Cranial Paraxial Mesoderm: Regionalisation of Cell Fate and Impact on Craniofacial Development in Mouse Embryos

Development 120, 2397-2408 (1994) 2397 Printed in Great Britain © The Company of Biologists Limited 1994

Cranial paraxial mesoderm: regionalisation of cell fate and impact on craniofacial development in mouse embryos

Paul A. Trainor1, Seong-Seng Tan2 and Patrick P. L. Tam1,* 1Embryology Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville NSW 2145, Australia 2Embryology Laboratory, Department of Anatomy and Cell Biology, University of Melbourne, Parkville, VIC 3052, Australia *Author for correspondence

SUMMARY

A combination of micromanipulative cell grafting and flu- the muscle plates. A dorsoventral regionalisation of cell fate orescent cell labelling techniques were used to examine the similar to that of the somitic mesoderm is also found. This developmental fate of the cranial paraxial mesoderm of the suggests evolution has conserved the fate of the murine 8.5-day early-somite-stage mouse embryo. Mesodermal cranial paraxial mesoderm as a multiprogenitor population cells isolated from seven regions of the cranial mesoderm, which displays a predominantly myogenic fate. Heterotopic identified on the basis of their topographical association transplantation of cells to different regions of the cranial with specific brain segments were assessed for their con- mesoderm revealed no discernible restriction in cell tribution to craniofacial morphogenesis during 48 hours of potency in the craniocaudal axis, reflecting considerable in vitro development. The results demonstrate extensive plasticity in the developmental fate of the cranial cell mixing between adjacent but not alternate groups of mesoderm at least at the time of experimentation. The dis- mesodermal cells and a strict cranial-to-caudal distribution tribution of the different groups of cranial mesoderm of the paraxial mesoderm to craniofacial structures. A two- matches closely with that of the cranial neural crest cells segment periodicity similar to the origins of the branchial suggesting the two cell populations may share a common motor neurons and the distribution of the rhombencephalic segmental origin and similar destination. neural crest cells was observed as the paraxial mesoderm migrates during formation of the first three branchial arches. The paraxial mesoderm colonises the mesenchymal Key words: cell fate, somitomere, craniofacial development, core of the branchial arches, consistent with the location of branchial arches, mouse embryo

INTRODUCTION order in the neural tube (Tan and Morriss-Kay 1986; Chan and Tam, 1988; Lumsden, 1990; Lumsden et al., 1991; Serbedzija Segmentation is a feature characteristic of the axial skeleton, et al., 1992; Wilkinson and Krumlauf, 1990; Hunt et al., musculature and nervous system of vertebrates (Keynes and 1991a,b,c). Furthermore, neural crest cells that originate from Stern, 1984; Lumsden and Keynes, 1989; Puelles and Ruben- specific segments of the hindbrain (e.g. rhombomeres r2, r4 stein, 1993). Craniofacial structures such as the branchial and r6) express a similar set of Hoxb genes characteristic of arches, pharyngeal pouches and cranial nerve ganglia are their rhombomeric origins (Hunt et al.,1991a). The emulation typically arranged into serially repeated patterns in mammalian of rhombomeric Hox gene expression by neural crest cells embryos (Hunt and Krumlauf, 1991; Hunt et al., 1991a; suggests that the early segmental organisation of the hindbrain Thorogood, 1993). The craniofacial tissues are composed prin- may have long-term effects on cell differentiation and pattern- cipally of neuroectodermally derived neural crest cells and the ing of the craniofacial structures. cranial paraxial mesoderm, both of which are segmentally Presently it is not known whether the cranial paraxial organised during early embryogenesis (Couly et al., 1993; mesoderm also exerts a similar segmental influence on cranio- Noden, 1986, 1988). Recent studies on the ontogeny of cranial facial morphogenesis. A meristic pattern of somitomeres neural crest cells have revealed that the neuromeric organisa- (Meier, 1979) has been described in head mesoderm of eight tion of the neural tube has a significant impact on neural crest vertebrate embryos, including the mouse (Tam and Trainor, differentiation (Wilkinson et al., 1989; Figdor and Stern, 1993; 1994). Somitomeres are clusters of loosely packed mesenchy- Fraser, 1993; Puelles and Rubenstein, 1993). In essence, neural mal cells that form in the paraxial mesoderm along the length crest cells destined for different branchial arches or cranial of the embryo (Meier and Tam, 1982). The somitomeres in the nerve ganglia always arise from specific neuromeres and their segmental plate or presomitic mesoderm have been regarded migration pattern faithfully follows their original metameric as the primordial structures for somites (reviewed by Tam and

