The complex embryonic phenotype of mutations in the faint little ball (flb) locus, encoding the Drosophila EGF receptor homolog (DER), was dissected by temperature shifts of a temperature-sensitive allele. We show that the phenotype can be resolved into at least five components, which are temporally and spatially distinct. Most notably, the central nervous system (CNS) phenotype is determined at two separate phases. A severe collapse results from early defects in the DER-expressing ectodermal cells from which neuroblasts and midline glial cells delaminate. We thus suggest that DER activity is crucial for interactions that occur in the ectoderm at an early stage, and determine the fate of neuronal and glial cell lineages. This finding explains how a severe CNS phenotype is generated in fib embryos, in spite of the absence of expression of the protein in neuronal cells. In a second phase, during germ band retraction, the fib function is required specifically in the three pairs of midline glial cells (MG). In the absence of a functional DER protein, these cells die or fail to differentiate correctly, resulting in a fused commissure phenotype.

Mutations in the faint little ball (flb) locus lead to embryonic lethality with a distinct phenotype (Nüsslein-Volhard et al., 1984; Price et al., 1989; Schejter and Shilo, 1989; Clifford and Schüpbach, 1990; reviewed in Shilo and Raz, 1991). Amorphic flb mutant embryos lack head structures, exhibit telson defects, fail to secrete ventral denticle belts and fail to retract their germ band. As a result, amorphic alleles give rise to a “curled” ball-like structure lacking ventral setae but retaining the typical dorsal hairs. Another aspect of the embryonic phenotype is the collapse of the scaffold of the embryonic central nervous system (Schejter and Shilo, 1989; Zak et al., 1990). The diversity of defective embryonic tissues in flb mutants complicates the elucidation of the basis for this phenotype. Although the embryonic phenotype is complex and involves several apparently independent defects, all hypomorphic alleles exhibit the full spectrum of phenotypic effects (Schejter and Shilo, 1989; Raz et al., 1991). Thus, it has not been possible to dissect the different flb embryonic defects by genetic means.

The flb locus was shown to encode the Drosophila epidermal growth factor (EGF) receptor homolog (DER) (Price et al., 1989; Schejter and Shilo, 1989).

DER is a transmembrane protein containing a tyrosine kinase domain at the cytoplasmic region, and an extracellular ligand-binding domain (Livneh et al., 1985; Schejter et al., 1986). The protein was shown to possess tyrosine kinase activity (Schejter and Shilo, 1989; Wides et al., 1990; Zak and Shilo, 1990). The transmembrane receptor structure of DER suggests that one should interpret the function of the fib locus in the context of cell-cell interactions. Indeed, the postem-bryonic functions of the DERfflb locus, revealed by the torpedo and Ellipse alleles, demonstrate that the receptor mediates cell-cell interactions in the ovary and in the eye imaginal disc, respectively (Schüpbach, 1987; Baker and Rubin, 1989).

The pleiotropic nature of the embryonic flb defects is in accordance with the wide range of tissues and developmental stages in which the gene is expressed (Zak et al., 1990). The protein is found in all cells at the cellular blastoderm and in all ectodermal cells at the gastrula stage. In extended germ band embryos, it is also found in the mesoderm. Finally, in retracted germ band embryos, it is localized to the sites of somatic muscle attachment and along the ventral midline of the CNS. The protein is expressed in the CNS only at late stages (after germ band retraction), and only in a subset of the midline glia cells, in the processes of the MG cells [midline glial cells - the MGA, MGM and MGP cell pairs, also termed midline ectodermal cells - MECs (Crews et al., 1988)]. No other cells in the developing CNS were shown to express the protein. This finding is intriguing, since flb mutant embryos exhibit a severe collapse of the CNS scaffold and discontinuities in the longitudinal axon tracts. This severe phenotype cannot be accounted for simply by defects in the MG cells in which DER is expressed, since the flb phenotype is more severe than that of embryos bearing mutations in which all midline cells are abolished, such as single minded (sim) (Thomas et al., 1988; Crews et al., 1988). These abnormalities may therefore stem from indirect effects inflicted on the CNS by other tissues in which DER is required. Alternatively, one may envisage the flb CNS defects as stemming from early defects in the ectoderm before it provides the progenitors of the central nervous system.

