Ectoderm Induces Muscle-Specific Gene Expression in Drosophila

Development 121, 1387-1398 (1995) 1387 Printed in Great Britain © The Company of Biologists Limited 1995

Ectoderm induces muscle-speciﬁc gene expression in Drosophila embryos

Rob Baker1,* and Gerold Schubiger2 1Department of Genetics and 2Department of Zoology, University of Washington, Seattle, WA 98195, USA *Author for correspondence at present address: Department of Anatomy, University of Wisconsin, 1300 University Avenue, Madison WI 53706, USA

SUMMARY

We have inhibited normal cell-cell interactions between mesoderm cells to express nautilus (a MyoD homologue) mesoderm and ectoderm in wild-type Drosophila embryos, and to differentiate somatic myofibers, whereas dorsal and have assayed the consequences on muscle development. ectoderm induces mesoderm cells to express visceral and Although most cells in gastrulation-arrested embryos do cardiac muscle-specific genes. Our findings suggest that not differentiate, they express latent germ layer-specific muscle determination in Drosophila is regulated by genes appropriate for their position. Mesoderm cells induction between germ layers during gastrulation. require proximity to ectoderm to express several muscle- specific genes. We show that ventral ectoderm induces Key words: Drosophila, induction, myogenesis, cell signalling

INTRODUCTION larval muscles, it is not known when muscle determination occurs. One possibility is that differential nuclear uptake of the Induction in amphibian embryos has been originally described dorsal morphogen within the presumptive mesoderm deter- by Spemann and Mangold (1924), and has since been studied mines different myogenic fates, just as it specifies the fate of in many species. In vertebrate embryos, cell-cell interactions the presumptive ectoderm, mesectoderm, and mesoderm between the mesoderm and ectoderm are required for the (Kosman et al., 1991; Ray et al., 1991; St. Johnston and proper development of both germ layers. For instance, the Nüsslein-Volhard, 1992). Conversely, it is possible that muscle mesoderm induces ectoderm to generate the neural plate during determination is regulated by inductive interactions between gastrulation (Nieuwkoop et al., 1985). Subsequent to this germ layers and/or between mesoderm cells themselves. For primary induction, the neuroectoderm controls somite organ- example, the adult male strap flight muscle acquires its identity ization (Nicolet, 1970; Lipton and Jacobsen, 1973). Studies on by being innervated by specific neurons (Lawrence and insect embryos have revealed similar inductive interactions Johnston, 1986). However, this is not a general mechanism for between the mesoderm and ectoderm. Seidel first demonstrated muscle determination; larval muscles differentiate normally in that ectoderm induces muscle development in the underlying prospero mutant embryos in which muscle innervation does mesoderm, and that mesoderm is required for organization, but not occur (Broadie and Bate, 1993). not histogenesis, of the ventral nerve cord (Seidel et al., 1940; Here we demonstrate that specific regions of the ectoderm Bock, 1942). Subsequent investigations have supported the induce the expression of different myogenic genes in paradigm that ectoderm, not mesoderm, behaves as the primary Drosophila embryos. We arrest gastrulation in wild-type inducer of insect organogenesis (Kühn, 1971; Counce, 1973). embryos so that presumptive mesoderm cells remain on the Upon cellularization of the Drosophila blastoderm, the ventral surface, preventing their interaction with ectoderm. The mesoderm invaginates into the interior of the embryo and then patterns of muscle morphogenesis and of muscle gene spreads uniformly along the basal surface of the ectoderm (Leptin expression in these arrested embryos indicate that: (1) and Grunewald, 1990). Gastrulation therefore allows interactions mesoderm must be near ectoderm cells to express muscle- between the ectoderm and mesoderm; however, it is not known specific genes; (2) hindgut and dorsal ectoderm independently to what extent such interactions regulate the expression of induce visceral muscle development; (3) dorsal ectoderm myogenic determinants within the mesoderm. During germ band induces heart development; (4) ventral ectoderm promotes extension, the mesoderm remains undifferentiated, but begins to somatic muscle development. differentially express muscle-specific genes (Michelson et al., 1990; Paterson et al., 1990; Azpiazu and Frasch, 1993; Bodmer, 1993). Meanwhile the mesoderm subdivides into three morpho- MATERIALS AND METHODS logically distinct sets of muscle progenitors: the somatopleura (presumptive somatic muscle), the splanchnopleura (presumptive Fly stocks, embryo collections, and developmental visceral muscle) and cardiac muscle precursors (Campos-Ortega staging and Hartenstein, 1985; Bate, 1993). Wild-type embryos were collected from the ‘Sevelen’ stock. Enhancer Because no one has yet elucidated a blastoderm fate map for trap line A490.2M3 (Bellen et al., 1989), which expresses lacZ in the 1388 R. Baker and G. Schubiger midgut, was kindly provided by W. Gehring. For all genotypes, eggs CYT blocks actin polymerization, and can inhibit microfila- were collected from females aged 3-10 days after eclosion. Overaged ment-dependent cell movements during a brief period of time embryos were eliminated by precollecting eggs on food bottles for prior to the onset of gastrulation; injection of CYT before 180 one hour, followed by three additional 20-minute precollections on minutes after egg laying (AEL) inhibits cellularization, and