracic spinal cord of a 12.5-day p.c. embryo probed for Hox-2.6. (D) Expression of Hox-2.6 in hindbrain and constituents of the peripheral nervous system of a 12.5-day p.c. embryo. Bright-field and dark-ground pictures are shown. (RBC) Red blood cells; (SC) spinal cord; (HB) hindbrain; (DRG) dorsal root ganglion; (NG) nodose ganglion; (PV) pre-vertebrae.

throughout the non-homeo-domain portion of the pro­of the Hox-1 and Hox-2 clusters reveal that at least five tein should generate a nonhelical structure. Some of members of each cluster are highly related to members these features are summarized in Figure 3C, which in the opposite cluster, suggesting a duplication event presents a generalized structure for these proteins. (Fig. 4). The relationship of other homeo box clusters to Hox-2.6 was found to be related to other mouse Hox-1 and Hox-2 is not well established. However, homeo-domain proteins[Hox-1.4 and Hox-5.1), based on based on the identity betweenHox-2.6 and Hox-5.1, sequence identity in several domains of the protein. some other genes in theHox-3 and Hox-5 clusters may This pattern of identity between different mouse genes also be related by duplication toHox-1 and Hox-2. Con­ appears to be the result of duplication and divergence of sequently, some mouse genes may form a subgroup with an ancestral homeo box cluster. Sequence comparisons as many as four highly related members.

1432 GENES & DEVELOPMENT Hox-2.6 structure and expression

Figure 8. Expression of Hox-2.6 in mesodermal derivatives, as shown by in situ hybridization, to embryo sections.[A] Hox-2.6 transcripts in the lung mesenchyme in transverse sections of 12.5-day p.c. embryo.[B] Lung of a 14.5-day p.c. embryo probed for Hox-2.6. (C) Hox-2.6 expression in the stomach mesenchyme of a 12.5-day p.c. embryo. (D) Expression ofHox-2.6 in the longitudinal muscle of a 14.5-day p.c. embryo. Bright-field and dark-ground pictures are shown. (E) Endoderm; (M) mesenchyme; (PV) pre-verte­ brae; (LM) longitudinal muscle; (CM) circular muscle.

In a larger context, one can note relationships between We suggest that these genes represent Da fd-like family, a number of homeo box genes from a range of different based on their multiple regions of similarity. However, organisms. Hox-2.6 not only shows similarity to two in view of the fact that there appear to be at least three mouse genes[Hox-1.4 and Hox-5.1) but also to a human murine genes that all show similar degrees of identity to gene, HHO.cl3 (Mavillo et al. 1986),Xenopus a gene, Dfd, it is clear that there is not a precise murine equiva­ Xhox 1A (Harvey et al. 1986), a zebra fish gene, ZF 13 lent to the Dfd gene. (Njolstad et al. 1988), and theDrosophila gene, Dfd (Re­ It may generally be possible to classify several of the gulski et al. 1987). High levels of sequence identity are nonlinked homeo box genes from different species into present in regions other than the homeo domain, in­ subgroups, based on conserved sequences in their cluding the hexapeptide and amino-terminal sequences. amino-terminal region (referred to as the conserved