2398 P. A. Trainor, S.-S. Tan and P. P. L. Tam

Trainor, 1994). In the mouse, the cranial mesoderm consists of develop according to their site of transplantation rather than their seven pairs of contiguous somitomeres, which maintain a con- origin. Transplantations and labelling of cells were performed by sistent topographical relationship with specific regions of the micromanipulating embryos that were explanted from the uterus. neural tube (Tam and Meier, 1982; Meier and Tam, 1982; Experimental embryos were cultured in vitro for the assessing the fate Jacobson and Meier, 1986). Somitomere I (Sm-I) underlies the of the cells. prosencephalon. Somitomeres II and III (Sm-II and Sm-III) Recovery and in vitro culture of host embryos reside lateral to the mesencephalon. Somitomere IV (Sm-IV) is associated with the metencephalon. Somitomeres V, VI and Early-somite-stage embryos were obtained from pregnant female mice of ARC/S strain at 8.5 days post coitum (p.c.), Whole concep- VII (Sm-V, Sm-VI and Sm-VII) underlie the myelencephalon tuses were dissected out from the uterus. The parietal yolk sac was (Fig. 1). However, to this date, lineage restriction and removed leaving the embryo proper with an intact visceral yolk sac, molecular heterogeneity of cells in different somitomeres com- amnion and ectoplacental cone. Previous studies on the migration of parable to those displayed by the neuromeres (Fraser et al., cranial neural crest cells in the mouse embryo have established that 1990; Puelles and Rubenstein, 1993) have not been found. the first population of neural crest cells to leave the neural tube are In the chick, different groups of voluntary cranial muscles those of the mesencephalon and it happens at about 5- to 6-somite and some components of the chondrocranium are found to be stage (Jacobson and Tam, 1982; Chan and Tam, 1986, 1988; Serbedz- derived from different portions of the cranial paraxial ija et al., 1992). To ensure that only the paraxial mesoderm and no neural crest cells were labelled or grafted, we applied a stringent mesoderm (Noden, 1983b, 1988; Couly et al., 1992). Such ≤ regionalisation of cell fate has been attributed to the partition- criterion that only embryos having 5 pairs of somites were used for the experiments. After cell grafting or labelling, embryos were ing of the different myogenic and skeletogenic precursors to cultured in vitro for 48 hours (Sturm and Tam, 1993). the cranial somitomeres. The chimaeric avian embryos analysed in these studies had developed beyond the stages of Isolating donor transgenic tissue and grafting branchial arch and facial primordia formation such that fusion 8.5-day embryos were obtained from matings of transgenic H253 and growth of these structures had already occurred. The early mice, which express a bacterial lacZ transgene under the control of pattern of distribution and the fate of individual cranial somit- the 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase omeres were not examined, leaving unresolved the question of promoter (Tam and Tan, 1992). Donor H253 embryos were first whether the difference in the myogenic fates between somito- bisected along their midline (Fig. 2A). Wedge-shaped fragments con- meres is a reflection of the regionalisation of cell fate or a taining the paraxial mesoderm and the associated neuroectoderm and restriction in cell potency. What is also lacking in previous surface ectoderm (Fig. 2B) were isolated by cutting the embryo trans- studies of cephalic mesodermal organisation is a clear appre- versely at various neuromeric junctions (Fig. 2A). ciation of the precise somitomeric origins of the branchial arch mesenchyme. It remains to be seen if, like the neural crest cells, there is a segmental allocation of paraxial mesoderm to craniofacial structures. In the present study, we have taken the first Mes step to analyse the role of somitomeres in craniofacial mor- Pro phogenesis by testing if there is any regionalisation of cell fate along the craniocaudal axis of the cranial paraxial mesoderm II regarding its contribution to serially repeated craniofacial I structures of the mouse embryo. Specifically, we aimed (i) to trace the distribution of cells in different segments of cranial III mesoderm as defined by the somitomeres and (ii) to determine if the craniocaudal order of cells in the cranial mesoderm is transposed into patterning of craniofacial structures such as the branchial arches. We have also examined the cranial mesoderm for evidence of restriction of cell fate by heterotopic trans- HT IV Met plantation of lacZ-tagged cells and by differential labelling of different parts of the paraxial mesoderm. V Mye MATERIALS AND METHODS VI Experimental strategy For tracking the fate of the cranial mesoderm, small groups of meso- VII 2 dermal cells were isolated from specific regions of the paraxial 1 mesoderm (Figs 1, 2A-C), and were then transplanted to an equivalent site in isochronic 8.5-day host embryos. The transplanted cells were marked by the β-galactosidase activity encoded by a lacZ Fig. 1. The subdivision of the cranial paraxial mesoderm into seven transgene, which is ubiquitously expressed during development (Tam (I to VII) somitomeres (Meier and Tam, 1982) in accordance to their and Tan, 1992). Alternatively, the mesodermal cells were labelled in topographical relationship to major segments of the prosencephalon situ with fluorescent carbocyanine dyes. Heterotopic transplantations (Pro), mesencephalon (Mes), Metencephalon (Met) and of mesoderm to different sites in host embryos were also performed Myelencephalon (Mye). The short dashes demarcate the boundaries to assess the prospective potency and state of commitment of the cells. between brain segments. The first 2 somites are numbered and HT In the absence of regional specification, the transplanted cells would denotes the heart.

Fate of cranial somitomeres 2399

The embryonic fragments were then incubated in a solution of 0.5% the donor mesoderm were performed. Somitomere III was chosen trypsin, 0.25% pancreatin, 0.2% glucose and 0.1% polyvinylpyroli- respectively as a source of donor mesoderm and a site for transplan- done in PBS for 20 minutes at 37¡C to loosen the mesoderm and tation because it is centrally located within the head. This allowed us ectoderm layers. Finely polished alloy and glass needles were used to to assess its development in ectopic positions in the head both rostral separate the ectoderm from the underlying mesoderm (Fig. 2C). The and caudal to its normal axial position. mesoderm was dissected into fragments containing approximately 30 cells which were then grafted into the cranial mesoderm of the host Analysis of the results of transplantation experiments embryo (Fig. 2D). For the purpose of this study, the mesoderm Host embryos were harvested after 48 hours of in vitro culture and isolated from different cranial fragments and the sites to which they grossly abnormal embryos were discarded. Embryos were fixed in were transferred are referred to by their somitomeric designation, i.e. 4% paraformaldehyde for 30 minutes and stained by X-gal histo- Sm I-VII (Fig. 1). chemistry to reveal the β-galactosidase activity (Tam and Tan, 1992). Embryos containing blue X-gal-stained cells were processed Transplantation experiments for wax histology and counterstained with nuclear fast red. The 1. Orthotopic transplantations of cranial mesoderm to location of labelled cells in structures such as the branchial arches isochronic hosts and craniofacial mesenchyme was determined on serial sections of Donor mesoderm derived from Sm I-VII was transplanted to the the embryo. equivalent somitomere in host embryos (Fig. 2E). Seven types of transplantation were performed, designated I→I, II→II, III→III, Preparation of fluorescent dyes for injection IV→IV, V→V, VI→VI or VII→VII, to denote the somitomeric 0.05% solutions (w/v) of DiI (1,1-dioctadecyl-3,3,3′,3′-tetra- origin of donor cells and the site of transplantation, respectively. methylindocarbocyanine perchlorate) and DiO (3,3′-dioctadecyloxa- carbocyanide perchlorate, Molecular Probes) were made from a 0.5% 2. Heterotopic transplantations of somitomeric mesoderm to stock ethanolic solutions. The stock was diluted 1:7.5 in 0.3 M sucrose isochronic hosts immediately before injection. Micropipettes were backfilled with dye In the first set of experiments, donor mesoderm from somitomeres I, solution and injection was done under positive pressure generated by II, III, IV, V and VI was transplanted to different somitomeres in host a deFonbrune oil pump (Alcatel). embryos. In the second set, somitomeres I, II, IV, V and VI were chosen to demonstrate how tissues both rostral and caudal to somito- Dye labelling of individual somitomeres mere III develop when ectopically grafted to somitomere III. Nine The same topographical landmarks employed for locating somito- experiments designated III→I, III→II, III→IV, III→V, I→III, II→III, meres in the transplantation experiments were also used for targeting IV→III, V→III and VI→III referring to the source and destination of the dye injection. Following insertion of the micropipettes into the

Table 1. Summary of the patterns of tissue colonisation in transplantation and labelling experiments Somitomere No. performed No. analysed FNM POM MXP MDP FM SA CV PTM TA FA a. Dye Injection I 22 17 + + − − − − − − − − II 30 25 − + + − − − − − − − III 27 21 − + + + + − − − − − IV 25 22 − − − − + + + − − − V 26 21 − − − − − + + + − − VI 20 15 − − − − − − − + + − VII 22 18 − − − − − − − + + + b. Orthotopic grafts I➞I 15 11 + + − − − − − − − − II➞II 13 10 − + + − − − − − − − III➞III 40 33 − + + + + − − − − − IV➞IV 32 26 − − + + + − − − − − V➞V 15 12 − − − − − + + + − − VI➞VI 18 13 − − − − − − − + + − VII➞VII 21 16 − − − − − − − − + + c. Heterotopic grafts III➞I 8 5 + + - − − − − − − − III➞II 10 6 − + + − − − − − − − III➞IV 11 8 − − − − + + + − − − III➞V 7 5 − − − − − + + + − − I➞III 8 5 − − + + + − − − − − II➞III 9 6 − − + + + − − − − − IV➞III 7 6 − − + + + − − − − − V➞III 8 5 − − + + + − − − − − VI➞III 10 7 − − + + + − − − − − d. Dorsal/Ventral Injections III dorsal 15 11 − − + − + − − − − − III ventral 15 11 − − + + − − − − − − IV dorsal 14 9 − − − − − − + + − − IV ventral 14 9 − − − − − + − − − −