agar plates flavored with cantaloupe juice and yeast pellets. Collec- later than 200 minutes AEL does not inhibit normal develop- tions were made during 10-minute intervals. ment (data not shown). The effectiveness of CYT varies Here we express the age of experimental and control embryos in terms of real time after egg laying (AEL). The standard classification between injected embryos, owing to variables inherent in our of embryos by 17 developmental stages (e.g. Campos-Ortega and methods (e.g. the volume of CYT injected, the distribution of Hartenstein, 1985) is based upon morphological characteristics that drug within the embryo, and the time of injection). Therefore, do not apply to gastrulation-arrested embryos; thus, we do not use a CYT-injected embryos may exhibit complete, partial, or no staging scheme to describe them. gastrulation movements, and accordingly can be classified into five distinct morphological groups (Fig. 1). Embryo injections and video recordings 24% (106/435) of CYT-injected embryos, which we term Synchronously staged embryos were manually dechorionated, aligned, arrested embryos, do not gastrulate and retain the general mor- and injected as described in Baker et al. (1993). Dechorionated embryos phology of a cellular blastoderm (Fig. 2A-D). We have were kept in humid chambers at room temperature (21.5-23.0¡C) until analyzed the development of arrested embryos in time-lapse 3 hours AEL, then they were desiccated for 5-7 minutes, covered with Halocarbon oil, and injected with 250 µg/ml cytochalasin D (Sigma) video recordings. Because CYT inhibits cytokinesis but not between 190 and 200 minutes AEL. For each injection slide, 3-5 control DNA synthesis (Whittaker, 1979; unpublished observations), embryos were not injected, but otherwise processed identically. blastomeres enlarge with time, making it impossible for all Embryos were incubated at 25¡C after injection until they reached the cells to remain at the cortex. Within minutes after cellulariza- desired age, then rinsed in heptane and fixed in 4% EM-grade tion is completed, individual cells throughout the embryo paraformaldehyde (Electron Microscopy Sciences) in PBS. Embryos were manually devitellinized with tungsten needles under a PBS. Time-lapse video recordings of live gastrulation-arrested embryos Morphological Class Frequency were made by placing injection slides on a Nikon microphot mounted with a MTI 65 camera hooked up to a Gyyr time-lapse video recorder. Embryos were observed under differential interference contrast (DIC) optics in order to distinguish the position of cortical nuclei. A Acellular 3.4 RNA and protein labeling We used digoxigenin-labeled DNA probes to detect RNA in whole embryos using the standard procedure (Ashburner, 1989). Probes were made and detected with the materials provided in the Genius kit (Boehringer-Mannheim). B Arrested 24.4 For antibody labeling, we blocked embryos in PBTB (PBS plus 0.01% Triton X-100 and 3% BSA) for 1 hour, then incubated them with primary antibodies overnight at 4¡C. Primary antibodies were diluted in PBTB in the following proportions: 1:1000 goat anti-horseradish peroxidase (USB); 1:500 mouse anti-β-galactosidase (Promega); 1:500 rat anti-elav (S. Robinow); 1:200 rabbit anti-nautilus C Invaginated 4.6 and 1:200 rabbit anti-twist (B. Paterson); 1:250 rabbit anti-β3-tubulin (R. Renkawitz-Pohl); 1:40 mouse anti-D4.1 (R. Fehon); 1:100 rabbit anti-vestigial (L. Maves). Embryos were rinsed in PBTB for an hour, then incubated with the appropriate biotin- or fluorochrome-conjugated secondary antibodies (Jackson ImmunoResearch) diluted 1:200 D Partially 61.4 in PBT for either 4 hours at 22¡C or overnight at 4¡C. Gastrulated Biotinylated secondary antibodies were labeled by the standard immunolabeling procedure (Ashburner, 1989) and photographed with a Nikon microphot. Fluorescently labeled embryos were dehydrated in methanol and mounted in a 1:2 mixture of benzyl benzoate and benzyl alcohol (Sigma Immunochemicals), and imaged on a Bio-Rad E Unaffected 6.2 MRC 600 confocal microscope. Photographic images were processed with Adobe Photoshop 2.5 (Adobe Systems Inc.) to normalize bright- ness and sharpen image resolution. Images were assembled and sized in Canvas 3.0.2 (Deneba Software), a program that we also used for Fig. 1. 435 CYT-injected embryos, fixed between 6 and 10 hours generating scale bars, arrows, and text. All photographs were printed AEL, were categorized into five different morphological classes. The by a Tektronix Phaser IISDX dye sublimation printer. percentage frequency for each class is given next to its schematic representation. (A) Acellular embryos fail to develop and show pycnotic nuclei and large yolk vesicles. (B) Arrested embryos RESULTS complete cellularization but fail to undergo morphogenetic movements. (C) Invaginated embryos form a ventral furrow and internalize their presumptive mesoderm. However, germ band Morphology of gastrulation-arrested embryos elongation and dorsal closure do not occur. (D) Partially gastrulated We arrested gastrulation movements in Drosophila embryos to embryos exhibit normal morphogenesis in some regions. varying degrees by injecting them with cytochalasin D (CYT). (E) Unaffected embryos appear to develop normally. Muscle induction in Drosophila 1389 ingress into the yolk, causing the cortex to thicken from a degree of physical interactions between mesoderm and monolayer to a multilayer of cells. However, cells do not shift ectoderm than do arrested embryos, which accounts for their positions relative to the axes, so that limited contact striking differences in