Segmentation and Specification of the Drosophila Mesoderm

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Segmentation and specification of the Drosophila mesoderm

Natalia Azpiazu, 1,3 Peter A. Lawrence, 2 Jean-Paul Vincent, 2 and Manfred Frasch 1'4 1Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029 USA; 2Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, UK

Patterning of the developing mesoderm establishes primordia of the visceral, somatic, and cardiac tissues at defined anteroposterior and dorsoventral positions in each segment. Here we examine the mechanisms that locate and determine these primordia. We focus on the regulation of two mesodermal genes: bagpipe (hap), which defines the anlagen of the visceral musculature of the midgut, and serpent (srp), which marks the anlagen of the fat body. These two genes are activated in specific groups of mesodermal cells in the anterior portions of each parasegment. Other genes mark the anlagen of the cardiac and somatic mesoderm and these are expressed mainly in cells derived from posterior portions of each parasegment. Thus the parasegments appear to be subdivided, at least with respect to these genes, a subdivision that depends on pair-rule genes such as even-skipped (eve). We show with genetic mosaics that eve acts autonomously within the mesoderm. We also show that hedgehog (hh) and wingless (wg) mediate pair-rule gene functions in the mesoderm, probably partly by acting within the mesoderm and partly by inductive signaling from the ectoderm, hh is required for the normal activation of hap and srp in anterior portions of each parasegment, whereas wg is required to suppress bap and srp expression in posterior portions. Hence, hh and wg play opposing roles in mesoderm segmentation. [Key Words: Mesoderm; segmentation; visceral musculature; fat body; heart; even-skipped; wingless; hedgehog] Received September 4, 1996; revised version accepted November 1, 1996.

How does the Drosophila mesoderm become subdivided dermal areas (Azpiazu and Frasch 1993; Staehling-Hamp- into different anlagen? The mesoderm is partitioned ton et al. 1994). bap is involved in the determination of along the dorsoventral axis: dorsally, cells found the pri- the anlagen of the midgut visceral mesoderm. Analogous mordia of the cardiac and visceral mesoderm and this events could activate tin target genes that would specify subdivision is triggered by inductive inputs from the ec- the anlagen of the heart and dorsal body wall muscula- toderm. Dpp is a TGF~ protein that is secreted from ture. dorsal ectodermal cells, and has been identified as the We now study patterning of the mesoderm along the inductive signal (Staehling-Hampton et al. 1994; Frasch anteroposterior axis of the embryo. The morphology of 1995). Dpp signaling is required for the formation of the the early mesoderm and the spatial domains of expres- anlagen of the heart and the midgut visceral mesoderm, sion of homeotic genes indicate that the mesoderm is and the dorsoventral extent of the visceral mesoderm is organized into parasegmental units (for review, see directly determined by the dorsoventral limits of dpp Lawrence 1992). Previous attempts to identify distinct expression in the ectoderm (Frasch 1995; Maggert et al. subdivisions within each mesodermal parasegment that 1995). An early response to the Dpp signal in mesoder- might be homologous to the anterior and posterior com- mal cells is the spatial restriction of expression of the partments of the ectoderm were negative (Lawrence and homeo-box gene tinman (tin) to dorsal portions of the Johnston 1984). However, more recently, the analysis of mesoderm, tin provides the dorsal mesoderm with the expression patterns of genes that control mesoderm de- competence to form midgut visceral mesoderm, dorsal velopment has suggested that mesodermal parasegments muscles, and the heart (Azpiazu and Frasch 1993; Bod- are indeed subdivided into anterior and posterior por- mer 1993). In combination with additional regulators tions with different developmental fates. A clear exam- that may include Dpp, tin activates the expression of a ple is the expression of the homeo-box gene bap which is second homeo-box gene, bagpipe (bap), in dorsal meso- restricted to metameric clusters of cells in the dorsal mesoderm (Azpiazu and Frasch 1993). Under the control of bap, these cell clusters develop into midgut visceral mesoderm, whereas cells in segmental portions lacking 3Present address: Centro de Biologia Molecular "Severo Ochoa," Facul- tad de Ciencias, Universita Autonoma de Madrid, Madrid 28034, Spain. bap expression form other mesodermal derivatives. The 4Corresponding author. expression of several marker genes in the anlagen of the

