Embryonic Transcription and the Control of Developmental Pathways

Eric Wieschaus Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

It appears that the initial steps up to the [sea urchin] selected to play unique, controlling roles in develop- blastula stage are independent of the quality of the nu- ment. In this admittedly extreme reformulation of BO- clear substance, even though it is essential that the nu- VERI’S observation, I will restrict consideration to those clear substance be of a kind capable of existing in the egg. The necessity for particular chromosomes becomes genes whose products must be supplied zygotically and apparent first with the formation of the primary mesen- to organisms that have minimized their transcriptional chyme and from then on shows up in all processes asfar requirements during embryogenesis. I will first present as development can be observed. . . . With respect to reasons why such a division of labor makes teleological those characters in which we are able to recognize indi- sense and then examine the data from Drosophila to vidual variations, the nuclear substance and not the cyte plasmic cell substance imposes its specific character on determine the extent to which gene activityactually the developing trait. obeys these expectations. Then I will discuss the excep- . . . Earlier stages, for which according to our results, tions, i.e., certain wellcharacterized cases where partic- specific chromosomes are not necessary, demonstrate a ular Drosophila genes are supplied zygotically but do purely maternal character. . . . I would like to ascribe to not seem to play controlling roles. Lastly, the Drosoph- the cytoplasm of the sea urchin egg only the initial and simplest properties responsible for differentiation. . . it ila data will be compared to that from other organisms. provides the most general basic form,the framework The simple model: In organisms where embryonic within which all specific details are filled in by the nu- development is rapid and occurs with no increase in size cleus. THEODORBOVERI 1902 before hatching from the egg, it will be advantageous to maximize maternal contributions,because the duration VEN before the molecular nature of the gene was of oogenesis is often much longer than embryogenesis E understood, embryos were known to get the prod- and the ovary provides a more sophisticated and effi- ucts they need from two different sources. In modern cient synthetic machinery. Evolutionary selection for terms, the unfertilized egg contains large stores of ma- efficiency will maximize such maternal contributions. ternal RNA and protein that arederived from transcrip- One major limitation to maternal supplies is the duration during oogenesis. Initially, these proteins provide tion of embryonic development. Maternal supplies the basic machinery for all cellular events. At some must be sufficient to last until a larva hatches and can point these maternally supplied products are supple- obtain additional nutrients from the environment. In mented by transcription from the embryo’sown ge- principle, maternal contributions might consist only of nome (the “nuclear substance” in BOVERI’Sformula- nutrient material (“yolk”) that could be converted by tion). This occurs at different stages in different the embryo to a wide variety of gene products. It may organisms, but significant transcription is usually de- be more efficient, however, thatthe “nutrients” in- tected by the blastula or blastoderm stage. Maternal clude RNA directly encodingthe individual compo- geneproducts persist after this point, however, and nents of most biological processes, including the cellu- most processes during embryogenesis involve both ma- lar machinery required for transcription, translation, ternal and zygotic components. energy utilization and basic cell structure. As the quote from BOVERI’S classicpaper indicates, On the other hand, all embryos have been shown to embryologists at the turn of the century were aware of require certain RNAs supplied by transcription of their both maternal and zygotic contributions to embryonic own genome. Early indications ofthis requirement development. They even considered the possibility that came from the seminal work of BOVERI, as well as spe- maternal components might play different roles in de- cies hybridization experiments and actinomycin and a- velopment than zygotic components. In theeighty years amantin studies (reviewed in DAVIDSON1986). If supply- that followed BOVERI’S work,it became clear that cer- ing gene products maternally is so advantageous, what tain zygotically activegenes play major controlling roles. can zygotic transcription do that just storing maternal Landmark studies were POULSON’S(1940) analysis of transcribed gene products can’t?One possibility is that chromosomal deletions in the Notch region and LEWIS’ zygotic transcription allows much more precise expres- (1978) characterization of the Bithorax complex. sion, putting particular gene products in one cell but In the following pages, I will address the generality not in its immediate neighbors. Such precision is proba- of a hypothesis only partly implied by the BOVERIquote, bly not necessary for most gene products. If a gene namely that in organisms where zygotically required product is needed in some cells but not others,may it be gene activities are rare, all such activitieshave been sufficient (although profligate) to supply it uniformly to