GENES & DEVELOPMENT 1433 Graham et al. amino-terminal subfamily domain in Fig. 3C). This con­ al. 1987; Gaunt 1987),Hox-1.6 (Baron et al. 1987), servation in several species supports the idea that these Hox-2.5 (Feinberg et al. 1987),Hox-2.3 (Deschamps et al. domains may be functionally significant.Hox-2.1 forms 1987),Hox-2.1 (Holland and Hogan 1988), and En-1 and another such subgroup with the zebra fish ZF 21 gene En-2 (Joyner and Martin 1987). These genes are ex­ (P.R. Njolstad et al., pers. comm.) and the Xenopus X1H pressed in an overlapping pattern along the anteropos­ box 4 gene (Fritz and De Robertis 1988). Some of these terior axis of the CNS, as illustrated for Hox-2.1 and subfamilies may also be related to other Drosophila Hox-2.6 in Figure 9, but they also exhibit subtle differ­ homeo domain proteins in a manner analogous to ences in their mediolateral and dorsoventral planes of Hox-2.6 and Dfd. expression. Hox-2.6 is expressed uniformly in cross sec­ The Hox-2.6 gene, as analyzed by in situ hybridization tions of the spinal cord (Fig. 5C), yet Hox-2.1 is ex­ and Northern blots, is expressed in fetal and adult pressed in a dorsally restricted manner (Holland and tissues. Major sites of expression are the lung, kidney, Hogan 1988). central and peripheral nervous systems, stomach, gut, Homeo box genes are clearly implicated in deter­ and testis. Within visceral organs, Hox-2.6 is expressed mining the identity of cells in the Drosophila blasto­ in derivatives of mesoderm but not endoderm. This is derm, and recent studies would also suggest that they shown in Figure 8, which illustrates that expression is may act in an analogous manner in theDrosophila restricted to the mesoderm of the stomach, gut, and nervous system (Doe et al. 1988). For example, it has lung, and is not seen in the endodermal epithelium. been reported that ftz in Drosophila and mec-3 in Caen- Within ectodermal derivatives, Hox-2.6 is expressed in orhabditis elegans (Way and Chalfie 1988) are necessary the CNS, in the spinal cord and hindbrain, and in the for specification of neuronal identity. Thus, homeo box PNS, in the dorsal root ganglia, and in the nodose genes in vertebrates, as well as in invertebrates, may ganglia, as shown in Figure 7. possibly act in controlling neuronal fate. The specifica­ The pattern of Hox-2.6 expression that we observe in tion of neuronal fate is certainly a very complex system, the 12.5-day embryo is complex and does not correspond which may require a family of proteins that can act in to any obvious biological or morphological signal. It concert to define cell fate/position. The large size of the seems probable that Hox-2.6 is responding to anteropos­ family of murine homeo box genes and the fact that they terior positional cues but is not doing so in any simple or all seem to be expressed in the CNS make them good exclusive fashion. It is clear that two structures derived candidates for performing such a role. from the same germ layer and sharing the same antero­ The three Dfd-related murine genes, Hox-2.6, posterior position do not necessarily both express this Hox-1.4, and Hox-5.1, may act potentially in an antago­ gene. This is illustrated well in the case of the lung mes­ nistic or synergistic manner. It will be of great interest enchyme, which does express this gene, and the limbs, to compare their patterns of expression and regulation in which do not. Both structures share a similar anteropos­ more detail. The one feature that may be shared is the terior position but exhibit differential expression of this extent of expression of these genes in the ectoderm gene. Thus, if position is of importance in determining along the rostrocaudal axis at comparable stages of de­ the patterns of Hox-2.6 expression, it is likely to have velopment. Dfd is expressed near the rostral end of the been modified by other cues to produce the observed Drosophila embryo (Martinez-Arias et al. 1987), and we pattern at 12.5 days. This is comparable to the patterns have shown that Hox-2.6 is expressed along the spinal of expression that are observed in later stage Drosophila cord into the hindbrain of the murine embryo. Hox-1.4 embryos, where the more simplistic earlier patterns of is also expressed in the murine hindbrain (Toth et al. expression have been modified during development 1987), as is Hox-5.1 (Featherstone et al. 1988); and (Martinez-Arias et al. 1987). Hence, a simple early pat­ Northern analysis shows HHO.cl3 transcripts in the tern of expression in mouse embryos may be masked brain but not in the forebrain (Mavillo et al. 1986). We later in embryogenesis. have also presented evidence illustrating thatHox-2.6 is There are similar regions of expression shared be­ expressed more anteriorly than the adjacent Hox-2.1 tween vertebrate and Drosophila homeo box genes in gene (Fig. 9), and it has been reported that Hox-2.5 is not comparable stages of development. It has been shown expressed in the hindbrain but is restricted to the spinal that a number of Drosophila homeo box transcripts ac­ cord (Feinberg et al. 1987). Therefore, it would appear cumulate to their highest levels in the embryonic CNS that there is a correlation between the anterior limit of (for review, see Doe and Scott 1988). Expression in the expression of a gene in the ectoderm and the relative po­ CNS is observed with a large number of homeo box sition within a cluster, as has also been observed inDro­ genes from a wide range of species. Expression of homeo sophila (Akam 1987). This correlation may also extend box genes in the CNS has been described for humans to the subfamily to which that given gene is a member. (Simeone et al. 1986), forXenopus (Carrasco and Mala- Methods cinski 1987; Condie and Harland 1987), and the zebra fish (Njolstad et al. 1988). With regard to the mouse, we Isolation of clones and sequencing have shown that Hox-2.6 is expressed in the CNS, and Cosmid clones were isolated from a mouse (129) genomic li­ this has also been demonstrated with Hox-1.2 (Toth et brary, prepared with the vector pcos2EMBL (a gift from Anna- al. 1987),Hox-1.3 (Dony and Gruss 1987),Hox-1.4 (Toth Marie Frischauf), by screening with subcloned probes from the et al. 1987; Wolgemuth et al. 1987),Hox-1.5 (Fainsod et Hox-2.1 gene (Krumlauf et al. 1987). The clones were mapped