their myogenic development (see between different germ layer primordia occurs in arrested below). embryos (indicated in Fig. 3B, D, and F). Cell enlargement and cortical thickening are most extensive in the lateral regions, or Germ layer-specific gene expression in arrested presumptive neuroectoderm; the smallest cells are situated at embryos the dorsal midline, or amnioserosa primordium. Thus, the Preliminary to investigating the role of induction between germ ultimate size of a cell appears to be proportional to the number layers during muscle development, it was necessary to verify of cell divisions it would normally undertake: neuroblasts that the developmental commitment of the germ layers is not divide upwards of eight times, while amnioserosa cells do not altered in gastrulation arrested embryos. Towards this end we divide (Campos-Ortega and Hartenstein, 1985). As a conse- probed arrested embryos for antigens uniquely expressed quence of nonuniform cell growth, arrested embryos progres- throughout either ectoderm, endoderm, or mesoderm. sively lose their blastoderm-like appearance with time. About 5% (20/435) of CYT-injected embryos invaginate a Ectoderm ventral furrow but fail to extend the germ band; we term these We have probed arrested embryos with antibodies against horse- invaginated embryos (Fig. 2E,F). Like control embryos, the radish peroxidase (HRP) and elav, which respectively label the mesoderm of invaginated embryos spreads dorsally beneath cell membranes and nuclei of all neurons (Jan and Jan, 1982; the ectoderm. Invaginated embryos therefore exhibit a greater Robinow and White, 1991). At 10 hours AEL, anti-HRP labels

Fig. 2. Blastoderm-stage and CYT-injected embryos were fluorescently labeled with rhodamine-conjugated phalloidin to outline cell surfaces. Lateral (A,C,E) and parasagittal (B,D,F) optical sections are shown for each embryo; dorsal side is up, anterior is left. Scale bar, 50 µm. (A,B) 3.5 hour AEL control embryo during the final stages of cellularization. Embryos are injected with CYT at this stage to inhibit gastrulation. (C,D) A 6 hour AEL arrested embryo retains a blastoderm-like morphology. The larger cells in the ventrolateral region are neuroblasts (compare with Fig. 3B). (E,F) 6 hour AEL invaginated embryo has completed only the initial stages of morphogenesis. The invaginated mesoderm (ms) and partially involuted posterior midgut (pmg) are indicated in the interior of the embryo. 1390 R. Baker and G. Schubiger the brain, ventral nerve cord, and lateral sensory organs of control arrested embryos, suggesting that the temporal program of embryos (Fig. 3A). In arrested embryos fixed 9-12 hour AEL, midgut development does not require gastrulation. anti-HRP labels two ventrolateral bands of cells, one on either side (Fig. 3B). Both neuronal bands extend roughly between 15% and 90% embryo length (EL), with an average width of 7 cells. The size and position of these bands match the domains of the neurogenic regions on the blastoderm fate map (Poulson, 1950; Campos- Ortega and Hartenstein, 1985). Arrested embryos labeled with anti-elav antibody show a similar lateral distribution of neuronal cells (Fig. 4D). We seldom observe neuronal cells within the dorsal ectoderm region, where the sensory neurons originate (Campos-Ortega and Harten- stein, 1985). 46 CYT-injected embryos were allowed to develop under oil for 30 hours, and analyzed for the presence of cuticle. One embryo generated a normal cuticle and 29 deposited varying amounts of disorganized cuticle consisting of either dorsal hairs only, or dorsal hairs and ventral denticles (data not shown). Because the method we used for visualizing cuticle destroys the soft tissues, we could not unam- biguously determine whether or not a given cuticle was derived from an arrested or an invaginated embryo. Nevertheless, the determination of dorsal epidermis clearly requires neither normal gastrulation movements nor the presence of mesoderm, as is indicated by the presence of dorsal cuticle in Fig. 3. Control (A,C,E) and arrested (B,D,F) embryos were labeled for various germ layer-specific ‘dorsalized’ mutant embryos markers. All embryos are shown in lateral perspective, dorsal side up. (A,B) 10-hour AEL embryos labeled with anti-HRP antibody. Control embryo (A) shows neural signal in the brain, ventral nerve (Nüsslein-Volhard et al., 1984). cord, and peripheral nervous system. The distribution of anti-HRP label in an arrested embryo (B) Endoderm corresponds to the blastoderm fate map positions of the ventral neurogenic region and brain (compare with G). (C, D) 7-hour AEL embryos collected from enhancer trap line A490.2M3 and labeled with Embryos derived from enhancer antibody against β-galactosidase (β-gal). This line expresses β-gal in the midgut of control embryos trap line A490.2M3 express β- (C), and at the poles in arrested embryos (D). These polar regions correspond to the fate map position of galactosidase (β-gal) within the the endodermal midgut primordia. (E,F) 12-hour AEL embryos labeled for mesoderm with antibody midgut (Bellen et al., 1989; Fig. against β3-tubulin. Control embryo (E) shows label in macrophages and somatic muscle; heart and 3C). In arrested A490.2M3 visceral muscle staining are not evident in this focal plane. Arrested embryo (F) expresses β3-tubulin in embryos, β-gal is expressed at the midventral region, corresponding to the fate map position of the presumptive mesoderm. The dorsal signal may represent presumptive neurons in the brain that normally express this protein. Scale bar, 50 the anterior and posterior poles µ (Fig. 3D), corresponding to the m. (G) Blastoderm fate map of a wild-type embryo, indicating the approximate positions of the following tissue primordia: amnioserosa (as), anterior midgut (amg), dorsal ectoderm (de), foregut (fg), blastoderm fate map positions hindgut (hg), mesoderm (ms), posterior midgut (pmg), procephalic neurogenic region (pNR) and ventral of the presumptive midgut. The neurogenic region (vNR). Illustration is based on fate maps constructed by Poulson (1950) and earliest we detect β-gal is 6 Campos-Ortega and Hartenstein (1985). 0% EL and 100% EL correspond to the posterior and anterior hours AEL in both control and poles, respectively. Muscle induction in Drosophila 1391