Genetics 142 5-10 (January, 1996) 6 E. Wieschaus the whole egg. A patterned expression only becomes from all these studies is that only a small number of important when the absence of a particular gene product loci need betranscribed in the embryo itself to establish is as significant as its presence. This might be the case normal morphology. Even if the Heidelberg screens if the gene product is used as a developmental switch, detected only half the zygotically important loci, the such that its expression in one cell causes that cell to number of such genes would increase only to 300, fewer assume a fate different from its neighbors. While it is than 2% of the estimated 20,000 molecularly defined possible to localize maternal gene products to particu- transcription units and only a small fraction of the tran- lar regions of the egg cytoplasm, localtranscription may scripts and proteins found in the embryo. The remain- allow more precision, and may therefore be more useful der of these proteins and RNAs, and thus the majority as spatial patterns become complex. of the components involved in any particular embry- This view has certain practical consequences. Over onic process, must be supplied maternally. the course of evolution, the efficiencies of maternal If early acting, zygotically transcribed genes are rare, contribution will reduce zygotic transcription to a mini- it should be possible to delete large portions of the mum. That minimum will define a set of genes, each genome with little effect on early stages ofdevelopment. of which is limiting for a specific process, such that its We tested this possibilityby examining the development presence or absence determines when and where in the ofembryos homozygous for variouscytologically de- embryo that process takes place. Not all controlling fined deletions, and eventually used translocations and elements will be zygotic; a process could be initiated by chromosomal rearrangements to generate embryos de- a localized maternal RNA or ligands (STJOHNSTON and leted for overlapping regions spanning the entire Dro- NUSSLEIN-VOLHARD1992). However, if there is a zygotic sophila genome (MERRILLet al. 1988; WIESCHAUSand component in a process, its expression is likely to play SWEETON1988). In general, such deficiency embryos a controlling role in all events downstream to it. In this show phenotypes that could be explained by point mu- view, selection during evolution has solved the research tations previously located to the deleted regions. There agenda of a large fraction of developmental biologists: are exceptions, notably a group of seven early acting in a complex process involving many gene products, it genes required for cellularization at the onset of cycle has sorted out which one actually plays the controlling 14. Ongoing work in my lab suggests the existence of role. Therefore, to understand how a particular process certain regions with previously undescribed effects on is controlled in the embryo, good candidate genes gastrulation and early morphogenetic movements (E. would be those whose products need to be supplied WIESCHAUS,unpublished results). In general, however, zygotically. For organisms where genetic analyses are such newly discovered loci have been rare, confirming possible, identification of candidates relativelyis the preponderanceof maternal geneproducts and scar- straightfornard; if the genes are removed by mutation, city of gene products that must be supplied by zygotic homozygous mutant embryos will develop abnormally. transcription. Consistentwith behavior as controllingelements, Transcripts that must be supplied to the embryo zy- genes that must be supplied zygotically represent only gotically appear to play special roles in development: a small fraction of the Drosophila genome: The muta- Mostof the mutations identified in theHeidelberg genesis screens that CHRISTIANENUSSLEIN-VOLHARD, screens produce discrete phenotypes, such that differ- GERDJURGENS and I began more than fifteen years ago entiation of most structures is normal and defects are in Heidelberg offered an initial opportunity to test limited to specific cell types or regions. Also, most phe- whether the expectations outlined above apply to Dro- notypes are locus-specific (ie., there is only one locus sophila (N~SSLEIN-VOLHARDWIESCHAUS and 1980;JUR- in the genome that can be mutated to produce that GENS et al. 1984; N~SSLEIN-VOLHARDet al. 1984; phenotype). Subsequent molecular analysessuggest WIESCHAUSet al. 1984). Of the 18,000 lethal mutations that this specificity is often reflected in their expression induced in those screens, only about 4500 (25%) cause patterns duringembryogenesis. On the Xchromosome, death during embryogenesis, and only 580 cause alter- for example, where 14 of the 20 loci have been cloned, ations in visiblemorphology