1434 GENES & DEVELOPMENT Hox-2.6 structure and expression

HOX 2.6

Hox 2.1

P

Figure 9. A comparison of the patterns of expression of Hox-2.6 (A) and Hox-2.1 [B] in the hindbrain of a 12.5-day mouse embryo. Near adjacent sections were probed with either Hox-2.6 or Hox-2.1 antisense probes. Both bright-field and dark-ground pictures are shown. (HB) Hindbrain; (RBC) red blood cells; (A) anterior; (P) posterior.

and flanking regions subcloned to be used for further screening medium (DMEM), supplemented w ith 10% fetal calf serum on of overlapping cosmids. Homeo box regions in the cosmids gelatinized plastic tissue culture dishes. The cells were induced were identified by hybridization with a Drosophila Antp (pro­ to differentiate by adding retinoic acid (5 x 10“8 m ), cAMP vided by Walter Gehring) and mouse Hox-2.1 ( ) homeo box (10“4 m ), and IMBX (10“4 m ) to cultures, and the cells were probe at reduced stringency according to Holland and Hogan maintained in this differentiation media for 1-5 days, with the (1986). Homeo box fragments were then subcloned and used to media being changed every 2 days. In the case where stem cells screen a mouse KgtlO cDNA library (Fahrner et al. 1987) pre­ were treated with the protein synthesis inhibitor cyclohexi­ pared from 8.5-day embryos for Hox-2.6 clones. All cosmid and mide, the inhibitor was added to the cultures to a final concen­ cDNA subcloning was performed using the vector Bluescript tration of 75 |xg/ml for a total of 6 hr before harvesting. Under pKSM13+ (Stratagene). Double-stranded DNA from the cDNA these conditions in control experiments, 95% of protein syn­ and genomic clones was sequenced by the dideoxy chain-termi- thesis is blocked and 60% of the cells can recover to reinitiate nation method, using the array of primers in the Bluescript protein synthesis when the inhibitor is removed. Cells were vector and the Sequenase polymerase (US Biochemical Corpora­ harvested for RNA at various time points by washing twice tion) according to manufacturer's directions. All sequencing with PBS and lysing them on the plate in LiCl urea. was performed in both directions using both the standard and dITP reactions to avoid the compressions in GC-rich regions.

RNA isolation and Northern hybridization

Cell culture Poly(A)+ mRNA was isolated from F9 cells or mouse tissues essentially as described in Krumlauf et al. (1987). Briefly, the F9 stem cells were maintained in Dulbecco's modified eagle's samples were homogenized in 3 M LiCl- 6 M urea on ice for 2