Mesoderm characteristic for somatic muscle precursors (Bate, 1990), We used antibodies against twist and β3-tubulin to identify although most are undifferentiated. mesoderm cells in arrested embryos. Mesoderm cells initially express twist before gastrulation begins (Thisse et al., 1988), Muscle development and myogenic gene expression and continue to do so until they differentiate (Bate et al., 1991). in arrested embryos As mesoderm cells differentiate, they express β3-tubulin (Leiss The three major larval myotypes (somatic, visceral, and cardiac et al., 1988; Fig. 3E). From 4 to 9 hours AEL, arrested embryos express twist protein in a midventral domain situated between 10% and 85% EL (Fig. 4A). From 10 to 14 hours AEL, β3-tubulin is expressed within this domain (Fig. 3F). Up to 10% of β3-tubulin-labeled cells lie beneath the surface of ventral and dorsal ectoderm. We presume that these cells had individually migrated beneath the ectoderm before assuming the spindle-shaped morphology of somatic muscle precursors (Fig. 4B) or syncytial myoﬁbers (Fig. 4C). However, the majority of presumptive mesoderm cells, particularly those within the midventral domain, do not differentiate. It has been shown, however, that cells committed to myogenic development express muscle myosin heavy chain (MHC), regardless of their ability to properly differentiate as muscle (Corbin et al., 1991; Bate et al., 1993). Thus, in order to identify myogenic cells in arrested embryos, we labeled them with anti-MHC antibody. We ﬁnd that MHC is primarily expressed in two broad, longitudinal bands of mesoderm cells situated near the mesoderm/ectoderm boundary (Fig. 4D). Some of these cells extrude projections

Fig. 4. Arrested embryos were examined at progressively later stages of development with mesoderm and muscle probes. Gradual and inevitable deviations from the blastoderm-like morphology accumulate with age. (A) Ventrolateral perspective of a 9-hour AEL arrested embryo labeled for twist. Mesoderm cells have remained on or beneath the ventral surface. (B,C) Lateral perspectives of older arrested embryos labeled with antibody to β3- tubulin. At 10 hours AEL (B), some mesoderm cells beneath the lateral ectoderm (black arrow) generate cone-shaped projections typical of somatic muscle precursors (Bate, 1990), while mesoderm cells closer to the ventral midline (towards the bottom of the figure) remain spherical (white arrow); most of these undifferentiated mesoderm cells do not express muscle myosin. By 12 hours AEL (C), some arrested embryos exhibit differentiated muscles beneath the ectoderm. (D) Ventral perspective of a 15-hour AEL arrested embryo double labeled for elav (red) and MHC (green). MHC is expressed mostly by lateral mesoderm cells near the ectoderm border. Scale bars, 50 µm; A,C and D are shown to the same scale. 1392 R. Baker and G. Schubiger muscle) become morphologically distinct several hours after Invaginated embryos aged 7.5-9.5 hour AEL express nau in gastrulation begins (Campos-Ortega and Hartenstein, 1985; 11 transverse bands of cells beneath the ventral ectoderm (Fig. Bate, 1993). Since most muscle cells do not differentiate in 5C). These bands are situated between 10% and 70% EL, and arrested embryos, we probed arrested embryos for myotype- correspond to the fate map positions of the 11 thoracic and specific markers. abdominal segments that normally express nau at this time. We Somatic muscle Somatic muscle differentiation begins as muscle founder cells fuse with neighboring, ‘fusion- competent’ mesoderm cells, which results in the formation of syncytial myotubes (Bate, 1990). During germ band retraction (7.5-9.5 hours AEL), nau is expressed in a subset of somatic muscle founders of the thorax and abdomen (Michelson et al., 1990; Paterson et al., 1990; Fig. 5A). In 7.5-9.5 hour AEL arrested embryos, nau is expressed in two narrow, punctuated rows of cells near the ectoderm border (Fig. 5B). This pattern is similar to that of MHC expression (Fig. 4D), except that there are fewer labeled cells. The distribution of nau-expressing cells is variable between arrested embryos, each row consisting of 5-15 cells located between 10% and 70% EL. Double-labeling with anti-nau and anti-HRP antibodies indicates that although some muscle founder cells directly abut neurons, most are one or two cell diameters from the apparent ectoderm/mesoderm border. Two explanations may account for the observation that nau is expressed only near the border with the ectoderm: (1) ectoderm cells induce mesoderm to express nau, or (2) somatic muscle precursors originate from presumptive mesoderm cells bordering the ventral ectoderm. To distinguish between these alternatives we have analyzed nau expression in invaginated embryos, the class of CYT- injected embryos that complete ventral furrow formation (Fig. 2E,F). If ectoderm induces nau expression, we would expect that the greater the degree of mesoderm Fig. 5. Ventral