Since the flb phenotype is complex and results from abnormalities in multiple embryonic tissues, it has not been possible to infer the basis for each of the defects by simply observing the final result in mutant embryos. We therefore decided to employ a temperature-sensitive allele, to dissect the, flb phenotype temporally. We show that the phenotype can be resolved into at least five aspects, which are temporally and spatially distinct. Most significantly, the CNS phenotype is determined at two stages. The severe collapse results from early defects in the ectodermal cells that express DER. This observation implicates the early cell-cell interactions that occur in the ectoderm in determination of the fate of neuronal and glial cell lineages stemming from the ectoderm. After germ band retraction, the DER protein is required once again for elaboration of the CNS architecture. In the absence of the protein in the three MG pairs of each segment, these cells die or fail to differentiate, resulting in a fused commissure phenotype.

Drosophila strains

The following alleles were used: flb1F26,twist1D96 (Nüsslein-Volhard et al., 1984) and flb1E1 (Schejter and Shilo, 1989). These strains were provided by J. O’Donnel flbJE1) and bv C. Niisslein-Volhard and E. Wieschaus (flb1F26 and twist1D96).

To identify the homozygous flb mutant embryos, a second chromosome balancer containing a PflacZ] element was used. The CyO P[A208.1M2] chromosome (obtained from H. Bellen and W. Gehring), can be easily detected from stage 9 and onwards by a striped staining pattern. It allows unambiguous identification of homozygous mutant embryos from the cross, which,do not stain with X-Gal.

Temperature shift protocols

Temperature shifts of flb1F26 embryos were carried out as follows: flies were allowed to lay eggs at the restrictive (29°C) or at the permissive (18°C) temperature. Egg collections of 2-3 hours at 18°C, or 45-75 minutes at 29°C were shifted up or down, accordingly. The chronological time of the shifts shown in Fig. 3 can be calculated as follows: for 18°C it is twice the time that is shown for 25°C. For 29°C, it is 0.8 of the time shown. A sample of live embryos from each point was taken for staging under Hallocarbon oil at the time of the shift. The embryos were then kept at the final temperature for a time corresponding to 48 hours of development at 25°C (for cuticle preparations), or until they reached the desired stage for antibody or X-Gal staining. At least 100 mutant embryos were analyzed for either cuticle or CNS phenotype, at each time point in the temperature-shift experiments. Cuticle preparations were made by dechorionating unhatched embryos in bleach, mounting in 1:1 Hoyer’s-lactic acid followed by an overnight incubation at 60°C (Wieschaus and Nüsslein-Volhard, 1986).

Fig. 1.

Cuticle phenotypes of flblF26 embryos at different temperatures. (A) Wild-type embryo; (B) flblF26 homozygous embryo grown at the permissive temperature of 18°C; (C) flblF26 embryo grown at the restrictive temperature of 29°C; (D) flblF26 embryo grown at 18°C until early stage 11 and then shifted up to 29°C; (E) flblF26 embryo grown at 29°C until early stage 11 and then shifted down to 18°C.

Fig. 1.

Cuticle phenotypes of flblF26 embryos at different temperatures. (A) Wild-type embryo; (B) flblF26 homozygous embryo grown at the permissive temperature of 18°C; (C) flblF26 embryo grown at the restrictive temperature of 29°C; (D) flblF26 embryo grown at 18°C until early stage 11 and then shifted up to 29°C; (E) flblF26 embryo grown at 29°C until early stage 11 and then shifted down to 18°C.

Fig. 2.