Abbreviations. FNM, frontonasal mass; POM, periocular mesenchyme; MXP, maxillary component of first arch; MDP, mandibular component of the first arch; FM, facial mesenchyme; SA, second arch; CV, cervical mesenchyme; PTM, periotic mesenchyme; TA, third arch; FA, fourth arch. 2400 P. A. Trainor, S.-S. Tan and P. P. L. Tam mesoderm, a small amount of DiI or DiO solution was expelled and reference point for a dorsal label. The two injection sites were always the labelled sites were visible through the dissecting scope as red (DiI) separated by a zone of unlabelled cells. or orange (DiO) patches of labelled mesoderm (Fig. 2F). For testing dorsoventral regionalisation, different parts of somitomeres III and IV Analysis of the results of dye labelling experiments were labelled with different fluorescent dyes (Fig. 2G). The ventral Embryos cultured in vitro for 48 hours were viewed without any region of the somitomere was labelled first, which provided the fixation as whole mount under a standard fluorescence microscope

Fig. 2. (A) The bisected halves of a 8.5-day (5-somite) mouse embryo showing the somitomeric boundaries (arrows). Transverse cuts made at these speciﬁc neuromeric landmarks resulted in isolation of single somitomeres. (B) Embryonic fragment containing the Sm-III and its associated surface ectoderm and neuroectoderm obtained by cutting the half-head at mid-mesencephalon and at the mesencephalon- metencephalon boundary. (C) The wedge-shaped somitomeric mesoderm (arrowhead) dissected free of the surface ectoderm and neural plate. (D) Grafting somitomeric cells to a 8.5-day early-somite-stage embryo by inserting the injection micropipette through the extraembryonic membranes into Sm-III. (E) A 8.5-day embryo examined 2 hours after transplantation to reveal the location of the tissue grafts (X-gal-stained blue cells), which were grafted into the dorsal region of Sm-I and the caudal-ventral region of Sm-IV (arrowheads). (F) A 8.5-day embryo examined immediately after DiI injection into Sm-I, Sm-III, Sm-IV and Sm-VI (arrows). (G) A 8.5-day embryo with the ventral and dorsal regions of Sm-III labelled by injection of DiO (arrow) and DiI, respectively. Abbreviation: hb, hindbrain; ht, heart; mb, midbrain. Bar, 100 mm. Fate of cranial somitomeres 2401 Fig. 3. For legend see p. 2402 2402 P. A. Trainor, S.-S. Tan and P. P. L. Tam