GENES & DEVELOPMENT 1435 Graham et al. min and stored overnight at 4°C to precipitate the RNA. The was supported by the Greek State Scholarship Foundation and RNA was harvested by centrifugation, redissolved in 10mM A.G. by a Medical Research Council studentship. Tris-HCl (pH 7.6), 1 m M EDTA, and 0.5% SDS and extracted several times with phenol: chloroform. Following ethanol pre­ cipitation, the poly(A)+ mRNA was isolated by oligo(dT) cellu­ References lose chromatography. The RNA samples were electrophoresed Akam, M.E. 1987. The molecular basis for metameric pattern in denaturing formaldehyde-agarose gels, transferred to Gene- in the Drosophila embryo. Development 101: 1-22. Screen (Dupont) in 20 x SSC, and coupled to the membrane by Awgulewitsch, A., M.F. Utset, C.P. Hart, W. McGinnis, and UV cross-linking and baking, as described in Krumlauf et al. F.H. Ruddle. 1986. Spatial restriction in expression of a (1987). The filters were hybridized using 32P-labeled single­ mouse homeo box locus within the central nervous sytem. stranded antisense RNA probes produced by in vitro transcrip­ Nature 320:328-335. tion of subcloned Hox-2.1 and Hox-2.6 cDNA clones with T7 or Baron, A., M.S. Featherstone, R.E. Hill, A. Hall, B. Galliot, and T3 RNA polymerase. Probes were selected from regions of the D. Duboule. 1987.Hox-1.6: A mouse homeo box containing genes that had a minimum identity with other subgroup gene member of the Hox-1 complex. EMBO J. 6: 2977-2986. members to minimize any cross hybridization. Hybridization Blochlinger, K., R. Bodmer, J. Jack, L.Yeh Jan, and Y. Nung Jan. conditions were 60% formamide, 5 x SCC, 0.1% bovine serum 1988. Primary structure and expression of a product from albumin, 0.1% Ficoll 400, 0.1% polyvinylpyrrolidine, 20mM cut, a locus involved in specifying sensory organ identity in sodium phosphate (pH 6.8), 1% SDS, 7% dextran sulfate, 100 Drosophila. Nature 333: 629-635. (xg/ml tRNA, and 10 p-g/ml poly(A) at 65°C for 16 hr. The filters Breier, G., G.R. Dressier, and P. Gruss. 1988. Primary structure were washed in 0.1 x SCC, 0.5% SDS at 80°C for 2 hr, and in and developmental expression of Hox3.1, a member of the the cases where high-stringency conditions were required to murine Hox 3 homeo box gene cluster. EMBO J. 7: 1329- ensure that the signal was not derived from related genes, the 1336. filter was treated further with RNase A. The filters were incu­ Breier, G., M. Bucan, U. Franke, A.M. Colberg-Poley, and P. bated in 2x SCC, 0.1% SDS at 52°C for 1 hr. Probe was re­ Gruss. 1986. Sequential expression of murine homeo box moved from filters (not treated with RNase A) for rehybridiza­ genes during F9 EC cell differentiation. EMBO f. 5: 2209- tion by washing in 70% formamide at 80°C for 30 min and 2215. reexposing the filter to ensure probe removal. Bucan, M., T. Yang-Feng, A.M. Colberg-Poley, D.J. Wolgemuth, J. Guenet, U. Franke, and H. Lehrach. 1986. Genetic and cy­ togenetic localisation of the homeo box containing genes on In situ hybridization mouse chromosome 6 and human chromosome 7. EMBO f. The protocol used was basically that of Wilkinson et al. (1987), 5: 2899-2905. with some modifications, and is essentially as follows. Mouse Carrasco, A.E. and G.M. Malacinski. 1987. Localisation of embryos were fixed in 4% paraformaldehyde in phosphate-buff­ Xenopus homeo box gene transcripts during embryogenesis ered saline (PBS) overnight at 4°C, and then embedded in par­ and in the adult nervous system.Dev. Biol. 121: 69-81. affin wax. Six-micrometer sections were cut and dried onto gel­ Cho, K.W.Y., J. Goetz, C.V.E. Wright, A. Fritz, J. Hardwicke, atin-subbed slides. The sections were dewaxed in xylene, and E.M. DeRobertis. 1988. Differential utilisation of the treated with 0.2 m HCl, refixed in paraformaldehyde, subjected same reading frame in a Xenopus homeo box gene encodes to proteinase K, and then treated with acetic anhydride. After two related proteins sharing the same DNA-binding speci­ rehydration, probes were redissolved at a final activity of ficity. EMBO J. 7: 2139-2149. 1.2 x 105 dpm/kb/p-l in hybridization buffer (50% formamide, Colberg-Poley, A.M., S.D. Voss, K. Chowdry, C.L. Stewart, E.F. 0.3 M NaCI, 20 m M Tris, 5 m M EDTA, 10 m M NaP04, 10% Wagner, and P. Gruss. 1985a. Structural analysis of murine dextran, 1 x Denhardt's, tRNA). Hybridization was overnight genes containing homeo box sequences and their expression at 50°C. The sections are washed at 65°C in 50% formamide, in embryonal carcinoma cells. Nature 314: 713-718. 2x SSC, 100 m M dithiothreitol (DTT) for 40 min, followed by . 1985b. Clustered homeo boxes are differentially ex­ incubation with RNase A at 20 |xg/ml in 0.5m NaCI, 10 m M pressed during murine development. Cell 43:39-45. Tris, 5 m M EDTA for 30 min. Sections were washed again in Condie, G.B.G. and R.M. Harland. 1987. Posterior expression of 50% formamide, 2x SSC, and 100 m M DTT for 30 min, fol­ a homeo box gene in earlyXenopus embryos. Development lowed by two 15-min washes in 2x SCC and 0.1 x SSC. Sec­ 101:93-105. tions were dehydrated through alcohol solutions containing 0.3 Deschamps, J., R. deLaaf, P. Verrijzer, M. deGuow, O. Destree, M NH4OAc. Slides were dipped in a mix of Ilford K5 nuclear and F. Meijlink. 1987. The mouseHox-2. 3 homeo box-con­ emulsion and glycerol/water (6 ml emulsion in 8.82 ml taining gene: Regulation in differentiating pluripotent stem H2O/0.18 ml glycerol) and kept at 4°C until developed. Expo­ cells and expression pattern in embryos. Differentiation sures were between 10 and 12 days. Sections were stained in 35: 21-30. 0.02% toluidine blue for 1 min and then mounted in Permount. Desplan, C., J. Theis, and P.H. O'Farrell. 1985. TheDrosophila developmental gene, engrailed, encodes a sequence specific Acknowledgments DNA binding activity.Nature 318: 630-635. Doe, C.G. and M.P. Scott. 1988. Segmentation and homeotic We thank Dr. Bernhard Hermann for help in screening the gene function in the developing nervous system Droso­of cosmid libraries and isolating pCos 5.4 and 3.1, and Drs. Denis phila. Trends Neurosci. 11: 101-106. Duboule, Mark Featherstone, Brigitte Galliot, Pal Njolstad, Jim Doe, C.Q., Y. Hiromi, W.J. Gehring, and C.S. Goodman. 1988. Garbem, Ward Odenwald, and Edoardo Boncinelli for sharing Expression and function of the segmentation gene fushi- unpublished sequence information and ideas about the organi­ tarazu during Drosophila neurogenesis. Science 239: 170- zation and evolution of homeo box genes. We also thank Drs. 175. David Wilkinson, Peter Holland, Frank Ruddle, Mike Snow, Dony, C. and P. Gruss. 1987. Specific expression of theHox-1.3 Andy McMahon, Dennis Summerbell, and Mai Har Sham for homeo box gene in murine embryonic structures originating valuable discussions and comments on the manuscript. N.P. from or induced by the mesoderm. EMBO f. 6: 2965-2975.