perspectives of 8- to 9-hour AEL embryos double labeled with anti-HRP (red) and invagination, the more somatic anti-nautilus (green) antibodies. (A) Control embryo shows dorsal (d) and ventral (v) clusters of muscle founder cells should appear. somatic muscle founder cells of the thorax and anterior abdomen; not all lateral (l) nau-expressing Conversely, if nau-expressing cells are evident in this focal plane. (B) Arrested embryo expresses nau in a small subset of mesoderm cells near the ventral ectoderm boundary (indicated by anti-HRP-labeled cells). muscle founder cells are derived Relatively few nau-expressing cells directly abut ectoderm, since mesoderm and ectoderm are from a fixed number of progenitor separated by a narrow row of mesectoderm cells (not shown). (C) A composite image of an cells, we would not expect different invaginated embryo shows neurons and muscle founders in two different focal planes; ventral numbers of these founder cells ectoderm occupies the ventral surface, while nau-expressing cells lie 3-5 µm beneath it. From this between the two classes of CYT- perspective 11 transverse bands of muscle founder cells (aligned with the yellow dashes) can be injected embryos. distinguished. Scale bar, 50 µm. Muscle induction in Drosophila 1393 counted between 140 and 185 nau-expressing cells per invagi- possess approximately the same number of cells, based on cell nated embryo (n = 12 embryos), which is approximately one- counts of α-tubulin-labeled injected embryos (data not shown). quarter the number present in uninjected controls (Michelson Therefore, the greater number of nau-expressing cells in et al., 1990; our own observations). To a large degree, this invaginated embryos (140-185) relative to arrested embryos numerical difference must reflect the greater number of (10-30), correlates primarily to their degree of contact between mesoderm and ectoderm cells in controls relative to CYT- ventral ectoderm and mesoderm. Moreover, the different injected embryos. Arrested and invaginated embryos, however, patterns of nau-expressing cells in these two classes of

Fig. 6. (A-D) 9- to 10-hour AEL embryos probed for visceral muscle markers. (A) Lateral perspective of a control embryo labeled for H2.0 RNA. Signals in visceral mesoderm (between asterisks) and around tracheal pits (arrows) are indicated. (B) Lateral perspective of an arrested embryo with H2.0 signal in posterior mesoderm, near the presumptive hindgut. Label in anterior mesoderm or in trachea progenitors is never detected in arrested embryos. (C) Horizontal perspective of a control embryo from enhancer trap line A490.2M3. β-gal is expressed in the visceral mesoderm of the midgut and in some neurons in head and peripheral nervous system. (D) Horizontal perspective of an invaginated embryo from line A490.2M3. β-gal is expressed in noncontiguous rows of visceral mesoderm beneath the dorsal epidermis. Note that posterior midgut cells (pmg) have migrated anteriorly. Anterior midgut (amg), hindgut (hg), and yolk (yk) are also indicated. (E-H) Embryos probed for tin RNA. (E) Lateral perspective of 6.0 hour AEL control embryo that expresses tin in visceral (v) and cardiac (c) muscle precursors in a segmentally reiterated pattern. Signal in the foregut (fg) is also evident. (F) Lateral perspective of a 5-hour AEL arrested embryo. tin RNA is detected in prospective mesoderm (m) and foregut ectoderm (fg). (G) Ventral perspective of 6.5-hour AEL arrested embryo shows tin signal in the prospective foregut. tin RNA can no longer be detected in the mesoderm. (H) Ventrolateral perspective of 6.5-hour AEL invaginated embryo; the line indicates the approximate position of the ventral midline. Expression is strong in the mesoderm cells beneath the dorsal ectoderm. The lower, out-of-focus row of tin-expressing cells lie on the far side of the embryo. Scale bars, 50 µm; all figures except D are shown to the same scale as A. 1394 R. Baker and G. Schubiger embryos (longitudinal rows versus transverse stripes) provides A176.1M2 (Bellen et al., 1989). This line expresses β-gal in further indication that ectoderm cells induce nau expression in the visceral muscle of the midgut, but not of the hindgut (Fig. the mesoderm. 6C). None of the arrested embryos from this stock (n = 87) express β-gal in the presumptive mesoderm. However, invagi- Visceral muscle nated A176.1M2 embryos express β-gal in mesoderm cells Although the visceral muscle of the hindgut and midgut situated beneath dorsal ectoderm (Fig. 6D). In some cases, develop independently and express different sets of genes posterior midgut cells had invaginated anteriorly along a track (Bodmer, 1993; Tepass and Hartenstein, 1994), both express of visceral mesoderm (Fig. 6D), as they do in control embryos the homeobox gene H2.0 (Barad et al., 1988). H2.0 RNA is (Tepass and Hartenstein, 1994). These results suggest that first detected about 5.5 hours AEL in mesoderm around the visceral muscle of the midgut is induced by dorsal ectoderm. hindgut, and later (beginning 6.5 hours AEL) is expressed in the visceral muscle of the midgut (Fig. 6A). In arrested Cardiac muscle embryos aged between 5.5 and 10 hours AEL, H2.0 transcripts tinman (tin) encodes a homeobox protein required for the are detected only in a few presumptive mesoderm cells situated differentiation of both cardiac and visceral muscles (Bodmer, around 10% EL (Fig. 6B). These H2.0-expressing cells are 1993; Azpiazu and Frasch, 1993). tin is initially transcribed positioned near the presumptive hindgut (compare with Fig. throughout thoracic and abdominal mesoderm of blastoderm- 3G), and begin