(Leitz). The DiI and DiO signals were detected using rhodamine and distal region of the first arch which gives rise to the mandibu- FITC filters respectively. Photographs were taken using Ektachrome lar prominence (Fig. 3B). Mesoderm derived from Sm-III 400 or 160 films directly with a Wild MPS 46/52 Photoautomat camera. colonised both the mandibular and the maxillary prominences of the first branchial arch (Fig. 3C). There was a widespread RESULTS distribution of grafted cells in the facial mesenchyme surrounding the proximal end of the first arch. Cells derived from Cranial somitomeric mesoderm contributes to Sm-III were also found in the maxillary component of the arch, branchial arch formation overlapping with that of Sm-II (Fig. 3D). Histology of the first Table 1 summarises the pattern of distribution of transplanted arch revealed that the graft-derived cells were localised in the cells and DiI/DiO-labelled cells to various craniofacial struc- central to medial core mesenchyme (Fig. 3E). Sm-II and Sm- tures in the mouse embryo after 48 hours of culture. Embryos III did not participate in morphogenesis of more caudal to be analysed were selected on the basis that (i) they are mor- branchial arches. The distribution pattern of Sm-III when phologically similar to the 30- to 36-somite-stage embryo in labelled with DiI resembled closely that observed for grafted vivo, (ii) the branchial arches were well formed and (iii) both mesoderm (Fig. 3F). The fluorescent mesoderm again the forelimb and hindlimb buds had developed. colonised the central regions of the maxillary and mandibular Results of the transplantation and labelling experiments prominences. The distribution of fluorescent cells, however, showed that the cranial mesoderm contributes cells to the first seemed broader than that observed for the grafted cells. The four branchial arches in a manner strictly in accordance with quantitative difference is due to the fact that more cells are their craniocaudal order in the axis (Table 1a,b). Each of the labelled through dye injection than are transplanted. There first three branchial arches, were colonised by cells from neigh- were no instances in which the mandibular epithelia or the bouring regions of the paraxial mesoderm that are defined by surface ectoderm were colonised with mesoderm derived from two consecutive somitomeres: namely Sm-II and III for first Sm-II and Sm-III. arch, Sm-IV and V for the second arch and Sm-VI and VII for The second branchial (hyoid) arch received contribution the third arch (Table 1a,b; Fig. 5). In contrast, only one somit- from the preotic mesoderm associated with the upper hindbrain omere participated in fourth arch development and the fifth and (Sm-IV and Sm-V). Mesoderm derived from Sm-IV, which sixth pairs of branchial arches did not receive any contribution underlies the metencephalon, contributed primarily to the from the cranial mesoderm. central region of the hyoid arch (Fig. 3G). The distribution of Both II→II grafts and Sm-II dye injection experiments IV→IV grafted cells was widespread in the rhombencephalic revealed that mesoderm derived from Sm-II extensively mesenchyme adjacent to the second arch but, within the arch, colonised the proximal maxillary prominence of the first the mesoderm colonised a central core region (Fig. 3H). branchial arch (Fig. 3A). In 8% (2/25) of cases where Sm-II Marking somitomere IV with DiI produced similar results to was labelled with either DiO or DiI and in 10% (1/10) of II→II IV→IV grafting experiments, with the labelled cells populat- grafts, a small number (5-10) of cells were displaced to the ing the hyoid arch (Fig. 3I). Sm-V, which underlies the rostral myelencephalon, also contributed to the hyoid arch. There was a clear segregation of donor mesoderm derived from the dorsal Fig. 3. Sm-II contribution to (A) the maxillary prominence of the and ventral regions of Sm-V respectively such that the dorsal first arch, caudal to the optic vesicle (arrow) and (B) to the distal mandibular region of the first arch (arrow), the mesenchyme lateral mesoderm was found in a more proximal position in the second to the diencephalon and the periocular mesenchyme (arrowhead). arch than the ventral mesoderm (Fig. 3J). The distribution of (C) Sm-III colonisation of the central core mesenchyme (arrowhead) Sm-V-derived cells overlapped significantly with that of Sm- of the mandibular arch and in the maxillary prominence. (D) Sm-III IV. There was no evidence of Sm-IV and Sm-V contributing distribution primarily to the maxillary (arrow) and mandibular to formation of either the first or third branchial arches. In one regions of the first arch. (E) A section through the facial instance only, the investing mesenchyme and epithelium of the mesenchyme and branchial arch region showing III→III graft- first pharyngeal pouch were populated with Sm-V-derived derived cells restricting to the mesenchyme core of the first arch but mesoderm (data not shown). are dispersed in the facial mesenchyme. (F) DiI-labelled cells in the Similar to the first two arches, the third arch received a dual core of the maxillary and mandibular portions of the first arch. (G) contribution from two adjacent somitomeres, Sm-VI and Sm- Non-overlapping distribution of Sm-IV and VI mesoderm cells to the → second and third branchial arches, respectively, after a double VII of the caudal myelencephalon. VI VI grafts showed that orthotopic grafting (IV→IV and VI→VI). Sm-IV colonised the core the mesodermal cells were mostly confined to the central of the second arch, the rostral periotic mesenchyme and the region of the third arch (Fig. 3G). Sm-VI did not contribute to mesenchyme at the base of the first arch. Some cells are found formation of first, second or fourth branchial arches. Sm-VII, dispersed rostrally to the facial mesenchyme. Sm-VI colonises the which is the caudal most cranial somitomere, participates core of the third arch and the ventral periotic mesenchyme (arrow). primarily in formation of the third arch. The distribution of (H) A section through the hyoid arch showing the distribution of VII→VII grafted cells in the third arch overlapped with cells IV→IV graft-derived cells in the core region of the hyoid arch and in derived from Sm-VI. VII→VII grafts also reveal that Sm-VII the adjacent paraxial mesenchyme. (I) DiI-labelled cells derived → cells were found in the central core region of the fourth from Sm-IV in the second arch. (J) V V graft-derived cells branchial arch. populating the hyoid arch (arrow) and the rostral and ventral periotic mesenchyme (arrowhead). Abbreviations: fb, forebrain; fm, facial Prosencephalic and mesencephalic mesoderm mesenchyme; ha, hyoid arch; ht, heart; ma, mandibular arch; mb, midbrain; md, mandibular prominence; mx, maxillary prominence; contribute to the periocular, frontonasal and facial np, nasal process; op, otic placode; ov, optic vesicle; ta, third arch. mesenchyme Bar, 100 mm for A-D,G and J. Bar, 300 µm for E,F,H,I. The two most rostral somitomeres (Sm-I and Sm-II), con- Fate