Central nervous system phenotypes of flblF26 embryos at different temperatures. (A) Wild-type embryo stained with BP102 monoclonal antibody; (B)homozygous embryo grown at the permissive temperature of 18°C; (C) flblF26 embryo grown at the restrictive temperature of 29°C. Note the non-retracted germ band and the disarrayed longitudinal axon tracts; (D) flblp26 embryo grown at 18°C until stage 12 and then shifted up to 29°C. Note that the longitudinal axon tracts are normal, but the commissures are fused. This is in contrast to embryos grown continuously at 18°C, in which the commissures are separated.

Fig. 2.

Central nervous system phenotypes of flblF26 embryos at different temperatures. (A) Wild-type embryo stained with BP102 monoclonal antibody; (B)homozygous embryo grown at the permissive temperature of 18°C; (C) flblF26 embryo grown at the restrictive temperature of 29°C. Note the non-retracted germ band and the disarrayed longitudinal axon tracts; (D) flblp26 embryo grown at 18°C until stage 12 and then shifted up to 29°C. Note that the longitudinal axon tracts are normal, but the commissures are fused. This is in contrast to embryos grown continuously at 18°C, in which the commissures are separated.

Fig. 3.

The temperature-sensitive periods for the flb1F26 c uticle and CNS defects. The time axis is drawn according to the developmental stage at which the embryos were shifted (as determined for 25°C). The percentage refers to embryos displaying a wild-type phenotype, with respect to the parameter tested. In panel C the % for the early shift up or down experiments refers to embryos with non-collapsed longitudinal axon tracts, but a fused commissure phenotype (as in Fig. 2D), while in the late shift up it refers to embryos displaying a wild-type CNS phenotype (as in Fig. 2B). Shift-up results are shown as open symbols while shift-down results are shown as filled circles.

Fig. 3.

The temperature-sensitive periods for the flb1F26 c uticle and CNS defects. The time axis is drawn according to the developmental stage at which the embryos were shifted (as determined for 25°C). The percentage refers to embryos displaying a wild-type phenotype, with respect to the parameter tested. In panel C the % for the early shift up or down experiments refers to embryos with non-collapsed longitudinal axon tracts, but a fused commissure phenotype (as in Fig. 2D), while in the late shift up it refers to embryos displaying a wild-type CNS phenotype (as in Fig. 2B). Shift-up results are shown as open symbols while shift-down results are shown as filled circles.

Antibodies and enhancer trap lines

The CNS was visualized by using anti-HRP antibodies (Cappel) or by utilizing monoclonal antibodies BP102 and BP104, provided by C. Goodman. Anti-slit antibodies were obtained from J. Rothberg and S. Artavanis-Tsakonas, and the anti-DER antibodies used were previously described (Zak et al., 1990). mAb 63 was developed in our lab by T. Volk and O. Leitner. We refer to it as an anti-fas III antibody because it gives a staining pattern identical to fasciclin in and recognizes the same molecular weight proteins on western blots (Fig. 5A; Patel et al., 1987; and data not shown). Anti-lacZ monoclonal antibodies were obtained from Promega.

The AA142 enhancer trap line expresses lacZ specifically in the three pairs of MG cells (MGA, MGM and MGP)(Klämbt et al., 1991), and the 242 line labels all or most of the midline cells (Nambu et al., 1990). Both were provided by C. Goodman. Enhancer trap lines used for following the fate of mesodermal lineages were provided by Y. N. Jan and L. Jan. Antibody and X-Gal stainings were carried out as previously described (Zak et al., 1990, Klämbt et al., 1991).

Embryo sectioning

Embryos (stained or nonstained) were dehydrated and infiltrated overnight with catalyzed JB-4 resin (Polysciences Inc.). The embryos were embedded, and the JB-4 blocks were allowed to polymerize under vacuum. 3 μm sections were then cut with a glass knife. Sections of embryos not stained with antibody were stained with Lee’s stain (0.26 mg/ml methylene blue, 0.26 mg/ml basic fuchsin, 25% ethanol, 70 mM phosphate buffer pH 7.5). The slides were immersed in the staining solution for 20 seconds, dipped briefly in deionized water, and twice in 70% ethanol. The slides were then dried over a hot plate and mounted.