sufficient to allow homozy- 11 show patterns of transcription corresponding to gous embryos to be distinguished from their heterozy- where the gene products are required in the embryo. gous siblings. These 580 mutations were assignedto 139 Genes that show uniform phenotypes affecting all cells complementation groups with an average hit frequency at a particular stage (the early acting cellularization of4.2. Identification of mutants in theHeidelberg genes ndlo, s?ya and bottleneck) show uniform transcript screens was based on examination of the larval cuticle. distribution, but are only expressed in a short period Later screens using other aspect of general morphology immediately preceding cellularization (JAMES and VIN- (EBERLand HILLIKER1988) or using molecular markers CENT 1986; ROSE and WIESCHAUS 1992; SCHEJTERand to follow the development ofspecific organ systems WIESCHAUS1993). (SEEGARet al. 1993; VAN VACTOR et al. 1993; mECKE Another feature of these genes that suggests special- and LENGYEL1995) identified additional genes, al- ized functions is that their transcription is generally not though their numberis not large. The major conclusion required during oogenesis. This behavior is different M edal Essay 1995 GSA Medal 7 from most genes in Drosophila and is best illustrated by havior (WIESCHAUSand NOELL1986). Such maternal examination of the X chromosome, where the wild-type transcripts may bring allcells close enough to the activities associated with many random lethal loci have threshold so that small differentials in zygotic transcrip- been studied using germ-line clones (PERRIMONet al. tion between neighboring cells can make an immediate 1984, 1989). Most of these lethals (35/48 and 133/211 difference. in two separate surveys) cause blocks during oogenesis At the other endof the spectrum are gene products or abnormalities in embryogenesis that cannot be cor- required throughout embryonic development in such rected by zygotic transcription. In contrast, only three large quantities that maternalsupplies are notsufficient of the 19 Heidelberg loci that have been tested are re- and additional zygotic transcription is required by later quired duringoogenesis ( JIMENEZ and COMPOSORTEGA stages in development. Such genes may represent the 1982; ZUSMANand WIESCHAUS1985; WIESCHAUSand bulk of embryonic-lethal mutationsthat make it NOELL 1986; EBERLet al. 1992). For the remaining 16 through the last major morphological events of Dro- loci, a single zygotic allele is sufficient to ensure normal sophila embryonic development(dorsal closure and development of the embryo, even when all maternal head involution), but die before hatchingwith no obvi- activity is removed by producing germline clones. In ous visible defects, a class that we found represented several cases (my,exd, Jog) alterations in the develop- about 25% of alllethal genes. In some cases the requirement of the resultantprogeny suggests that thewild-type ment for additionalzygotic transcription may set in ear- genes may normally be transcribed during oogenesis. lier, so that mutations cause visible defects in late em- Unlike the average vital gene, however, such transcrip- bryonic processes. The major cytoplasmic myosin gene tion is not necessary for embryonic survival, as long as (zipper) may provide an example (YOUNG et al. 1993). the embryo has at least one normal copy of the gene. Although cytoplasmicmyosin is required throughout The precision of these expression patterns mightsim- development,mutations that block zygotic transcrip- ply reflect an economy of synthesis, the embryo only tion cause abnormalities only during dorsal closure and making gene products where it needs them. In a few head involution. Zygotic transcription of zipperdoes not cases this possibility has been excluded by expressing appear to play a regulatory role; zipper transcripts are the geneectopically. This was initially accomplished for uniformly distributed in normal embryos, and the mu- the ftz segmentation gene using a heat-shock promoter tant defect can be rescued by ubiquitous zipper tran- (STRUHL1985), but alternative systems such the Ga14/ scripts supplied from a heat-shock promoter (YOUNG et UAS system of BRANDand PERRIMON(1993) have been al. 1993). Presumably the early normal development of designed to produce more specific patterns of expres- homozygous mutants reflects maternal myosin RNA sion. If the presenceof a particular gene productdirects and protein that begin to run out shortly before dorsal a cell into a fate different from that of its neighbors, closure. then it should be possible to direct its neighbor (or any More puzzling, however, are cases where the absence other cell) into the same fate by forcing expression of of zygotic transcription produces an early phenotype the same gene product.Although only a few genes have (within the first two hours after the blastoderm stage), been tested, the results have confirmed the significance yet the relevant gene product is transcribed uniformly of most of the spatial expression patterns associated in the embryo. Examples include am(RIGGLEMAN et al. with genes affecting