to express H2.0 at the same time as the visceral stage embryos (3.5 hours AEL). During gastrulation, however, mesoderm of the hindgut does in control embryos. During tin RNA disappears first in mesodermal cells beneath the normal development, the visceral musculature of the midgut is ventral ectoderm (5 hours AEL), and later in visceral muscle derived from mesoderm cells situated throughout the precursors (6.5 hours AEL). During germ band retraction and metameric germ band (Azpiazu and Frasch, 1993), whereas the dorsal closure (7-12 hours AEL), tin RNA is transcribed in visceral muscle of the hindgut appears to be derived only from cardiac muscle precursors. tin is also expressed in the foregut posterior mesoderm cells. Thus, the failure of the presumptive between 4 and 8 hours AEL (Fig. 6E). mesoderm to express H2.0 throughout most of the germ band We have assayed for tin RNA in arrested embryos at various suggests that hindgut-specific, but not midgut-specific, visceral times. Between 4.5 and 6 hours AEL, tin is expressed in pre- muscle is determined in arrested embryos. sumptive foregut and in a strip of midventral presumptive To test this, we arrested embryos from the P[lacZ] stock mesoderm (Fig. 6F). Around 6 hours AEL, the tin signal dimin-

Fig. 7. (A) 12-hour AEL Toll10B embryo labeled for MHC, shown in two different optical planes. MHC-expressing cells are generally undifferentiated. (B) Same embryo as in A, focused in deeper optical plane. MHC is depicted in green; the red counter stain is intended to highlight the yolk. Note that the yolk is clearly surrounded by gut (which is not labeled); in turn, the gut is surrounded by mesoderm. The arrow points to differentiated visceral muscle that adheres to the anterior edge of the gut. (C) 11-hour AEL wild-type embryo labeled for D4.1, shown in a midsagittal optical section. D4.1 is strongly expressed by ectoderm epithelia, including the foregut (fg), hindgut (hg), and epidermis (cells on the embryonic surface). The midgut (mg) weakly expresses this antigen, and mesoderm cells do not express it. (D) 11-hour AEL Toll10B embryo double labeled for D4.1 (red) and vestigial (green). Numerous epidermal cells occupy the apical surface. vestigial is normally expressed in a subset of somatic muscles and neurons (Williams et al., 1991). Arrow and arrowhead indicate syncytial vestigial-expressing cells with 3 and 4 nuclei, respectively, which may be syncytial muscle cells. Scale bar, 50 µm. Muscle induction in Drosophila 1395 ishes in arrested embryos, and is undetectable in the presump- hemisegment is cauterized, the mesoderm interacts only with tive mesoderm by 7 hours AEL, although foregut-specific ventral ectoderm, and generates somatic muscle; these signal remains strong (Fig. 6G). Conversely, mesodermal tin embryos do not differentiate heart or visceral muscle. If the expression persists until 9 hours AEL in invaginated embryos. ventral ectoderm is ablated instead, heart and visceral muscles Between 7 and 9 hours AEL, tin RNA is present in bilateral, develop beneath the dorsal ectoderm, and few somatic muscles punctuated rows of cells lying beneath the dorsal ectoderm differentiate. (Fig. 6H). In one collection of CYT-injected embryos, three The distribution of cells that express nau, H2.0, and tin in invaginated embryos show a pattern of tin expression similar gastrulation-arrested Drosophila embryos similarly suggests to the embryo in Fig. 6H, whereas 34 arrested embryos show that dorsal and ventral ectoderm differentially induce undeter- tin signal only in the presumptive foregut. These observations mined mesoderm cells to make specific myotypes. This idea indicate that proximity to the dorsal ectoderm is necessary for was previously suggested by the results of cell transplantation sustaining tin expression in the mesoderm after 7 hours AEL. experiments of Beer et al. (1988). They removed dye-labeled mesoderm cells from the midventral furrow of early gastrulat- ing embryos and individually transplanted them into cellular DISCUSSION blastoderm-stage hosts, and monitored their subsequent development. In these experiments, clones of mesoderm cells peri- The role of dorsal and ventral ectoderm as inducers of somatic odically gave rise to mixed populations of somatic and visceral and splanchnic mesoderm, respectively, appears to be muscle, or of somatic muscle and fat. This result suggests that conserved between distantly related insect species. This at the onset of gastrulation, muscle versus nonmuscle determi- paradigm was originally demonstrated by experiments on pre- nation has not yet occurred. Furthermore, the types of muscles gastrula stage Chrysopa (lacewing fly) embryos (Seidel et al., generated by these clones strongly correlated with the site of 1940; Bock, 1942). When the dorsal ectoderm of a given transplantation into the host, irrespective of their original