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Azpiazu et al. fat body and studies on the regulatory gene twist also (Fig. 1D) show that these cells are at the same antero- indicate that cells at different anteroposterior positions posterior positions and lie just ventrolateral to the pri- within each mesodermal parasegment give rise to differ- mordia of the midgut visceral mesoderm. ent mesodermal derivatives (Abel et al. 1993; Hoshizaki In summary, the mesoderm of stage 10-11 embryos et al. 1994; Dunin-Borkowski et al. 1995; Baylies and appears to be organized into parasegmental repeats that Bate 1996). are in exact register with the ectodermal parasegments. By analogy to dorsoventral patterning, inductive sig- Each parasegment is subdivided into two domains along nals from the segmented ectoderm could help segment the anteroposterior axis. In keeping with the nomencla- the mesoderm. Alternatively, the mesoderm could seg- ture used for the ectoderm (Lawrence 1992), we term the ment itself autonomously. For example, wingless (wg) mesodermal domains below the anterior compartments acts in heart and muscle development and can function "A domains" and those below the posterior compart- either from within the ectoderm or from the mesoderm ments "P domains." As shown schematically in Figure (Baylies et al. 1995; Lawrence et al. 1995; Wu et al. 1995; 1E, the P domains express bap and srp and give rise to Ranganayakulu et al. 1996). However, there must be ad- visceral mesoderm derivatives that include midgut mus- ditional segmental regulators, because both in the ab- culature and the fat body. The A domains include the sence of wg and in the presence of uniformly distributed primordia of the cardiac mesoderm and the bulk of the Wg protein the mesoderm is still segmentally patterned. somatic mesoderm and give rise to the heart and most of Our experiments show that the mesoderm forms seg- the body wall muscles. ments autonomously, although induction from the ectoderm also contributes. We show that wg and hedgehog (hh), which are candidates for inductive signals from the A mesoderm-autonomous role of pair rule genes in ectoderm, play opposite and antagonistic roles in the segmentation and specification of the visceral subdivision of mesodermal parasegments, wg is mainly mesoderm involved in the formation of derivatives from posterior portions of each parasegment, including somatic muscu- The close alignment between mesodermal and ectoder- lature and the heart, whereas hh functions mainly in the mal parasegments suggests that the two germ layers formation of derivatives in anterior parasegmental por- might be segmented by the same genes. Indeed, mutations, in particular the visceral mesoderm of the midgut tions in pair rule genes have similar effects in both the musculature and the fat body. ectoderm and the mesoderm. For example, fushi tarazu (ftz) mutant embryos retain only six out of normally 13 en stripes between parasegments 2 and 14 in wild-type embryos, and the number of bap patches is similarly Results reduced from normally 11 to 5 (Fig. 2A; note that parasegments 13 and 14 lack bap expression even in wild- Segmental organization of mesodermal primordia type embryos; see Fig. 1A). srp patches are also reduced The expression patterns of several genes reveal that the in ftz mutant embryos (Fig. 2B; wild-type expression early mesoderm is segmented. Notably, the homeo-box only occurs between parasegments 4 and 12; see Fig. 1C). gene bap is expressed in 11 metameric clusters of dorsal In spite of their aberrant patterns, the normal spatial mesodermal cells that develop into visceral musculature relationships between the bap or srp domains and the en of the midgut (Azpiazu and Frasch 1993). The anterior stripes are maintained in ftz mutants, indicating that ftz borders of each of the bap patches coincide with the mutations cause identical alterations in the mesoderm parasegmental borders of the ectoderm--thus the pri- and ectoderm. Concomitant alterations of bap and en mordia of the midgut visceral mesoderm are largely po- stripes are observed in mutants for all other pair-rule sitioned below the posterior compartments of the ecto- genes tested, including hairy (h; Fig. 2C), runt (run; Fig. derm (Fig. 1A; Azpiazu and Frasch 1993). The homeo-box 2D), paired (prd; Fig. 2E), odd-paired (opa; Fig. 2F), and gene even-skipped (eve) marks heart progenitors that odd-skipped (odd; data not shown). Pair-rule mutations will form pericardial cells (Frasch et al. 1987). Double that cause mirror-image arrangements in the ectoderm, stainings for bap and eve demonstrate that these heart such as runt (Nhsslein-Volhard and Wieschaus 1980), re- progenitors form between the bap patches at the dorsal sult in mirror-image arrangements of the bap patches in crest of the mesoderm (Fig. 1B). In addition, the progen- the mesoderm (Fig. 2D). itors of the cardioblasts are formed in close juxtaposi- The importance of pair-rule genes to segmentation of tion, just anterior to the pericardial progenitors (K. Jagla, the mesoderm is particularly obvious in embryos that pets. comm.). Thus, it appears that the anlagen of the are mutant for eve. In embryos lacking eve function, the cardiac mesoderm alternate with the anlagen of the mid- ectoderm is unsegmented and no en stripes are formed in gut visceral mesoderm. There are also primordia of the the trunk region (Nhsslein-Volhard et al. 1985; Harding fat body in each segment; these are marked by the ex- et al. 1986; Macdonald et al. 1986). Similarly, bap and srp pression of the serpent (srp) gene that is required for fat patches are completely lost in the trunk region of eve body formation (srp encodes the GATA transcription fac- mutant embryos, indicating that the mesoderm is unseg- tor ABF; Abel et al. 1993; Rehorn et al. 1996). Double mented as well (Fig. 2G, H). As a consequence, neither stainings for srp and engrailed (en; Fig. 1C) or srp and eve midgut visceral mesoderm nor fat body visceral meso-

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Drosophila mesoderm segmentation

Figure 1. Intrasegmental and dorsoventral arrangement of mesodermal anlagen. All panels show wild-type embryos with anterior to the left and dorsal sides up. White boxes serve as a reference to indicate the location of the bap domains in parasegments 5. CA) Stage 10 embryo stained for bap mRNA (purple) and En protein (brown). The anterior margins of bap expression coincide with the ectodermal parasegmental borders, but are broader than the posterior compartments marked by en. (B) Early stage 11 embryo stained for bap mRNA (purple) and Eve protein (brown). The heart (pericardial} progenitors marked by eve arise at positions between the bap patches at the dorsal margin of the mesoderm. (C) Stage ll embryo stained for srp mRNA (purple) and En protein (brown). The srp-stained fat body primordia are found at similar intrasegmental, but more ventrally located, positions as compared with the bap-stained visceral muscle primordia. (D) Stage 11 embryo stained for srp mRNA and Eve protein to compare the relative positions of heart and fat body primordia. Bottom right: Schematic summary of the spatial arrangement of mesodermal anlagen (top of panel: dorsal margin; bottom of panel: ventral midline). The primordia of the visceral mesoderm {vm) are located below the posterior compartments of the ectoderm (marked by en in yellow) and extend posteriorly into the first half of each adjacent anterior compart- ment. The primordia of the midgut visceral mesoderm (mgvm; purple) are located in dorsal portions and those of the fat body visceral mesoderm (fbvm; blue-green) immediately adjacent in lateral portions of the mesoderm. The primordia of the cardiac mesoderm are located between the vm primordia at the dorsal margin (cm: cardiac mesoderm; solid red indicates Eve-stained pericardial progenitors, stippled red indicates the presumed location of cardial progenitors). Most of the rest of the mesoderm will give rise to somatic mesoderm (sm; grey). (A,P) Mesodermal A and P domains; (PS) parasegment; (pc) pericardial cell progenitors; (CNS) neuronal progenitors of the central nervous system.