of cranial somitomeres 2403 tributed extensively to the periocular mesenchyme. I→I and distal mandibular and maxillary regions of the first branchial II→II grafts showed that the rostral and ventral portions of the arch. Progressively more dorsal mesoderm derived from Sm- periocular mesenchyme were derived from Sm-I, whereas the III contributed to the maxillary and more proximal regions of dorsal and caudal periocular mesenchyme came primarily from the first arch, and to the facial mesenchyme (Fig. 4H). Sm-II (Fig. 4A,B). There was, however, some overlap in the Similarly, the ventral Sm-IV mesoderm was confined ulti- distribution of Sm-I and Sm-II cells. In 9% (1/11) of I→I trans- mately to the hyoid arch proper and the dorsal mesoderm con- plantations, the dorsal and caudal periocular mesenchyme were tributed to the cervical mesenchyme and to the proximal region colonised by graft-derived cells. In 10% (1/10) of II→II grafts, of the hyoid arch (data not shown). Although some cell mixing Sm-II-derived cells contributed to the rostral periocular mes- occurred at the interface between the DiO- and DiI-labelled enchyme and also the ventral periocular mesenchyme in the populations, mesoderm derived from the dorsal region of the vicinity of the primitive oral cavity. Similar but broader dis- somitomere seldom colonised the distal region of the branchial tribution patterns were observed for Sm-I and Sm-II, respec- arch. Conversely ventrally derived mesoderm never con- tively, when they were directly injected with DiO (Fig. 4C). In tributed to the facial mesenchyme adjacent to the neural tube. addition to the periocular mesenchyme, Sm-I and Sm-II con- These results indicate that regionalisation of cell fate may exist tributed to the endothelium of blood vessels surrounding the in the cranial mesoderm. The dorsoventral partition within optic epithelium (data not shown). These two somitomeres also somitomeres is transposed into a proximal-distal disposition of contributed extensively to the facial mesenchyme lateral to the cells in the branchial arches and adjacent paraxial mes- telencephalon and diencephalon. The frontal area of the enchyme. embryo associated with the lateral and medial nasal processes was colonised by Sm-I only (Fig. 4A). The fate of the cranial paraxial mesoderm is not Sm-III colonised the facial mesenchyme and some vascular positionally restricted and endothelial tissue lateral to the caudal midbrain and meten- Heterotopic transplantations showed that mesoderm derived cephalon (Fig. 4E). The distribution of Sm-III cells overlapped from any axial level, when grafted to a heterotopic site would with Sm-II cells in the facial mesenchyme lateral to the develop according to its new position and not its site of origin. midbrain and with Sm-IV cells in the region lateral to the Table 1c summarises the results of several types of heterotopic rostral hindbrain, adjacent to the mandibular and hyoid arches. transplantation. Sm-III due to its central location in the head, III→III grafts showed that Sm-III colonised the branchial arch was chosen as the source of donor cells for most of the grafts artery, the connective tissue surrounding the truncus arteriosus (45%; 24/53). Sm-III mesoderm was grafted into somitomeres and the endothelium of the primary head vein (anterior cardinal both rostral and caudal to its origins (III→I, III→II, III→IV, vein). III→V). The final distribution of graft-derived cells was always characteristic of the site of transplantation. For Rhombencephalic mesoderm contributes to the example, in III→V transplantation experiments, the mesoderm periotic and cervical mesenchyme cells contributed to the hyoid arch mesenchyme and to the The otic placode develops superficially to Sm-VI and VII. The rostral periotic mesenchyme (Fig. 4I). This was almost periotic mesenchyme was derived from Sm-IV, V, VI and VII. identical to the distribution pattern observed from V→V grafts IV→IV and V→V grafts revealed that Sm-IV and Sm-V con- (Fig. 3J). Similarly in III→II transplantations, the pattern of tributed significantly to both the rostral and rostroventral distribution resembled very closely II→II grafts: the mesoderm periotic mesenchyme, respectively, and to the facial mes- contributed to the caudal and ventral periocular mesenchyme, enchyme associated with the rostral hindbrain (Figs 3J, 4F). to the mesenchyme lateral to the diencephalon and upper VI→VI and VII→VII grafts showed that the ventral and the midbrain, and also to the maxillary component of the first caudal periotic mesenchyme were colonised by mesoderm branchial arch (Table 1c). As expected there was no colonisa- derived from Sm-VI and Sm-VII, respectively (Figs 3G, 4G). tion of the mandibular component of the first arch, previously Sm-VI and Sm-VII also contributed to the cervical mes- shown to be derived from Sm-III (Fig. 3C and 3D). Hetero- enchyme associated with caudal segments of the hindbrain and topic transplantations were also performed for Sm-I. The dis- to the dorsal periotic mesenchyme, which is bounded by the tribution of cells observed after I→III grafts showed that the otic placode and neuroectoderm. In one instance, Sm-VI- ectopic mesoderm cells colonised the maxillary prominence derived mesoderm transplanted into Sm-VI (VI→VI graft) and facial mesenchyme (Fig. 4J). This pattern of colonisation colonised the otic epithelium. This was probably the result of was characteristic of III→III grafts. The grafted mesoderm did grafting cells too superficially to the surface ectoderm. A not contribute to the mesenchyme of the frontal area, nor to the summary of the distribution of somitomeric mesoderm to cran- rostral and ventral periocular mesenchyme, which were deriv- iofacial structures is presented in Table 2 and Fig. 5. atives of I→I grafts (Fig. 4A). Mesoderm cells derived from Sm-IV (IV→III), V (V→III) and VI (VI→III) were also het- Evidence for dorsoventral constraints erotopically grafted into Sm-III. The grafted mesoderm exten- The relative positioning of cells within cranial somitomeres sively colonised the facial mesenchyme, the maxillary and was maintained during their deployment to craniofacial struc- mandibular prominences and the perivascular tissues of the tures, such that the dorsal and ventral regions of a somitomere truncus arteriosus (Fig. 4K,L). In each case where cranial have different fates with respect to craniofacial morphogene- mesoderm was derived from different craniocaudal levels and sis. Table 1d summarises the results of experiments in which grafted into Sm-III, it always developed in a manner charac- the ventral and dorsal mesoderm of Sm-III and Sm-IV were teristic of III→III grafts. These results suggest that the normal labelled with DiO and DiI, respectively. There was a strong fate of the cranial paraxial mesoderm is not positionally or tendency for the ventral mesoderm of Sm-III to colonise the somitomerically restricted at least at 8.5 days p.c. when the het- 2404 P. A. Trainor, S.-S. Tan and P. P. L. Tam Fate of cranial somitomeres 2405