Fig. 8. Schematic representation of transverse sections of Drosophila embryos at different developmental stages. Embryos are oriented dorsal surface up; for simplification, the morphological contortions caused by germ band extension are not depicted. See text for details. (A) During stage 9 (4.5 hours AEL) the invaginated mesoderm spreads laterally and dorsally along the basal surface of the ectoderm. Mesoderm juxtaposed to the dorsal ectoderm are induced by dpp to maintain tin expression (blue arrows). Mesoderm juxtaposed to the ventral ectoderm are not induced in this manner, and eventually cease to express tin. (B) In stage 11 (6.0 hours AEL), some dorsal ectoderm (lateral epidermis) cells express an unidentified signal that induces ventral tin-expressing mesoderm cells to develop as midgut-specific visceral muscle (green). These cells soon separate from the lateral epidermis and discontinue to express tin. (C) By stage 12 (7.5 hours AEL) dpp-expressing cells in the dorsal ectoderm induce underlying mesoderm cells to maintain tin expression and become cardioblasts. Meanwhile, a subset of the somatopleura is induced (black arrows) by the ectoderm to develop as somatic muscle founders (red cells). These cells in turn laterally inhibit (red crossbars) their ‘fusion competent’ neighbors (yellow cells) from likewise becoming muscle founders via a Notch-dependent signal transduction pathway. 1396 R. Baker and G. Schubiger position within the donor embryo. For example, mesoderm protein (Staehling-Hampton et al., 1994; M. Frasch, personal cells transplanted into the dorsal ectoderm region of the host communication). dpp is a member of the TGF-β family of most frequently gave rise to visceral muscle, while those trans- secreted growth factors that induce dorsal mesoderm in planted into the anterior midgut primordium predominantly Xenopus embryos (Green et al., 1990). Although dpp is gave rise to esophageal muscle. These observations, like those expressed throughout the dorsal ectoderm during germ band presented here, indicate that muscle determination is largely extension, around 7 hours AEL it becomes restricted to two dependent on signals provided by the ectoderm. narrow stripes of ectoderm: one longitudinal row of dorsal cells In order to test our hypothesis by genetic means, we that border the amnioserosa, and a row of ventrolateral cells analyzed myogenesis in embryos derived from heterozygous bordering the neurogenic ectoderm (St. Johnston and Gelbart, Toll10B females (henceforth called ‘Toll10B embryos’), which 1987; Ray et al., 1991). It remains unclear why maintenance reportedly lack ectodermal derivatives (Erdélyi and Szabad, of dpp expression in the dorsal stripe, but not the ventrolateral 1989). If inductive signals from the ectoderm induce myogen- stripe, induces latent dpp expression in the underlying esis, then Toll10B embryos should neither express myogenic mesoderm. genes nor differentiate muscle. We observed, however, that at Another signal transduction pathway governed by the neu- 12 hours AEL Toll10B embryos express MHC in a large number rogenic genes (e.g. Notch and Delta) plays a critical role in of cells, some of which are differentiated as muscle (Fig. Drosophila myogenesis. Embryos lacking function for any of 7A,B). This observation prompted us to assay these embryos these genes exhibit developmental abnormalities in all germ for the presence of ectoderm. For this purpose we used an layers (Poulson, 1945; Hartenstein et al., 1992). The most antibody against D4.1 (Fig. 7C), the product of the coracle obvious defects are present in the ectoderm, which develops a gene, which is normally expressed in the epidermis, hindgut, grossly hypertrophied nervous system at the expense of ventral and foregut (Fehon et al., 1994). We found that all Toll10B epidermis (Lehmann et al., 1983). These defects seem to be embryos assayed express D4.1 throughout the apical surface mirrored in the underlying mesoderm, which exhibits a hyper- beginning at 9 hours AEL (Fig. 7D), indicating a substantial trophy of somatic muscle founder cells and a paucity of fused presence of ectoderm in these embryos. Furthermore, roughly myotubes (Corbin et al., 1991; Bate et al., 1993). One 30% of Toll10B embryos (17/56) deposit some larval cuticle important question is whether these muscle-specific defects are (data not shown), which is secreted by the epidermis. The due to the loss of neurogenic gene function within the abnormal development of both ectoderm and mesoderm in mesoderm itself, or whether the altered ectoderm fails to these embryos is consistent with the hypothesis that ectoderm provide the appropriate signals for muscle development. To induces muscle development in the underlying mesoderm; address this question, we have investigated the germ layer- however, further investigation is necessary to verify a causal specific requirements for Notch protein. In embryos that relationship in this case. possess Notch function in the mesoderm but not in the ectoderm, only certain aspects of myogenic development are Signal peptides that induce mesoderm in normalized relative to mutant embryos (manuscript in prepa- vertebrates may induce muscle in Drosophila ration). For instance, these embryos do not exhibit a hypertro- Although somatic muscle founders normally abut ectodermal phy of somatic muscle founders, and myoblast fusion occurs cells directly (Bate, 1990), in arrested embryos they generally to a significant degree (Baker, Ph.D dissertation), suggesting lie 10 µm from the apparent ventral ectoderm boundary (Fig. that Notch is required in the mesoderm for muscle differen- 5B). Mesectoderm cells most likely occupy the space between tiation. However, the distribution of both muscle founders and the nau-expressing cells and the neurogenic region; arrested fused myotubes are abnormal in these embryos, which suggests embryos express the mesectoderm-specific gene slit in two that Notch function in the ectoderm (and, by extension, normal rows of single cells along the ventral edge of the neurogenic ectodermal development) is required for proper muscle organ- ectoderm (unpublished observation). It therefore seems likely ization. that ectoderm induces somatic muscle precursor development by a secreted signal with an effective range of two or three cell A model for muscle induction in Drosophila