derm is formed in the absence of eve function (Fig. 2I,K, expressed in stripes in both ectoderm and mesoderm. cf. wild-type embryos in Fig. 2J and L}. Therefore, they could regulate bap expression and me- It appears that segmentation of the mesoderm is al- soderm segmentation through either of two possible ready set up by the pair-rule genes during gastrulation, routes. The first could be direct and within the mesoder- that is, at least 2 hr prior to the time when the dorsal bap real cells proper. The second route could be via induc- patches appear. Upon overstaining of embryos hybrid- tion from the overlying ectoderm. To find out which ized with bap antisense RNA probes, weak stripes of bap route is used, we made embryos that were genetically expression can be observed throughout the dorsoventral mosaic for eve; eve was chosen because it is essential for extent of the mesoderm as early as stage 8. Initially these bap expression and development of the midgut visceral stripes display a pair-rule distribution, but at stage 9, a mesoderm. Nuclei from wild-type donor embryos regular pattern of weak transverse stripes is detected marked with a lacZ reporter gene (Vincent et al. 1994) (Fig. 3A). This shows that mesoderm segmentation is were transplanted into eve embryos that were stained initiated even before the the mesoderm spreads under at stage 10 for bap mRNA and f~-galactosidase. Figure 4A the ectoderm and therefore that these early segmenta- demonstrates that bap expression is rescued near to tion events occur without any influences from the ecto- wild-type cells in an otherwise eve mutant embryo. The derm. It has been proposed that the segmental expression restriction of the bap domains to the area of wild-type of twist is required for visceral mesoderm specification cells indicates that signals emanating from these cells, if (Baylies and Bate 1996). However, double stainings of any, are unable to act at a long distance to activate bap in stage 10 wild-type embryos for Twist protein and bap adjacent eve- areas. We then prepared sections of the mRNA show that twist cannot determine the segmental mosaic embryos. Figure 4B shows an embryo with two bap pattern, because Twist protein is still evenly distrib- separate wild-type patches of cells, one restricted to the uted along the anteroposterior axis at the time when the ectoderm (left side) and another extending into the me- bap patches are fully developed (Fig. 3B). soderm (right side). Importantly, bap expression is only Prior to and during gastrulation, pair-rule genes are observed in the mesodermal cells that are wild-type for

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Figure 2. Regulation of mesoderm segmentation and specification by pair-rule genes, bap and srp mRNAs are stained purple; En, fasciclin III, and [3-galactosidase proteins are stained brown. Em- bryos shown in A-H are in stage 10. {A)ftz mutant embryo stained for hap and En. (B) ftz mutant embryo stained for srp and En. (C) hairy mutant stained for bap and En. {D) runt embryo stained for bap and En. The wedges demarcate examples for segments arranged in a mirror image fashion. (E) paired mutant stained for bap and En. Although the number of fully developed bap patches is reduced from normally 11 to 6, weak bap signals are detected in intervening areas that lack detectable en stripes. (F) opa mutant stained for bap and En. Both the en and bap stripes are reduced in number and irregularly spaced. {G) bap expression in a stage 10 eve ~ mutant embryo. No bap expression in the midgut visceral mesoderm primordia is detected. (H) Stage 11 eve ~ embryo stained for srp (purple) and En (brown). srp is not expressed in the trunk region. (I) eve ~ mutant, and {J) wild-type embryo at early stage 12, both stained for fasciclin III. No midgut visceral mesoderm is formed in eve mutants. (K) eve ~ and {L) wild-type embryo at stage 13, both containing an enhancer trap insertion expressing ~-galactosidase in the fat body. The fat body is missing in the eve mutant embryo. (mgvm) Midgut visceral mesoderm; (fb) fat body.

eve, but not in eve mutant cells that underly and contact in regulating these two genes must be indirect and in- wild-type cells of the dorsal ectoderm (Fig. 4B). The fail- volve intermediates. Here we examine whether segment ure of wild-type ectoderm to induce bap in the underly- polarity genes, which mediate pair-rule gene functions in ing eve mutant mesoderm is not attributable to the the ectoderm, are also required for the segmentation of small size of the wild-type patch in this particular em- the mesoderm. The spatial overlap between the stripes of bryo, because embryos with more extensive areas of en or hh expression and the patches of bap or srp sug- wild-type cells in the ectoderm also fail to activate bap gests that these two segment polarity genes are candi- (Fig. 4C). These results demonstrate that at least one dates (Figs. 1A, C and 5A; note that segment polarity function of eve is required within the mesoderm itself gene expression is predominantly ectodermal at this for activating bap and for mesoderm segmentation. stage). Both hh and en are indeed required for full activation of bap. In hh mutant embryos, the bap domains are sig- Opposing roles of hh and wg in establishing nificantly reduced in size (Fig. 5B). There is also some mesodermal A and P domains reduction in embryos deficient for en (Fig. 5C). In Because the pair-rule gene products disappear prior to the Df(en);hh double mutants, there is an even stronger re- stage when bap and srp are fully activated, their function duction of bap expression than in mutants for either hh

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Figure 3. Dynamics of bap expression in wild-type embryos. (A) Wild-type embryo at stage 9, overstained with a bap RNA probe. Weak transverse stripes are detected transiently even in ventral portions of the mesoderm. (This ventral expression ceases at stage 10 when dorsal mesodermal expression reaches high levels.) (B) Stage 10 wild-type embryo, stained for bap and Twist. Twist protein is still evenly distributed at the time when the mature bap pattern appears.