all three levelsof segmentation 1990), Notch ( JOHANSEN et al. 1989), exd (RAUSKOLB et ( PARKHUFSTand ISH-HOROWICZ1991; CAPOVILLA et al. al. 1993) odd-paired (BENEDYKet al. 1994) and jlb/DER 1992; MANOUKIAN and KRAUSE 1992; NOORDEMEERet (LEVet al. 1985; ZAK et al. 1990). Theuniform distribu- al. 1992; TSAIand GERGEN1994), as well as neuronal tion of these transcripts makes it difficult to see how development (RHYU et al. 1994), cell proliferation (ED- theirtranscription could play acontrolling role in GAR and O'FARRELL1990) and gastrulation (P. MORIZE, choosing between pathways. Some of these genes M. COSTA,S. PARKSand E. WIESCHAUS,unpublished (Notch,jlb/DER) encode cell surface receptors or (ama- results). One potential exception (patched) is discussed dillo) other molecules required for theresponse of cells below. to local signals. Many of these signals are known; they Do allzygotically requiredgenes playcontrolling are encoded by other zygotically active genes that are roles? The observations presented above suggest that expressed in restricted patterns, such as wingless (BAKER most transcriptionally required genesplay specificregu- 1987). It is natural to assume that it is the expression latory roles in the Drosophila embryo. There areexcep- of these latter genes that controls cell fate. The early tions, of course, but many of the apparent exceptions stage of development when signalling is required makes can be integrated into the model with little modifica- it hard to understandwhy their receptors could not be tion. As mentioned above, some of these genes are also supplied maternally. transcribed during oogenesis. In many such cases, the In some cases, a large supply of maternal receptor maternal products appear tobe supplied in insufficient protein may be disadvantageous if that receptor can quantities, so that patterned zygotic transcription still cross-react with or interfere with processes that involve provides the critical difference in establishing cell be- similar components. The Drosophila EGF receptor ( jlb/ 8 E. Wieschaus

DER), for example, is required in the follicle cells but both of whichundergo substantial increases in size dur- not in the oocyte during oogenesis (SCHUPBACH1987). ing early stages when spatial patterns are being estab- It also utilizes many of the same downstream elements lished. The model would predict that the average gene (e.6, Ras, Raf) as certain maternally supplied receptor in mammals or plants would showsubstantial transcrip tyrosine kinases (torso) that pattern the embryo during tional requirements in the embryo, because the in- cleavage; see DUFFY and PERRIMON(1994). To avoid crease in size makes it impractical to supply any gene cross-reactions, it may be simplest for the organism not product in sufficient quantities from purely maternal to express jZb/DER transcripts in maternal germ cells. sources. In contrast to Drosophila, most random lethal This would explain why$b/DER is not expressed mater- mutations would cause death during embryogenesis; nally and only expressed zygotically after the blastoderm see JURGENS et al. (1991) and ROSSANTand HOPKINS stage. This notion could be tested by determining (1992) for adiscussion of thispoint. In theother organ- whether maternally supplied jZb/DER product has dele- isms, transcription would stillbe required foronly a few terious effect on development or, alternatively, can res- genes, but these would be predicted to play controlling cue mutant embryos. In any case, it is more difficult to roles in specific developmental processes. apply such explanations to genes like armadillo, which It was the discrete phenotypes produced in sea urchin act early and are expressed in abundant quantities mater- embryos when particular chromosomes wereelimi- nally and yet are also expressed zygotically. nated that led BOVERIto conclude that “the nuclear Potentially more damaging to the model are genes substance imposes its specific character on the devel- that behave like thesegment polarity gene patched. oping trait.” (In fact, had the early zygotic defects not patched transcripts are not supplied maternally and zy- been specific, BOVERIwould not have been able to con- gotic transcripts accumulate in complicated patterns clude that individual chromosomes carry distinct devel- (NAKANOet al. 1989), consistent with a controlling role opmental properties.) His observations suggest that the in development. However, no effects are observed when model may apply to sea urchins and other marine or- these patterns are altered by ectopic expression (ING ganisms. A more concerted genetic approachis possible HAM et al. 1991; SAMPEDROand GUEFXERO 1991). One in other invertebrates, such as nematodes. In a survey possibility is that the informational content of the ex- of temperature-sensitive lethal mutations, HIRSH et al. pression patterns is redundant. Various genes in the (1978) identified 25 ts mutants affecting genes required segment polarity subgroup are expressed in overlap- for normalembryogenesis. The mutations had not been ping patterns and final cell fates are regulated by cell selected based on their