diameters. embryos One candidate for this signal is fibroblast growth factor On the basis of observations presented here and in previous (FGF), which induces mesoderm formation in vertebrate reports, we suggest a working model for muscle induction in embryos (Kimelman et al., 1988). Although a homologue of Drosophila (summarized in Fig. 8). The mesoderm is initially FGF has yet to be discovered, Drosophila embryos express a pluripotent (Beer et al., 1988) until about 4.5 hours AEL (stage homologue of the FGF receptor, called DFR1, in the somato- 9), when it forms a uniform layer of cells beneath the ectoderm pleura preceding and during myoblast fusion (Shishido et al., (Fig. 8A). Shortly afterwards, the ventral mesoderm terminates 1993). DFR1 is expressed in large clusters of mesoderm cells tin transcription, since its transcriptional maintenance requires in a pattern similar to the wild-type distribution of nau-express- a signal from the dorsal ectoderm (Fig. 6), which has been pos- ing clusters. A deficiency of the 90D-E region, which includes itively identified as dpp (Staehling-Hampton et al., 1994; M. the DRF1 locus, results in greatly reduced nau expression and Frasch, personal communication). somatic muscle differentiation (Shishido et al., 1993). The subsequent divergence of splanchnic and cardiac pre- Our results also suggest that a signal from the dorsal cursors during stage 11 probably involves another inductive ectoderm induces latent expression of tin; only mesoderm cells event (Fig. 8B). Around 6.5 hours AEL (stage 11), the ventral- that are juxtaposed to dorsal ectoderm continue to express tin most tin-expressing cells ingress towards the yolk, cease to beyond 7 hours AEL (Fig. 6). Recent evidence strongly express tin, and differentiate as visceral muscle. Meanwhile, suggests that this inductive signal is the decapentaplegic (dpp) the dorsal-most tin-expressing cells maintain tin expression Muscle induction in Drosophila 1397 and remain undifferentiated until the completion of dorsal R. and Gehring, W. J. (1989). P-element-mediated enhancer detection: a closure, when they assemble into the heart. The proximate versatile method to study development in Drosophila. Genes Dev. 3, 1288- cause for this differential behavior of visceral and cardiac 1300. Bock, E. (1942). Wechselbeziehung zwischen den Keimblättern bei der muscle does not appear to be the inactivation of tin transcrip- Organbildung von Chrysopa perla. Wilhelm Roux Arch. EntwMech. Org. tion, since the splanchnic muscle precursors still possess tin 141, 159-247. RNA after they segregate away from the lateral epidermis Bodmer, R. (1993). The gene tinman is required for specification of the heart towards the gut (Bodmer, 1993). and visceral muscles in Drosophila. Development 118, 719-729. Broadie, K. and Bate, M. (1993). Muscle development is independent of Between 8 and 10 hours AEL (stage 12), prospective muscle innervation during Drosophila embryogenesis. Development 119, 533-543. founder cells are induced by short-range signals derived from Campos-Ortega, J. A. and Hartenstein, V. (1985). The Embryonic the ectoderm, possibly an FGF homologue (Fig. 8C). DFR1, an Development of Drosophila melanogaster. Berlin: Springer-Verlag. FGF-receptor homologue, is expressed in a segmentally reiter- Corbin, V., Michelson, A. M., Abmayr, S. M., Neel, V., Alcomo, E., ated pattern within the mesoderm that prefigures the ventral, Maniatis, T. and Young, M. W. (1991). A role for the Drosophila neurogenic genes in mesoderm differentiation. Cell 67, 311-323. lateral, and dorsal somatic muscle clusters (Shishido et al., Counce, S. J. (1973). The causal analysis of insect development. In 1993), and may be necessary for founder cell determination. Developmental Systems: Insects (Vol. 2), (ed. S. J. Counce and C. H. However, the final arrangement and number of founders are Waddington), pp. 1-156. London: Academic Press. apparently regulated by cell-cell interactions between prospec- Erdélyi, M. and Szabad, J. (1989). Isolation and characterization of dominant tive muscle founder cells themselves (R. Baker, Ph.D disserta- female sterile mutations of Drosophila melanogaster. I. Mutations on the third chromosome. Genetics 122, 111-127. tion). Thus, somatic muscle development probably involves at Fehon, R. G., Dawson, I. A. and Artavanis-Tsakonas, S. (1994). A least three different inductive pathways: first, ectoderm induces Drosophila homologue of membrane-skeleton protein 4.1 is associated with somatopleura with the competence to generate muscle founders; septate junctions and is encoded by the coracle gene. Development 120, 545- second, potential muscle founders limit their own numbers by 557. Green, J. B., Howes, G., Symes, K., Cooke, J. and Smith, J. C. (1990). The lateral inhibition; third, signaling between muscle founders and biological effects of XTC-MIF: quantitative comparison with Xenopus epidermis is necessary for the fusion of syncytial myotubes and bFGF. Development 108, 173-183. for their arrangement and attachment to the epidermis (Bate, Hartenstein, A. Y., Rugendorgg, A., Tepass, U. and Hartenstein, V. (1992). 1990; Volk and VijayRaghaven, 1994). The function of the neurogenic genes during epithelial development in the Drosophila embryo. Development 116, 1203-1220. We are grateful to Rolf Bodmer, Bruce Paterson, Renate Jan, L. Y. and Jan, Y. N. (1982). Antibodies to horseradish peroxidase as Renkawitz-Pohl, Steve Robinow, and Barbara Taylor for their specific neuronal markers in Drosophila and grasshopper embryos. Proc. Natl. Acad. Sci. 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