or en alone (Fig. 5D), therefore the function of en in al- lowing full bap activation is not mediated soley through hh. The lack of either hh or en function alone has only minor consequences for visceral mesoderm formation (data not shown), whereas the simultaneous loss of function of both genes results in a clear reduction of visceral Figure 4. Mesoderm-autonomous requirement for eve func- mesoderm derivatives. As shown in Figure 5E, the nor- tion to activate bap. Shown are mosaic embryos where host mally continuous band of the midgut visceral mesoderm tissues are eve ~ Donor-derived tissues are wild-type and carry (see Fig. 2J) is interrupted in Df(en);hh double mutants, an arm-lacZ reporter gene insertion. Embryos are stained for and resembles that of mutant embryos with strongly re- bap mRNA {purplel and ~-galactosidase (brown). (A) bap expres- duced bap function (Azpiazu and Frasch 1993). The num- sion is strictly confined to a wild-type patch of cells {brown) in bers and sizes of cell clusters of the fat body visceral the otherwise eve mutant embryo. (B,C) Cross-sections of mesoderm are both reduced in the double mutants (Fig. stained mosaic embryos, bap expression is seen only seen when 5E). wild-type patches encompass dorsal mesoderm (B, right), but The requirement for hh and en for normal bap activa- not when eve function is provided in the dorsal ectoderm only tion could partially explain the absence of bap expres- (B, lefL, and C). sion in eve mutant embryos, because these embryos lack hh and en expression in their trunk region. To test suggest that hh and en participate in the establishment whether hh or en could rescue bap expression in the of the mesodermal P domains that express both bap and absence of eve, we ectopically expressed each of these srp and form visceral mesoderm derivatives. two genes in the mesoderm of eve mutant embryos (see bap is expressed in the mesodermal P domains, Materials and Methods). bap is indeed activated upon whereas the secreted protein Wg is synthesized adjacent mesodermal expression of hh in eve mutant embryos, in the A domains and, more prominently, in the A com- although the number of rescued bap patches is less than partments of the ectoderm (Fig. 6A). The alternating in the wild-type (Fig. 5F). The striped expression of bap stripes of expression abut at the parasegmental borders, in these embryos indicates that the mesoderm of eve but small areas posterior to each bap patch lack detect- mutant embryos retains some periodic properties, which able levels of Wg protein. Wg appears to act negatively on could reflect the contributions of other pair-rule genes to bap, because bap expression is expanded in wg mutant mesoderm segmentation. There is also a partial rescue of embryos (this interaction may not be direct, however; midgut visceral mesoderm formation. Ubiquitous ex- see Discussion). In posterior regions of these embryos, a pression of en in the mesoderm of eve mutant embryos continuous rather than periodic pattern of bap expres- activates bap expression as well, albeit less efficiently sion is observed in the dorsal mesoderm (Fig. 6B). More than hh (data not shown). Taken together, these results anteriorly, some periodic bap expression is still ob-

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Figure 5. Positiveregulation of bap by hh and en. Embryo in E is at stage 12, all others shown are at stage 10. mRNA detection appears purple and protein detection appears brown. CA) Wild-type embryo stained for bap mRNA and Hh protein. The bap and hh stripes are largely congruent. (B) hh mutant stained for bap mRNA and Wg protein. The bap patches are reduced in size. (C) Df(en) embryo stained for bap mRNA (and En protein). A minor reduction of bap expression is observed. (D) Df(en);hh double mutant embryo, stained as in C. bap expression is strongly reduced. (E) Df(en);hh double mutant embryo, stained for fasciclin III protein and srp mRNA. The visceral mesoderm of the midgut musculature and fatbody are substantially reduced, iF) eve ~ mutant embryo with ectopic mesodermal hh expression (using a twist-GAL4 driver) and stained for bap mRNA and Hh protein. Partial rescue of bap expression is observed.

served. Mutations in genes downstream of wg such as entirely blocks bap expression in the midgut visceral armadillo (arm; Fig. 6C) and dishevelled (dsh; Fig. 6D) mesoderm primordia (Fig. 7I, J). These results indicate have a similar phenotype. The expression of srp is also that overexpression of hh in wg-, and of wg in hh-, continuous in wg mutant embryos (Fig. 6E). produce unsegmented mesoderm. The patterns of bap expression suggest that the mesoderm has opposite iden- The subdivision of the mesoderm into A and P tities in these two experimental situations. Ectopic hh in domains involves antagonistic interactions between wg- appears to result in a uniform P character, whereas hh and wg the lack of bap expression upon ectopic wg expression in hh- signifies a uniform A character of the resulting me- Because the results described above indicate a positive soderm. Ectopic hh and wg can produce these alterations function of hh and a negative function of wg in bap reg- only if the other gene is not functional, but are unable to ulation, we tested whether ectopic expression of hh or do so in wild-type embryos. Thus, we conclude that wg wg would affect bap expression in opposite directions. and hh normally antagonize each other's function. This Ectopic expression of hh in the Krfippel domain of the antagonism is likely to be important for the proper sub- ectoderm (Fig. 7A; Frasch 1995), in the whole ectoderm division of the mesoderm into A and P domains. (data not shown), or in the whole mesoderm (Fig. 7B) of Analysis of wg- embryos with ectopic hh, and of hh- wild-type embryos results in a minor expansion of the embryos with ectopic wg, confirmed that the mesoder- bap domains only. By contrast, ectopic hh expression in mal fate maps are shifted in opposite directions in these either the ectoderm (Fig. 7C) or mesoderm (Fig. 7D) of wg two situations. For example, the fat body in wg-/ectopic mutant embryos causes massive overexpression of bap, hh embryos is expanded as compared with wild-type and which comes to resemble the expression pattern of tin at wg- embryos (Fig. 8A-D). The midgut visceral meso- this stage (Bodmer et al. 1990; Azpiazu and Frasch 1993). derm appears also to be enlarged (Fig. 8C, D) at the ex- If hh is ectopically expressed together with dpp in the pense of heart precursors and somatic musculature (data ectoderm of wg mutant embryos, bap expression ex- not shown). The latter phenotypes are also observed in pands also ventrally and is observed in the whole meso- wg- embryos (Baylies et al. 1995; Wu et al. 1995), and it derm between parasegments 2 and 12 (Fig. 7F; cf. ectopic is difficult to judge whether the reduction of somatic expression of dpp in wild-type embryos, Fig. 7E). This muscles is enhanced upon ectopic hh expression. In wg clearly illustrates the combinatorial roles of anteropos- mutant embryos with ectopic ectodermal expression of terior and dorsoventral cues in mesoderm patterning. both hh and dpp, the visceral midgut mesoderm is even Ectopic expression of wg has opposite effects to those more expanded. It occupies the whole mesoderm be- observed for ectopic hh. Whereas wg expression either in tween parasegment 2 and 12, and no other mesodermal the ectoderm (within the Krfippel domain, Fig. 7G, or in derivatives are formed in this region (Fig. 8E). the whole ectoderm, data not shown) or in the mesoderm In stark contrast to wg-/ectopic hh embryos, hh-/ec- of wild-type embryos (Fig. 7H) results only in a slight and topic wg embryos show a massive reduction of visceral partially penetrant reduction of bap expression, similar mesodermal tissues (Fig. 8G). Ectopic wg expression in ectopic expression of wg in hh mutant embryos almost wild-type embryos causes a minor reduction of visceral