Temporal dissection of the flb phenotype

The mutant flb alleles that were previously studied exhibit a complex embryonic phenotype affecting several tissues, which were presumably affected at different stages of embryonic development. To understand the basis for each one of these aspects, it was necessary to dissect the phenotype into its independent temporal and spatial components. This was done by temperature shifts of a temperature-sensitive flb allele designated flb1F26. This allele was shown to result from a missense mutation within the kinase domain where proline1112 is replaced by leucine (Raz et al., 1991). At the permissive temperature (18°C), the homozygous 1F26 embryos do not hatch, but their cuticle is similar to wild-type cuticle (Fig. 1B). In, addition, the CNS of these embryos appears almost normal (Fig. 2B). In contrast, when grown at the restrictive temperature (29°C), both the cuticle and CNS exhibit a severe phenotype, which is comparable to null flb alleles (Figs 1C, 2C). flblF26 thus provides a unique tool to dissect the embryonic phenotype by the utilization of temperature shifts.

flblF26 homozygous embryos were laid at 18°C and shifted at various times to 29°C. The embryos were allowed to develop at the restrictive temperature and scored for their cuticle phenotype. The time axis in the results of the temperature shifts will be presented throughout this work according to the developmental stage of the embryos at each point [in hours at 25°C, in concordance with the stages of Campos-Ortega and Hartenstein, (1985)], rather than by the chronological time elapsed. The results of this temperature-shift experiment show that the cuticle phenotype can be dissected into an early component affecting germ band retraction and a late component affecting ventral cuticle pattern. flblF26 embryos, shifted up as early as stage 8 (corresponding to 3 hours at 25°C) and onwards, begin to show a normally retracted germ band. Most mutant embryos which develop for at least 6 hours at the permissive temperature display a retracted germ band (Fig. 3A). Shift-down experiments gave a reciprocal result, exhibiting retraction defects in embryos shifted down from 3.5 hours and onwards (Fig. 3A). This result shows that in spite of the late time in embryogenesis in which germ band retraction takes place (beginning at 7.5 hours), DER function is required as early as 4 hours into embryogenesis to allow proper germ band retraction.

A second cuticular defect observed in flb embryos is the absence of ventral denticle bands. The involvement of DER in this process appears to occur later than in germ band retraction. Only embryos that were shifted up from 6 hours onwards show formation of the ventral denticle belts (Fig. 3B). The fact that the involvement of flb in retraction precedes the function of the protein in ventral epidermis differentiation, could be clearly demonstrated by temperature shifts at the proper time windows. Embryos that were shifted up at 5 –6 hours retracted their germ band, but failed to secrete ventral cuticle (Fig. 1D). Conversely, embryos that were shifted down in the same time window failed to undergo retraction, but secreted normal ventral cuticle (Fig. 1E).

In contrast to the retraction and denticle phenotypes, which showed a sharp response to the temperature shifts, the head phenotype of flb could not be easily dissected, due a wide gradation of phenotypes following the temperature shifts.

The central nervous system phenotype of flb was similarly analyzed by temperature shifts of the flblF26 allele, revealing two temporally distinct components of the CNS phenotype. At the permissive temperature, homozygous 1F26 embryos develop a CNS that displays only subtle defects, manifested by a slightly thinner spacing between the longitudinal axon tracts and between the commissures in each segment (Fig. 2B). At the restrictive temperature the CNS exhibits severe defects similar to that of null flb alleles (Fig. 2C). Shift-up and shift-down protocols were applied, and the mutant embryos were scored for their CNS phenotype. Fig. 3C shows that the severe CNS phenotype is determined as early as 3–5 hours, and shows a profile comparable to that of the germ band retraction defect. This result was surprising since at this early stage, DER is not expressed in cells of the developing CNS - neuroblasts and glia cells.