Table 2. The developmental fate of cranial mesoderm during craniofacial morphogenesis. A comparison of the developmental fate of the cranial somitomeres in the chick (Noden, 1983b; Couly et al., 1992) and the mouse (this study) Tissue distribution: chick Tissue distribution : mouse Prechordal mesoderm muscles: rectus ventralis unknown rectus medialis obliquus ventralis Somitomeres I II & III muscles: rectus dorsalis I lateral and medial nasal process obliquus dorsalis periocular mesenchyme rectus lateralis intermandibularis dorsalis intermandibularis ventralis II dorsal and caudal periocular mesenchyme skeleton: corpus sphenoidalis maxiliary component of branchial arch 1 orbitosphenoid III maxilliary componenet of 1st branchial arch mandibular component of 1st branchial arch mandibular arch artery truncus arteriosus IV & V muscles: pterygoideus medialis IV 2nd (hyoid) arch pterygoideus lateralis hyoid arch artery adductor mandibulae externis rostral periotic mesenchyme adductor mandibulae caudalis cervical mesenchyme pseudotemporalis superﬁcialis pseudotemporalis profundus V 2nd (hyoid) arch protractor pterygoidei hyoid arch artery protractor quadrati ventral and rostral peri-otic mesenchyme adductor mandibulae caudalis skeleton: orbitosphenoid corpus sphenoidalis dorso lateral mesencephalic meninges VI & VII muscles: depressor mandibulae, VI 3rd branchial arch skeleton: otic capsule ventral periotic mesenchyme posterior corpus sphenoidalis ventral mesencephalic meninges ventral metencephalic meninges VII 3rd branchial arch 4th branchial arch ventral and caudal periotic mesenchyme

erotopic grafting was performed. In 11% (6/54) of heterotopic Fig. 4. (A) Sm-I contribution to the rostral-ventral periocular transplantations, the grafted mesoderm failed to disperse. The mesenchyme and lateral nasal process (arrow). (B) Orthotopic graft cells stayed together as a clump of tissue at the injection site. (II→II) resulting in the distribution of Sm-II-derived cells predominantly in the dorsal and caudal periocular mesenchyme (arrow) and in the facial mesenchyme lateral to the diencephalon (arrowhead). (C) DiO-labelled cells derived from Sm-II contributes to the DISCUSSION dorsocaudal periocular mesenchyme. (D) III→III graft-derived cells populating the facial mesenchyme (arrow) lateral to the midbrain The impact of regionalisation of cell fate in the extending from the maxillary prominence to the dorsal neural tube. cranial paraxial mesoderm on craniofacial (E) A section through the midbrain region showing the contribution of patterning III→III graft-derived cells to the endothelium of the primary head vein. Results of cell transplantation and fluorescent dye marking (F) IV→V graft resulting in colonisation of the rostral periotic → experiments reveal that the cranial paraxial mesoderm partici- mesenchyme (arrowhead). (G) VII VII graft-derived cells contribute pates extensively in craniofacial morphogenesis and in partic- to the caudal periotic mesenchyme (arrowhead). (H) The proximodistal separation of cells labelled by DiI (yellow fluorescence) in the ular impacts upon the development of the branchial arches maxillary and mandibular regions of the first arch and DiO (green (Table 2). There is an orderly craniocaudal distribution of cells fluorescence) in the facial mesenchyme with considerable mixing of from different regions of the cranial mesoderm to the cranio- the two labelled cell populations in the maxillary prominence. (I) II→V facial tissues in the mouse embryo (Table 1a,b). A previous graft-derived cells in the hyoid arch (arrowhead) and the mesenchyme morphological study has revealed that the murine cranial rostral to the otic vesicle (arrow). (J) V→III graft-derived cells colonise mesoderm is segmented into clusters of cells known as somit- extensively the mesenchyme associated with the lower mesencephalon omeres (Meier and Tam, 1982). Each of the seven somitomeres (arrow) and maxillary prominence (arrowhead). (K) IV→III grafted are related topographically to a particular segment of the brain cells contributing to the maxillary (arrowhead) and mandibular regions → (Jacobson, 1993; Tam and Trainor, 1994). Our discovery of a of the first arch. (L) I III graft-derived cells in the core of the regionalisation in cell fate may argue for the presence of a mandibular arch (arrowhead) and the perivascular tissues of the truncus arteriosus (arrow). Abbreviations: fb, forebrain; fm, facial segmental prepattern in the cranial mesoderm although the mesenchyme; hb, hindbrain; ht, heart; ma, mandibular arch; mb, findings neither require the existence of somitomeres nor give midbrain; md, mandibular prominence; mx, maxillary prominence; ne, support for their existence. For clarity of discussion, however, neural epithelium; np, nasal process; oe, optic epithelium; op, otic we shall refer to the different fragments of cranial mesoderm placode; ov, optic vesicle; ta, third arch; ts, truncus arteriosus. Bar, 100 by their somitomeric address. mm for A,B,E-H,J-L. Bar, 300 µm for C,D,F and I. By following the distribution of cells from individual somit- 2406 P. A. Trainor, S.-S. Tan and P. P. L. Tam

enchymal derivatives from these somitomeres (Tam and Trainor, 1994).