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number of cells with somatic mesodermal cell fates, as shown by the larger number of MyoD expressing cells. Whereas, in stage 13 wild-type embryos, there are segmental clusters of MyoD-expressing cells in the somatic mesoderm (Fig. 8H; Michelson et al. 1990; Paterson et al. 1991 ), hh-/ectopic wg embryos have uniform MyoD expression in the dorsal mesoderm, and the more ventral clusters appear expanded (Fig. 8I). Taken together, these results show that expansion of the mesodermal P domains at the expense of the A domains, as exemplified by wg /ectopic hh embryos, causes the mesoderm to form mostly visceral organs. By contrast, fate map shifts in the opposite direction in hh-/ectopic wg embryos result in the exclusive formation of somatic muscles and heart.

Discussion Anteroposterior subdivisions of mesodermal parasegments and the mesodermal fate map Our analysis of mesodermal gene expression patterns shows that the early mesoderm is organized into parasegments that are in exact register with the ectodermal parasegments. The existence of stable parasegmental mesoderm boundaries is supported by our observation that the stripes of eve-lacZ reporter genes perdure in the mesoderm with sharp anterior borders that coincide with the anterior borders of the bap patches (M. Frasch, unpubl.). Each mesodermal parasegment is subdivided along the anteroposterior axis into a P and an A domain that express different control genes, have different developmental fates, and respond differently to mutations in segmentation genes. The P domains comprise the expression domains of bap plus those of srp, genes that mark the anlagen of the transverse midgut muscles and of the fat body, respectively. The A domains contain the anlagen of the heart and the bulk of the body wall muscles. This picture conforms with the morphological analysis of Dunin-Borkowski et al. (1995), except we observe that the anlagen of the fat body are located ventrally rather than dorsally to those of the midgut musculature. Our assignment is further supported by an analysis of bap reporter genes showing that all bap expressing cells Figure 6. Negative regulation of bap and srp by the wg path- from the dorsal mesoderm will form midgut visceral me- way. (A) Wild-type embryo stained for bap mRNA and Wg pro- soderm (Z. Yin and M. Frasch, unpubl.). We are not sure tein. The bap patches abut the wg stripes at their posterior of the fate of those mesodermal cells located between the margins but do not appear to fill the entire wg interstripe re- fat body anlagen and the ventral midline in the P do- gions. (B) wg mutant embryo stained for bap mRNA and En mains of each parasegment (Fig. 1). Although it is possi- protein, bap expression expands and becomes continuous, par- ble that some of them give rise to mesodermal glial cells ticularly in the posterior segments. (C) Embryo lacking both the (Gorczyka et al. 1994), others probably generate specific maternal and zygotic contributions of arm, stained as in B. (D) body wall muscles. Thus, although most of the somatic Embryo lacking both the maternal and zygotic contributions of mesoderm is derived from the A domains, some comes dsh, stained as in B. bap expression in arm and dsh mutants is from the P domains and therefore could be subject to altered in a similar fashion as in wg-. (E) wg mutant embryo stained for srp mRNA expression, which becomes almost con- different control. This could explain the presence of tinuous (cf. Fig. 1C). some residual somatic mesodermal cells and muscles in embryos with genetic backgrounds that appear to produce a uniform "P" character in the mesoderm. mesoderm formation only, but the number of heart pro- The subdivision of the mesodermal parasegments into genitors is clearly increased (Fig. 8F; Lawrence et al. P and A domains is reminiscent of the subdivision of 1995). The reduction of visceral mesoderm in hh mu- ectodermal parasegments into posterior and anterior tants with ectopic wg is accompanied by an increased compartments. At present, we do not know whether