The flblF26 embryos that were shifted up after 5 hours did not show the near-wild-type CNS phenotype of embryos that were kept continuously at 18°C. Instead, while the CNS appeared to have non-collapsed longitudinal axon tracts, the commissures were found to be completely fused (Fig. 2D). This more subtle defect suggests that, after the early function of DER has been carried out, the protein is required once again at a later stage. The phenotype of embryos shifted up after 5 hours resembles the CNS phenotype of the spitz, rhomboid and Star alleles (Meyer and Nüsslein-Volhard, 1988; Klâmbt et al., 1991), and will be discussed below. The kinetics of appearance of the fused commissure phenotype suggest that in the second phase, DER is not required beyond 10.5 hours (Fig. 3C). Since DER is not expressed in any neuronal cells, and its expression in the midline glia is first detected only at 9 hours (Zak et al., 1990), the time window of the late requirement for DER in the CNS should be 9–10.5 hours.

Early localization of the DER protein

Since the DER protein is a receptor tyrosine kinase, we would expect it to affect the tissues in which it is expressed in a cell autonomous fashion. It was thus crucial to localize the protein at the time point corresponding to the temperature-sensitive period (TSP) for the retraction and severe CNS defects. This should allow us to focus on the candidate tissues that could be responsible for the manifestation of these defects later in development. Visualization of anti-DER staining in whole mounts and sections shows that, at the cellular blastoderm stage DER is expressed in all cells. As gastrulation commences DER is expressed in ectodermal cells and in mesodermal cells after their invagination (Fig. 4; Zak et al., 1990). However, no expression of the protein is seen in the neuroblast cell layer. The retraction and CNS defects should thus be accounted for by a requirement for DER in the ectoderm or in the mesoderm.

Fig. 4.

The expression of DER in stage 10 embryos. Wild-type (stage 10) embryos were stained with anti-DER antibodies and sectioned. Staining is observed only in the ectoderm (e) and mesoderm (m). (A) Cross-section; (B) parasagittal section.

Fig. 4.

The expression of DER in stage 10 embryos. Wild-type (stage 10) embryos were stained with anti-DER antibodies and sectioned. Staining is observed only in the ectoderm (e) and mesoderm (m). (A) Cross-section; (B) parasagittal section.

Fig. 5.

Fasciclin III staining in flb embryos. (A) A wild-type embryo stained with anti-fas III antibodies. Note the staining in the ectodermal (e) and neuronal (n) patches, the visceral mesoderm (vm), several glial cells (g) and in the stomodeum and hindgut. (B) An embryo homozygous for the severe flbJE1 allele. Note that the epidermal and neuronal staining disappears while all other tissues stain normally. Some epidermal staining at the segmental grooves is observed. This staining normally appears after the patched expression in the epidermis, and is also unaltered in the flb embryos. (C) A ovist1D96 homozygous embryo. Note that there is no visceral mesoderm staining, while all other aspects are normal.

Fig. 5.

Fasciclin III staining in flb embryos. (A) A wild-type embryo stained with anti-fas III antibodies. Note the staining in the ectodermal (e) and neuronal (n) patches, the visceral mesoderm (vm), several glial cells (g) and in the stomodeum and hindgut. (B) An embryo homozygous for the severe flbJE1 allele. Note that the epidermal and neuronal staining disappears while all other tissues stain normally. Some epidermal staining at the segmental grooves is observed. This staining normally appears after the patched expression in the epidermis, and is also unaltered in the flb embryos. (C) A ovist1D96 homozygous embryo. Note that there is no visceral mesoderm staining, while all other aspects are normal.

Mesoderm development

The development of the mesoderm of fib embryos was followed, using the severe flbjEi allele, which was maintained over a second chromosome balancer containing an enhancer trap marker. Homozygous mutant embryos were thus unambiguously identified by the absence of X-Gal staining. This method was used to identify the fib embryos throughout this work. Staining with anti-DER antibodies, at the extended germ band stage, shows that in the mutant the mesoderm is less uniform than in wild-type embryos. It is arranged in clusters of cells that are not always connected, due to disruptions in the regions opposite the parasegmental grooves (not shown).