morphological phenotypes and interactions that occur continuously throughout devel- can be assumed to represent a randomsample of gene opment. This means that patterned expression of a sin- activities required during that period. In 22 of the 25 gle component may contribute to the efficiency with cases, a wild-type allele in the mother was sufficient to which the final pattern is achieved, but does not func- rescue homozygous mutant embryos, arguing for the tion as an absolutely essential determinant of that pat- numerical predominanceof maternal gene productsin tern. Effects of ectopic expression may therefore be that organism. The three loci whose products must be very subtle or transient. supplied zygotically do not represent a very large sam- In summary, limited expression patterns seem to play ple; still, it would be interesting to know whether their an important role in many of the gene activities that zygotic phenotypes suggest specific roles in develop- must be supplied to the embryo by zygotic transcription. ment. In any case, concerted mutagenesis screens for In the majority of cases, gene products are transcribed genes whose transcription is strictly required in C. ele- in spatial patterns. In many cases where they have been guns embryos should eventually identify all genes with tested, misexpression leads to at least transient abnor- specific morphological effects in that organism. malities indevelopment. This behavior is consistent In vertebrates, less is known genetically about mater- with a role for their expression in controlling where nal us. zygotic requirements. In frogs, maternal contri- and when specific developmental pathways are initi- butions appear to be substantial. Although few mutants ated. While real and significant exceptions exist, they are known, embryos homozygously deletedfor ribo- are rare. Inmany of the exceptional cases it is not clear somal DNA develop to young tadpoles, even though whether the analysis was sufficient to exclude subtle new rRNA normally begins to be made at the blastula roles for the expression pattern, particularly when tem- stage (BROWNand GURDON1964). This indicates that poral as well as spatial aspects are considered. rRNA and probably many cellular components are ma- How much is this extrapolatable to otherspecies? In ternally supplied in quantitiessufficient to last the dura- principle, one might expect similar selective pressures tion of embryogenesis. Data for other vertebrates are to maximize maternal contributions in all organisms not much more extensive. In principle, zebrafish has that develop rapidly with little growth or increase in size all the hallmarks of a species that should have minimal before larval stages. Manyorganisms fit this description, and thereforeregulatory zygotic transcription. Develop- including Caernorhabditis elegans, frogs, sea urchins and ment is rapid, the basic form being established in 48 fish. The obvious exceptions are mammals and plants, hours (KIMMEL 1989). The volume of the embryo in- 1995 GSA Medal Essay 9 creases only after hatching. Zygotically active mutations BROW, D. D., and J. B. GURDON,1964 Absence of ribosomal RNA synthesis in the anucleolate mutant of Xenopus laeuis. Proc. Natl. are known that significantly alter the pattern of the Acad. Sci. USA 51: 139-146. embryo. The real test will involve comparing the fre- CAPOVILLA, M., E. ELDONand V. PIROTTA, 1992 The giant gene of quency of such mutations with the general behavior Drosophila encodes a b-ZIP DNA-binding protein that regulated the expression of other segmentation genes. Development 114 of random lethals. If the situation in zebrafish is like 99-112. Drosophila, one would predict thatonly a small fraction DAVIDSON,E., 1986 Gene Activityin Early Developmat. Academic of vital genes have transcripts that must be supplied to Press, Orlando, FL. Dum,J. B., and N. PERRIMON,1994 The torso pathway in Drosoph- the embryo by its own transcription. Such genes should ila: lessons on receptor tyrosine kinase signalling and pattern produce discrete phenotypes suggestive of specificroles formation. Dev. Biol. 166: 380-395. in development. Results from the ongoing zebrafish EBERL,D. F., and A. J. HILLIKER,1988 Characterization of X-linked recessive lethal mutations affecting embryonic morphogenesis screens (MULLINS et al. 1994; SOLNICA-KREZELet al. in Drosophila melanogaster. Genetics 118: 109-120. 1994) will be interesting in this respect. EBERL,D. F., L. A. PERKINS, M. ENGELSTEIN,A. J. HILLIKERand N. However, the major advantage of any mutagenesis PERRIMON,1992 Genetic and developmental analysis of poly- tene section 17 of the Xchromosome of Drosophila melanogaster. screen is not the characterization of general behaviors Genetics 130: 569-583. of genes and their relative maternal and zygotic contri- EDGAR,B. A. and P. H. O’FARRELL,1990 The three postblastoderm butions. The real reason anyone does a screen is to cell cycles of Drosophila embryogenesis are regulated in G2 by string. Cell 62 469-480. identify interesting genes that offer entry points into HARBECKE,R., and J. A. 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