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Figure 7. Effects of ectopic hh and wg expression on bap expression. (A) Wild-type embryo with ectopic ectodermal hh expression (ZKrGAL driver, see Frasch 1995) and stained for bap mRNA and Hh protein. (B) Wild-type embryo with ectopic mesodermal hh expression (twist-GAL4 driver) and stained as in A. In both situations, the bap patches are only slightly expanded. (C) wg mutant embryo with ectopic ectodermal hh expression (E22cGAL4 driver), stained as in A. bap expression is strongly expanded and becomes continuous along the anteroposterior axis. (D) wg mutant embryo with ectopic mesodermal hh expression (twist-GAL4 driver), stained for bap mRNA and En protein. The bap pattern is similar to that seen with ectodermal overexpression of hh. (E) Wild-type embryo with ectopic ectodermal expression of dpp (E22cGAL4 driver), stained for bap mRNA and Eve protein. The bap stripes extend from the dorsal margin (d) to the ventral midline (v). (F) wg mutant embryo with ectopic expression of hh and dpp (driver as in E), showing uniform bap expression between parasegments 2 and 12. (G) Wild- type embryo with ectopic ectodermal expression of wg (ZKrGAL driver as in Fig. 7A), stained for bap mRNA and Wg protein. A weak suppression of bap expression is observed, mostly in the patches #4 and #5. (H) Wild-type embryo with ectopic mesodermal expression of wg (twist-GAL4 driver), stained as in A. Some suppression of bap expression is observed, particularly in more anterior parts of the mesoderm. Note that the examples shown in E and F are among the most strongly affected embryos found, and usually the reduction of bap expression is less conspicuous. (I) hh mutant embryo with ectopic ectodermal expression of wg (E22cGAL4 driver as in C) and {1) hh mutant embryo with ectopic mesodermal expression of wg, stained as in E. In both situations, bap expression in the midgut visceral mesoderm primordia is almost completely abolished. there are lineage restrictions in the mesoderm prior to or (gut muscle and fat body) P compartments from somatic/ during the stage when this subdivision becomes visible cardiac A compartments. through the expression of genes such as bap and srp. Regulation of mesoderm patterning However, it is likely that any such restrictions would occur before the onset of the third wave of mitotic divi- The segmentation of the mesoderm and the anteropos- sions in the mesoderm. By that time the outer borders of terior subdivision of the parasegments are controlled by the bap patches form sharp boundaries and all bap-ex- gene systems that are similar, but not identical, to those pressing cells start segregating into the interior to form that segment the ectoderm. In particular, the pair-rule midgut visceral mesoderm (Azpiazu and Frasch 1993). genes and their upstream regulators have similar roles in Our observation that most body wall muscles are derived both ectoderm and mesoderm and their mutations cause from the A domains may explain why it has not been identical alterations of the number and polarity of seg- possible to detect A and P compartments within somatic ments in the two germ layers. However, these genes may musculature (Lawrence 1982). If there are lineage bound- not act through an identical set of downstream genes. aries between A and P domains in the gastrulating me- Indeed, previous experiments with genetic mosaics for soderm, they would be expected to demarcate visceral en suggested that this gene is not required for normal

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Drosophila mesoderm segmentation

Figure 8. Effects of ectopic hh and wg expression on mesoderm differentiation. (A) Stage 14 wild-type embryo and (B) wg mutant embryo with uniform ectopic hh expression in the ectoderm as in Fig. 7C. Staining for srp mRNA shows enlargment of the fat body in the mutant as compared with the wild-type situation. (C,D) Sec- tioned stage 12 embryos with genotypes as in A and B, stained for fasciclin III protein and srp mRNA. The embryo in D shows enlarged fat body and midgut visceral mesoderm. (E) wg mutant embryos with ectopic expression of hh and dpp as in Fig. 7F, stained for fasciclin III and Eve. All mesodermal cells between parasegments 2 and 12 are specified as midgut visceral mesoderm. (F) Wild-type embryo with ectopic ectodermal wg as in Fig. 7I, stained for fasciclin III (brown) plus Eve (purple).There are supernumerary pericardiat progenitors but visceral mesoderm formation is largely normal. (G) hh mutant embryo with ectopic ectodermal wg expression and stained as in F. Visceral mesoderm formation is largely abolished. (H) Stage 13 wild- type embryo, stained for MyoD (brown) and Eve (black). The muscle precursors expressing MyoD are arranged in segmental clusters. (I) hh mutant embryo with ectopic ectodermal wg, stage and staining as in H. In the dorsal mesoderm, MyoD is expressed in a continuous band of cells, and the more ventrally located MyoD clusters are enlarged. Eve staining of pericardial cells was used to identify embryos with ectopic wg expression.

mesoderm development (Lawrence and Johnston 1984). ceral mesoderm primordia (P) in the absence of wg func- This is consistent with our finding that absence of en tion could cause the loss of somatic and cardiac meso- causes only minor disruptions of bap expression and has derm (A). This could explain why wg is required long no obvious effects on visceral mesoderm formation. before these cell types are specified (Wu et al. 1995; Ran- Thus, even though en is expressed in the early mesoderm ganayakulu et al. 1996). In addition, wg could continue (Lawrence et al. 1994), the pair-rule genes do not seem to to provide inputs for the development of muscle and act through it. heart precursors after mesoderm segmentation has been More important mediators are wg and hh which are established. The lack of heart progenitors in hh mutant expressed at opposite sides of the parasegmental borders. embryos, which is a result of the decay of wg expression, Whereas wg is involved in defining A (i.e., somatic and could reflect this continued requirement for wg in heart cardiac) identities of each parasegment, hh is involved in specification (Park et al. 1996). defining P (i.e., mostly visceral) identities. One function The experiments involving ectopic expression of wg of wg is to prevent the expression of P genes in the A and hh show that these two genes can antagonize each domains. Whereas bap is activated in the dorsal meso- other during mesoderm segmentation. Uniform expres- derm by dpp and tin, wg appears to block this activation. sion of either of them does not dramatically affect the Likewise, wg blocks the expression of srp ventrally to periodic pattern of bap, provided that the other functions the tin domains. These are negative effects, but wg also normally. Similar interactions between wg and hh occur acts positively to specify the cell fates of the heart pro- during the segmentation of the ectoderm (Bejsovec and genitors and body wall muscle precursors; ectopic ex- Wieschaus 1993; Heemskerk and DiNardo 1994; note pression of wg can even generate supernumerary cells that these negative, functional interactions are distinct with these identities (Baylies et al. 1995; Lawrence et al. from the positive interactions between wg and hh that 1995; Wu et al. 1995; D'Alessio and Frasch 1996; Ran- serve to maintain the expression of both genes in the ganayakulu et al. 1996; this paper). The two actions of ectoderm after stage 9). Because wg and hh are expressed wg could be related: Specifically, the expansion of vis- in adjacent cells and encode secreted molecules, this