II Cranial mesoderm does not display any positional identity in developmental fate I OV III The orderly and predictable distribution of cells from specific somitomeres to different craniofacial structures may imply that Mx IV a stringent specification of developmental fate has been endowed on individual somitomeres. A comparison of the dis- Md V position of cells from within the dorsal and ventral components OT of the paraxial mesoderm to different parts of the branchial VII VI arches further reinforces the view that some form of regional HT restriction in potency may be present. Cells in the ventral A B region of the cranial mesoderm contribute to the distal mesenchyme of the mandibular and hyoid arches, respectively. Progressively more dorsal regions contribute to tissues in the Fig. 5. Schematic diagrams showing the spatial distribution of cells proximal regions of the arches and preferentially to facial mes- derived from different regions of the cranial paraxial mesoderm, as defined by somitomeres, to the craniofacial structures of the mouse enchyme. Whether the maintenance of the dorsoventral segre- embryo at about 10. 5 days p.c. Fig. 5A shows those originating from gation of these two cell populations is the result of diminished odd-numbered somitomeres and Fig. 5B for those from even- cell mixing or an intrinsic regional difference in tissue’s fate numbered somitomeres. Abbreviations: HT, heart; Md, mandibular has not been tested. It has been shown in the chick, however, prominence; Mx, maxillary prominence; OT, otic vesicle; OV, optic that regionalisation of cell fate does exist between the medial evagination. and lateral cell populations in the cranial mesoderm (Couly et al., 1992). Cell fate analysis following heterotopic transplantation omeres to the branchial arches, it was found quite unexpect- revealed that cells taken from any axial level of the cranial edly that each branchial arch is colonised by cells from a set mesoderm can contribute to formation of the maxillary and of two consecutive somitomeres. The mandibular arch is mandibular prominences of the first arch in a manner typical derived form somitomeres II and III. Somitomeres IV and V of the caudal mesencephalic mesoderm to which they are trans- contribute to hyoid arch development and somitomeres VI and planted. Similarly mesoderm ectopically grafted into Sm-II or VII form part of the visceral arch mesenchyme. The fourth arch Sm-IV will migrate in a manner characteristic of tissues at the receives a cranial mesoderm contribution from somitomere VII native site. These results indicate that in the 8.5-day early- only. However, an additional contribution by the most rostral somite-stage mouse embryo, the differentiative pathway that cervical somites cannot be ruled out. The fifth and sixth pairs could be taken by cranial mesoderm is not positionally of branchial arches were not colonised by any somitomeric restricted. Our results agree with previous studies in the chick, mesoderm cells. It is interesting to note that this pairwise which demonstrate remarkable morphogenetic plasticity of the restriction of cell distribution only occurs in the branchial paraxial mesoderm (McLachlan and Wolpert, 1980; Noden arches. Outside the arches, cells from adjacent somitomeres 1982; Cauwenbergs et al., 1986; Butler et al., 1988). mixed extensively in the periocular, facial and cervical mesenchyme. Cell mixing, however, is minimal between alternate Co-distribution of cranial mesoderm and the neural somitomeres. crest cells The contribution by dual somitomeres to each branchial arch The distribution of cranial mesoderm to the frontonasal and is reminiscent of the so-called ‘two segment periodicity’ asso- branchial regions (this study) is generally similar to that of the ciated with the derivation of the branchial arch motor neurons neural crest cells originating from the same axial level from pairs of rhombomeres (Lumsden and Keynes, 1989). The (Lumsden and Keynes, 1989; Serbedzija et al., 1992). motor nerves innervating the three arches (trigeminal, facial Mesoderm from the first two somitomeres and the forebrain and glossopharyngeal) develop from nuclei found in consecu- and upper mesencephalic neural crest cells contribute primarily tive pairs of rhombomeres (r2 and r3 for trigeminal; r4 and r5 to the periocular region in a similar pattern to the somitomeric for facial; r6 and r7 for glossopharyngeal; Lumsden, 1990; mesoderm. The maxillary region of the first arch is colonised Lumsden et al., 1991; Hunt et al., 1991a). The segmentally by mesenchyme derived from Sm-II and also the mesen- repeating patterns of rhombomeres and motor neurons are cephalic crest (Lumsden et al., 1991; Serbedzija et al., 1992; associated with the differential expression of genes such as Osumi-Yamashita et al., 1994). Neural crest cells from the Krox-20, Hoxb-1 and Sek (Wilkinson et al., 1989; Nieto et al., caudal mesencephalon and the mesoderm of Sm-III both 1991). Differential expression of a similar set of genes is also migrate into the distal region of the mandibular arch. The found in the migrating neural crest derived from specific rhom- preotic or metencephalic neural crest cells and mesoderm bomeres (Hunt et al., 1991a). All the known genes for derived from Sm-IV and Sm-V both contribute to hyoid arch embryonic mesoderm however, such as Mox1, Mox2 (Candia morphogenesis. A similar concordance of cell contribution is et al., 1992), M-twist (Wolf et al., 1991), Sek (Nieto et al., also found for the postotic or myelencephalic neural crest cells 1992) and S8 (de Jong and Meijlink, 1993) do not display any and Sm-VI and SM-VII, which collectively colonise the third spatially restricted patterns of expression that may reflect the branchial arch. Therefore cranial neural crest cells and the initial somitomeric organisation or the distribution of the mes- somitomeric mesoderm generally share a common segmental Fate of cranial somitomeres 2407 origin and final destination. This raises the question of how the neural crest cells (Le Lievre, 1978; Noden, 1978; Le Douarin, two tissue populations are spatially distributed at their final 1982). Finally, the murine cranial mesoderm also contains destinations. The Dlx-2 gene, which is a vertebrate homologue vascular or endothelial tissue precursors just as in the avian of the Drosophila Distal-less gene, is uniformly expressed embryos (Peault et al., 1983; Pardanaud et al., 1987; Coffin throughout the arches excluding a core of mesenchymal cells and Poole, 1988; Noden, 1988; Poole and Coffin, 1988). (Bulfone et al., 1993). These mesenchymal arch cores, which Somitomeres differentiate to form supporting tissues of the are devoid of Dlx-2 gene expression, are consistent with the primitive cephalic vein and also contribute frequently to the location of the mesodermally derived muscle plates. Migrating endothelium of branchial arch arteries. The cranial paraxial neural crest cells also express Dlx-2. This suggests that, in the mesoderm therefore consists of a multiprogenitor mesodermal branchial arches, the muscle plates, which are mesodermally cell population. derived become enveloped by neural crest cells. This could be important as it has been shown that, in the limb and the face We thank Peter Rowe and Gabe Quinlan for discussion and of the chick embryo, the region-specific organisation of muscle comments on the manuscript. This work was supported by the groups depends critically upon the connective tissue pattern set National Health and Medical Research Council of Australia and the up locally by the neural crest cells (Chevallier, 1979; Cheval- Clive and Vera Ramaciotti Foundation. lier and Kieny, 1982; Noden, 1982, 1983a, 1986). The co-distribution of the craniofacial precursors could therefore be REFERENCES important for morphogenetic interactions that lead to the establishment of tissue patterns during craniofacial development. Bulfone, A., Hee-Joong, K., Puelles, L., Porteus, M. H., Grippo, J. F. and The somitomeric organisation of the paraxial mesoderm, if it Rubenstein, J. L. R. (1993). The mouse Dlx-2 (Tes-1) gene is expressed in does exist, may be a part of the metameric patterning process spatially restricted domains of the forebrain, face and limbs in midgestation that operates during the establishment of the neuromeres and mouse embryos. Mech. Dev. 40, 129-140. the cranial neural crest cells. Butler, J., Cosmos, E. and Cauwenbergs, P. (1988). Positional signals: evidence for a possible role in muscle fibre-type patterning of the embryonic avian limb. Development 102, 763-772. A presumptive myogenic fate of cranial mesoderm Candia, A. F., Hu, J., Crosby, J., Lalley, P. A., Noden, D., Nadeau, J. H. and in the branchial arches Wright, C. V. E. (1992). Mox-1 and Mox-2 define a novel homeobox gene Cells derived from the cranial somitomeres colonise the mes- subfamily and are differently expressed during early mesodermal patterning in mouse embryos. Development 116, 1123-1136. enchymal core of the branchial arches. Interestingly, mes- Cauwenbergs, P., Bulter J. and Cosmos, E. (1986). Intraspecific chick/chick enchyme at the core of newly formed branchial arches has been chimaeras: dystrophic somitic mesoderm transplanted to a normal host forms found to express muscle-specific genes such as Myf5 (Ott et muscles with a dystrophic phenotype. Neurosci Letters 68, 149-154. al., 1991) and Hlx (R. Harvey, per comm) at 9.25 to 10.0 days. Chan, W. Y. and Tam, P. P. L. (1986). The histogenetic potential of neural This mesenchyme later condenses into a myogenic mass plate cells of early-somite-stage mouse embryos. J. Embryol. Exp. Morph. 96, 183-193. known as the muscle plate of the branchial arch (Bulfone et Chan, W. Y. and Tam, P. P. L. (1988). A morphological and experimental al., 1993). In the chick, the cranial mesoderm displays a pre- study of the mesencephalic neural crest cells in the mouse embryo using dominantly myogenic fate (Noden, 1983b). More significantly, wheat germ agglutin-gold conjugates as the cell marker. Development 102, the precursors of different groups of craniofacial muscles and 427-442. Cheah, K. S. E., Lau, E. T., Au, P. K. C. and Tam, P. P. L. (1991). skeletogenic cells are regionalised craniocaudally in specific Expression of the mouse a1(II) collagen gene is not restricted to cartilage sets of somitomeres (Table 2). For example, the myoblasts of during development. Development 111, 945-953. the extrinsic ocular muscles in the chick were derived from Chevallier, A. (1979). Role of somitic mesoderm in the development of the Sm-I and Sm-II and the jaw opening and hyobranchial muscles thorax in bird embryos. II. Origin of thoracic and appendicular musculature. came from Sm-VI and Sm-VII. Although the mouse culture J. Embryol. Exp. Morph. 49, 73-88. Chevallier, A. and Kieny, M. (1982). On the role of the connective tissue in system only permits in vitro development up to about 10.5 the patterning of the chick limb musculature. Wilhelm Roux’ Archiv. Ent d.p.c. and therefore does not allow a definitive elucidation of Mech. Org. 191, 277-280. the final histogenetic fate of the cranial somitomeres, parallel Coffin, J. D. and Poole, T. J. (1988). Embryonic vascular development: findings in the chick and the initial localisation of somitomeric Immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos. Development cells to the myogenic regions of the branchial arch are strongly 102, 735-748. suggestive of a myogenic fate for the cranial somitomeres in Couly, G. F., Coltey, P. M. and le Douarin, N. M. (1992). The developmental the mouse. fate of the cephalic mesoderm in chick-quail chimaeras. Development 114, 1- However, other genes not specific for the myogenic lineage, 15. such as the type II collagen gene (Col2a-1, Cheah et al., 1991; Couly, G. F., Coltey, P. M. and le Douarin, N. M. (1993). The triple origin of the skull in higher vertebrates: a study in chick-quail chimeras. Development Ng et al., 1993) and the murine homologue of the human 117, 409-429. Hox11 gene (Tlx-1, Raju et al., 1993) are also expressed in the de Jong, R. and Meijlink, F. (1993). The homeobox gene S8: mesoderm- so-called muscle plate. It has been shown recently in the avian specific expression in presomite embryos and in cells cultured in vitro and embryo that cranial somitomeres also participate, in addition modulation in differentiating pluripotent cells. Dev. Biol. 157, 133-146. Figdor, M. C. and Stern, C. D. (1993) Segmental organisation of the to the differentiation of craniofacial muscle, in the formation embryonic diencephalon. Nature 363, 630-634. of skull bones such as the orbitosphenoid, sphenoidalis and otic Fraser, S. E. (1993) Segmentation moves to the fore. Curr. Biol. 3, 787-789. capsule (Couly et al., 1992). The expression of Col2a-1 gene Fraser, S. E., Keynes, R. J. and Lumsden, A. (1990). Segmentation in the in the mesenchyme colonised by the somitomeric cells chick embryo hindbrain is defined by cell lineage restrictions. Nature 344, therefore suggests that cranial somitomeres may also con- 431-435. Hunt, P. and Krumlauf, R. (1991). Deciphering the Hox code: Clues to tribute to the formation of at least part of the facial skeleton patterning branchial regions of the head. Cell 66, 1075-1078. which previously was thought to be entirely derived from Hunt, P., Wilkinson, D. and Krumlauf, R. (1991a). Patterning the vertebrate 2408 P. A. Trainor, S.-S. Tan and P. P. L. Tam