1436 GENES & DEVELOPMENT Hox-2.6 structure and expression

Doyle, H.J., K. Harding, T. Hoey, and M. Levine. 1986. Tran­ Holland, P.W.H. and B.L.M. Hogan. 1986. Phylogenetic distri­ scripts encoded by a homeo box gene are restricted to dorsal bution of Antennapaedia like homeo boxes. Nature tissue of Drosophila embryos. Nature 323: 76-79. 321:251-253. Duboule, D., A. Baron, P. Mahl, and B. Galliot. 1986. A new . 1988. Spatially restricted patterns of expression of the homeo box is present in overlapping cosmid clones which homeo box containing gene Hox-2.1 during mouse embryo­ define the mouse Hox-1 locus. EMBO /. 5: 1973-1980. genesis. Development 102: 159-174. Fainsod, A., L.D. Bogarad, T. Ruusala, M. Lubin, D.M. Crothers, Jackson, I.J., P. Schofield, and B.L.M. Hogan. 1985. A mouse and F.H. Ruddle. 1986. The homeo domain of a murine pro­ homeo box gene is expressed during embryogenesis and in tein binds 5' to its own homeo box. Proc. Natl. Acad. Sci. adult kidney. Nature 317: 745-748. 83: 9532-9536. Joyner, A.L. and G.R. Martin. 1987. 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1438 GENES & DEVELOPMENT