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Azpiazu et al.

functional antagonism could help ensure that wg func- mental stripes in the early mesoderm (Bopp et al. 1989). tion is limited to the A domains and hh function to the The expression of twist becomes striped, with high-pro- P domains of the mesoderm. tein levels present in the A domains, and this periodic modulation is required to allow visceral mesoderm development (Baylies and Bate 1996). However, twist can- Mesoderm-autonomous vs. inductive regulation of not be involved in organizing the mesodermal A and P mesoderm segmentation domains, because its periodic expression appears only The close contact between ectoderm and mesoderm after after the segmental expression of bap has been estab- gastrulation could allow indirect regulation of meso- lished. It is more probable that twist could be controlled derm segmentation by pair-rule genes, through their role at a similar level of the regulatory hierarchy as bap and in the segmental subdivision of the ectoderm. Inductive its disappearance in the A domains could be required for signals from ectodermal cells could transmit this posi- the function, rather than the regulation, of bap. tional information to the mesoderm and instruct meso- In conclusion, our current view of mesodermal seg- dermal cells to assume either A or P properties. Such a mentation and patterning can be summarized as follows mechanism would be analogous to the dorsoventral sub- (see Fig. 9): (1) During gastrulation, pair-rule genes con- division of the mesoderm, which is induced by dorsal trol the expression of target genes in segmentally re- ectodermal cells through the secreted signal molecule peated stripes in the mesoderm. This process is autono- Dpp (Staehling-Hampton et al. 1994; Frasch 1995). Both mous to the mesoderm and establishes the A and P do- wg and hh act in the mesoderm and their ectopic expres- mains. Mesodermal targets of the pair-rule genes include sion in the ectoderm can indeed alter the pattern of me- hh and en (in P domains) and wg (in A domains), en plays sodermal bap expression and induce changes in visceral only a marginal role, and therefore we believe that there mesoderm development. Similarly, ectopic expression of are as yet unidentified pair-rule target genes, M and N, wg in the ectoderm affects the development of somatic that are important for the allocation of mesodermal cells muscle precursors (Baylies et al. 1995; Ranganayakulu et to the A and P domains {Fig. 9, left). Gene M could be al. 1996; this paper). However, embryos that are geneti- required to activate bap and srp in P domains, whereas N cally mosaic for wg show that the function of wg in heart could activate target genes in A domains that are information can be provided either from the ectoderm or volved in somatic and cardiac mesoderm formation. Nei- from within the mesoderm (Lawrence et al. 1995). We ther M nor N is sufficient to activate their target genes; believe that this function of wg is, at least in part, a they both would later require localized coregulators that consequence of its general role in determining the A do- are not active in the early mesoderm. mains of each parasegment. Therefore, it is likely that its (2) After gastrulation, the pair-rule gene products fade function to promote somatic mesoderm development away and the maintenance of the striped expression of M and to suppress visceral mesoderm in the A domains is and N requires hh and wg function. The mesodermally also provided from both ectoderm and mesoderm. Note expressed hh and wg products can only maintain normal that the gradual decrease of wg and hh expression in the A and the P domains (i.e., normal M and N expression) mesoderm during gastrulation suggests that the ectoder- transiently as they are not expressed beyond stage 8 or 9. mal contributions of both genes, which persist at high In the ectoderm, hh and wg maintain each other's ex- levels, are most significant for normal mesoderm devel- pression after this stage. Therefore, the contribution of opment (Baker 1988; Lee et al. 1992; Mohler and Vani hh and wg products secreted from the ectoderm is likely 1992; Tabata et al. 1992; Baylies et al. 1995). Importantly, our results with eve mosaic embryos demonstrate that pair-rule genes act within the mesoderm to segment it. Thus, wg and hh signals provided by ectodermal patches of wild-type cells are not sufficient to mediate normal mesoderm segmentation and bap activation. Probably, the pair-rule genes establish first a prepattern of gene expression within the mesoderm and later inductive inputs from the ectoderm ensure that the mesodermal and ectodermal parasegments remain in exact register. What are the candidates for genes that are regulated by the pair-rule genes in the mesoderm? We postulate that there exist as yet unidentified downstream genes in addition to hh and wg that are expressed in striped patterns Figure 9. Model of mesoderm segmentation and the specification of mesodermal tissues. We propose that mesoderm seg- in the early mesoderm. Such genes could be responsible mentation occurs in two phases; an early, mesoderm-autono- for the residual segmental expression of bap in wg;hh mous phase (left) and a subsequent phase that involves meso- double or wg, en;hh triple mutant embryos (M. Frasch, derm-autonomous as well as inductive regulation (right). unpubl.) and the faint stripes of bap expression in the During the second phase, developmental control genes are ac- early wild-type mesoderm. A potential candidate is the tivated in quadrants at the intersections of transverse and lon- paired-domain gene pox meso, which is expressed in seg- gitudinal domains of patterning genes (see text for details).