head: murine Hox 2 genes mark distinct subpopulations of premigratory and Noden, D. M. (1983b). The embryonic origins of the avian cephalic and migrating neural crest. Development 112, 43-50. cervical muscles and associated connective tissues. Amer. J. Anat. 168,257- Hunt, P., Gulisano, M., Cook, M.-H., Faiella, A., Wilkinson, D., Boncinelli, 276. E. and Krumlauf, R. (1991b). A distinct Hox code for the branchial region Noden, D. M. (1986). Patterning of avian craniofacial muscles. Dev. Biol. 116, of the vertebrate head. Nature 353, 861-864. 347-356. Hunt, P., Whiting, J., Muchmore, I., Marshall, H., and Krumlauf, R. Noden, D. M. (1988). Interactions and fates of avian craniofacial mesenchyme. (1991c). Homeobox genes and models for patterning the hindbrain and Development 103 Supplement, 121-140. branchial arches. Development 113 Supplement, 187-196. Osumi-Yamashita, N., Ninomiya, Y., Doi, H. and Eto, K. (1994). The Jacobson, A. G. (1993). Somitomeres: Mesodermal segments in the head and contribution of both forebrain and midbrain crest cells to the mesenchyme in trunk. In The Vertebrate Skull vol. 1 (ed. J. Hanken and B. Hall), pp. 42-76. the frontonasal mass of mouse embryos. Dev. Biol. 164 (in press). Chicago: University of Chicago Press. Ott, M., Bober, E., Lyons, M., Arnold, H. and Buckingham, M. (1991). Jacobson, A. G. and Tam, P. P. L. (1982). Cephalic neurulation in the mouse Early expression of the myogenic regulatory gene, myf-5, in precursors of embryo analyzed by SEM and morphometry. Anat. Rec. 203, 375-396. skeletal muscle in the mouse embryo. Development 111, 1097-1107. Jacobson, A. G. and Meier, S. (1986). Somitomeres: The primordial body Pardanaud, L., Altman, C., Kitos, P., Dieterlen-Leivre, F. and Buck, C. A. segments. In Somites in Developing Embryos. (ed. R. Bellairs and J. W. (1987). Vasculogenesis in the early quail blastodisc as studied with a Lash). NATO ASI series 118, 1-16. monoclonal antibody recognising endothelial cells. Dev. Biol. 100, 339-349. Keynes, R. J. and Stern, C. D. (1984). Segmentation in the vertebrate nervous Peault, B. M., Theiry, J. P. and Le Douarin N. M. (1983). Surface marker for system. Nature 310, 786-789. hemopoeitic and endothelial cell lineages in quail that is defined by a Le Douarin, N. M. (1982). The Neural Crest. Cambridge: Cambridge monoclonal antibody. Proc. Nat. Acad. Sci. USA 80, 2976-2980. University Press. Poole, T. J. and Coffin, J. D. (1988). Developmental angiogenesis: Quail Le Lievre, C. (1978). Participation of neural crest-derived cells in the genesis embryonic vasculature. Scanning Microscopy 2, 443-448. of the skull in birds. J. Embryol. Exp. Morph. 347, 17-37. Puelles L. and Rubenstein, L. R. (1993) Expression patterns of homeobox and Lumsden, A. (1990). The cellular basis of segmentation in the developing other putative regulatory genes in the embryonic mouse forebrain suggest a hindbrain. Trends Neurosci. 13, 835-840. neuromeric organisation. Trends Genet. 16, 472-479. Lumsden, A. and Keynes, R. J. (1989). Segmental patterns of neuronal Raju, K., Tang, S., Dube, I. D., Kamel-Reid, S. Bryce, D. M. and Breitman development in the chick hindbrain. Nature 337, 424-428. M. L. (1993). Characterisation and developmental expression of Tlx-1, the Lumsden, A., Sprawson, N. and Graham, A. (1991). Segmental origin and murine homologue of HOX11. Mech. Dev. 44, 51-64. migration of neural crest cells in the hindbrain region of the chick embryo. Serbedzija, G. N., Bronner-Fraser, M. and Fraser, S. E. (1992). Vital dye Development 113, 1281-1291. analysis of cranial neural crest cell migration in the mouse embryo. McLachlan, J. and Wolpert, L. (1980). The spatial pattern of muscle Development 116, 297-307. development in the limb. In The Development and Specialisation of Muscle Sturm, K. and Tam, P. P. L. (1993). Isolation and culture of whole (ed. D. F. Goldspink), pp. 1-17. Cambridge: Cambridge University Press. postimplantation embryos and germ layer derivatives. Meth. Enzymol. 225, Meier, S. (1979) Development of the chick mesoblast. Formation of the 164-189. embryonic axis and the establishment of the metameric pattern. Dev. Biol. Tam, P. P. L. and Meier, S. (1982). The establishment of a somitomeric 38, 73-90. pattern in the mesoderm of a gastrulating mouse embryo. Amer. J. Anat. 164, Meier, S. and Tam, P. P. L. (1982). Metameric pattern development in the 209-225. embryonic axis of the mouse I. Differentiation of the cranial segments. Tam, P. P. L. and Tan, S. S. (1992). The somitogenic potential of cells in the Differentiation 21, 95-108. primitive streak and the tail bud of the organogenesis-stage mouse embryo. Ng, L. J., Tam, P. P. L. and Cheah, K. S. E. (1993). Preferential expression of Development 115, 703-715. alternatively spliced mRNAs encoding Type II procollagen with a cysteine- Tam, P. P. L. and Trainor, P. A. (1994). Segmentation and specification of the rich amino-propeptide in differentiating cartilage and non-chondrogenic paraxial mesoderm. Anat. Embryol. 189, 275-307. tissues during early mouse development. Dev. Biol. 159, 403-417 Tan, S. S. and Morris-Kay, G. M. (1986). Analysis of cranial neural crest cell Nieto, M. A., Bradley, L. C., and Wilkinson, D. G. (1991). Conserved migration and early fates in post-implantation rat chimaeras. J. Embryol. segmental expression of Krox-20 in the vertebrate hindbrain and its Exp. Morphol. 98:21-58. relationship to lineage restriction. Development 1991 Supplement 2, 59-62. Thorogood P. (1993) The problems of building a head. Current Biology 3, 705- Nieto, M. A., Gilardi-Hebenstreit, P., Charnay, P. and Wilkinson, D. G. 708. (1992). A receptor protein tyrosine kinase implicated in the segmental Wilkinson, D. G., Bhatt, S., Cook, M., Boncinelli, E. and Krumlauf, R. patterning of the hindbrain and mesoderm. Development 116, 1137-1150. (1989). Segmental expression of Hox-2 homeobox-containing genes in the Noden, D. M. (1978). The control of avian cephalic neural crest developing mouse hindbrain. Nature 343, 405-409. cytodifferentiation. I. Skeletal and connective tissues. Dev. Biol. 67, 313- Wilkinson, D. G. and Krumlauf, R. (1990). Molecular approaches to the 329. segmentation of the hindbrain. Trends Neurosci. 13, 335-339. Noden, D. M. (1982). Patterns and organisation of craniofacial skeletogenic Wolf, C., Thisse, C., Stoetzel, C., Thisse, B., Gerlinger, P. and Perrin- and myogenic mesenchyme: A perspective. In Factors and Mechanisms Schmitt N (1991) The M-twist gene of Mus is expressed in subsets of Influencing Bone Growth (ed. A. Dixon and B. Sarnat), pp. 167-203. New mesodermal cells and is closely related to the Xenopus X-twi and Drosophila York: Alan R. Liss. twist genes. Dev. Biol. 143, 363-373. Noden, D. M. (1983a). The role of the neural crest in patterning of the avian cranial skeletal, connective and muscle tissues. Dev. Biol. 96, 144-165 (Accepted 30 May 1994)