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Drosophila mesoderm segmentation to become significant later. Functional antagonism be- toderm was achieved with the driver lines ZKrGAL8 (in the tween hh and wg contributes to the formation of sharp Kr0ppel domain; Frasch 1995} or E22c-GAL4 (made by K. borders between A and P domains (Fig. 9, right). Yoffe, Harvard Medical School, Boston, MA). The expression (3) After the mesoderm has spread below the ectoderm, pattern of E22c-GAL4 was tested by crossing it with Bg4-i-2 (Brand and Perrimon 1993). Araldite sections of embryos stained the cells in the dorsal portions of each transverse stripe for f3-galactosidase demonstrated that the expression from this receive Dpp signals from the dorsal ectoderm and there- driver is restricted to the embryonic ectoderm (M. Frasch, un- fore express high levels of tin. This combination of trans- publ.). The line UASdpp-5 (third chromosome insertion) was verse and longitudinal activities (gene M plus Tin/Dpp 1 used for dpp misexpression (Frasch 19951. Embryos from UAS- is sufficient to activate high levels of bap in a dorsal GAL4 crosses were collected at 27~ The enhancer trap line quadrant of each P domain. Similarly, different combi- E7-3-63 (provided by V. Hartenstein, University of California, nations could activate developmcntal control genes in Los Angeles) was used to analyze fat body formation. other quadrants and thereby define other anlagen. Nuclear transplantation and analysis of mosaic embryos (4) bap specifies the midgut visceral mcsoderm, whereas genes such as srp determine the fat body vis- Nuclear transplantations were done as described previously ceral mesoderm. Both types of visceral mesoderm segre- (Lawrence et al. 1994; Vincent and Lawrence 1994), using do- gate into the interior and their development becomes nors marked with arm-lacZ. Hosts were an eve 127 mutant strain maintained over a CyO,hb-lacZ balancer. Mosaic em- independent from the ectoderm. However, the mesodcr- bryos were processed for staining with ~-gal antibodies, fol- real cells of the A domains remain in contact with the lowed by hybridization with digoxygenin-labeled bap cDNA ectoderm and wg could therefore continue to influence probes as described in Azpiazu and Frasch (1993). Nine of the the development and patterning of the somatic and car- obtained mosaic embryos were analyzed in serial sections. Of diac mesoderm. these, three had mesodermally restricted wild-type cells showing hap expression, four had ectodermally restricted wild type Materials and methods cells and lacked bap expression, and in two embryos both ectoderm and the underlying mesoderm was wild-type for eve. Drosophila mutant strains Antibody staining and in situ hybridization of embryos The following segmentation mutants were used in this study: eve L27, ftz 9H34, h 7A94, prd 2.45, odd 1.36.1~, o[,TaIlp32, rlln LBF', The following antibodies were used for the phenotypic analysis: armHS 6; Df(2R)en c Ideleting both en and invected (inv); Gustav- Monoclonal mouse-anti En (4D9, provided by C. Doe, Univer- son et al. 19961, Df(2R)gsb and ClI)ga-mlA (both of which had sity of illinois, Urbana), rabbit-anti Eve (Frasch et al. 1987), normal hap expression, data not shown), dsh 477, hh l'<:>), and rabbit-anti Hh (rabbit 1255, provided by J. Knight and T. Korn- Wc~cx4. Mosaic germlines for arm and dsh were generated by berg, University of California, San Francisco), rabbit-anti Wg using the yeast recombinase-based FLP-DTS system (Chou and (provided by R. Nusse, Stanford University, CA), polyclonal Perrimon 1992). Recombination was induced in femalcs ho- mouse-anti 6-galactosidase (Sigma), monoclonal mouse-anti mozygous for an FRT insertion on the X chromosome (FRT mr) fasciclm III (7G 10, provided Developmental Studies Hybridoma and heterozygous for the dominant female sterile mutation Bank), rabbit-anti Twist (provided by S. Roth, Max Planck In- ovo TM, as well as the mutation to be studied. The source of FLP stitut for Entwicklungsbiologie, Tfibingen, Germany), rabbit- recombinase was an autosomal insertion of FLP recombinase anti Dmyd (1184, provided by B. Paterson, National Institute of under the control of the heat shock promoter (FLp~S). The FLP/ Health, Bethesda, MD}. Antibody double stainings and anti- FRT stocks were provided by E. Siegfried (Pennsylvania State body-in situ hybridization double stainings were performed as University, State College). Homozygous pair-rule mutant em- described in Azpiazu and Frasch (1993). bryos were identified through altered e, patterns, hh mutant embryos through the use of a TM3,lacZ balancer or their de- Acknowledgments creased wg expression, and wg mutants through their decreased We thank Zhizhang Yin for technical assistance and Patrick Lo en or hh expression. for bap RNA in situ hybridizations. We thank Esther Siegfried, Andrea Brand, Tom Kornberg, Volker Hartenstein, Alan Mich- UAS/GAL4 strains elson, Leslie Pick, Jym Mohler, and Ken Yoffe for fly strains, and Generation of UASwg: wg cDNA c14 (provided by N. Baker, Chris Doe, Jonathan Knight, Tom Kornberg, Roel Nusse, Bruce Albert Einstein College of Medicine, Bronx, NY) was cloned as Paterson, Siegfried Roth, and the Developmental Studies Hy- a BamHI-XbaI fragment into BglII-Xbal of pUAST (Brand and bridoma Bank for antibodies. We appreciate the helpful com- Perrimon 1993). An insertion of this P element on the third ments of Hanh Nguyen on the manuscript. This work was sup- chromosome (UASwqg2) was used throughout the experiments. ported by National Institutes of Health grant HD30832 and a Generation of UAShh: hh cDNA 11 (provided by Phil Beachy, Pew Scholarship in the Biomedical Sciences to M.F. and by a Johns Hopkins University, Baltimore, MD) was made by first fellowship from the Basque Country to N.A. cloning a ftindIII-EcoRI insert from hhll-pNB40 into pBlue- The publication costs of this article were defrayed in part by script KS+ and then a KpnI-Xbal insert was recloned into payment of page charges. This article must therefore be hereby pUAST. The line UAShhl with an insertion on the third chro- marked "advertisement" in accordance with 18 USC section mosome was used in all experiments described. 1734 solely to indicate this fact. 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Segmentation and specification of the Drosophila mesoderm.

N Azpiazu, P A Lawrence, J P Vincent, et al.

Genes Dev. 1996, 10: Access the most recent version at doi:10.1101/gad.10.24.3183

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