The subtle morphological defects in the structure of the mesoderm did not affect the differentiation of the tissues that it normally forms. Staining of enhancer trap lines marking the fat body or visceral mesoderm in flb mutants, did not show any defects (not shown). An additional antibody was used to analyze the development of the mesoderm and ectoderm. mAb 63, a monoclonal antibody that was generated in our lab, gives an identical staining pattern to fasciclin III (fas III) and recognizes the same molecular weight proteins (Patel et al., 1987). It will thus be regarded as a fas III antibody. In wild-type embryos, it stains several tissues including the visceral mesoderm, several glial cells, a patch of cells at the hindgut and stomodeum, and segmental patches of cells in the ectodermal and neuronal cell layers (Fig. 5A). In embryos homozygous for the severe flbJE1 allele, the visceral mesoderm staining of fas III seemed normal (Fig. 5B). Note, however, that due to the abnormal mesoderm organization, the visceral mesoderm lies more ventrally in the mutant than in wild-type embryos at this stage.

Somatic muscles also appear to develop in flb embryos as judged from anti-myosin antibody staining and from observations of muscle contractions in living embryos. Interestingly, the muscles are not connected to the body wall (not shown). This effect is likely to be a result of the absence of DER activity in the muscle attachment sites, in which it was shown to be expressed at stage 13 (Zak et al., 1990).

Early functions of DER in the CNS

Using a variety of antibodies to follow the early development of the ectoderm mflb embryos (including anti-engrailed and anti-wingless), no abnormalities could be identified (Schejter and Shilo, 1989; and data not shown). However, using the anti-fas III antibodies as markers for ectoderm differentiation, a striking defect could be identified in the mutant embryos. Normally, fas III is expressed in the ectoderm at 6 hours in segmentally repeated patches (about 4 cells long by 4 cells wide) located between the segmental grooves (Patel et al., 1987; Fig. 5A). In flb embryos, this segmentally repeated ectodermal staining of fas III is absent (Fig. 5B). This defect appears to be specific, since other patterns of fas III expression are intact, including the expression in the stomodeum, hindgut, a subclass of glial cells and in the visceral mesoderm.

fas III staining is missing not only from the ectodermal cells, but also from a group of neuronal cells lying parallel to them (Fig. 5B). This result is especially surprising, since at this time and all earlier times, DER is not expressed in neuronal lineages. As a receptor tyrosine kinase, DER is thought to function in a cell-autonomous manner. Thus, the absence of fas III expression in the neuronal cells must result from defects in tissues expressing DER at an earlier time - the mesoderm or the ectoderm.

Although a direct role for the mesoderm in neuro-blast specification is considered unlikely in Drosophila (Doe and Goodman, 1985; Leptin, 1991; Rao et al., 1991), mesodermal control of neuronal pattern was clearly demonstrated in leech neurogenesis (Torrence et al., 1989). It is thus formally possible that the mesoderm in flb mutants does not provide the neural lineage with signals required for proper differentiation, as monitored by fas III expression. To examine this possibility, twist embryos, which do not develop any mesoderm, were tested for their fas III expression profile (Fig. 5C). While the visceral mesoderm expression of fas III is absent, twist embryos show staining in the segmentally repeated patches of ectodermal cells and in the specific underlying neuronal lineages. The mesoderm is thus not required for the expression of fas III in the developing CNS. Therefore, the CNS phenotype of flb should be attributed to defects in the ectoderm.

The precise alignment between fas Ill-expressing cells in the ectoderm and in the neuronal cell layer of wild-type embryos suggests that the basis for the coordinated expression is the common spatial origin of the two tissues: the neuronal cells delaminate from the ectoderm as neuroblasts. After delamination, they undergo an asymmetric division, giving rise to new neuroblasts, which will continue to divide, and to ganglion mother cells. The latter divide once and form the neuronal cells. The absence of fas III expression in the neural lineages of flb embryos is thus likely to reflect an early defect in the ectoderm from which these cells delaminated as neuroblasts. This observation may be the key to understand the early CNS defects of flb embryos, as it links them with the development of the ectoderm, a tissue in which DER is expressed at high levels.

The absence of fas III expression at 6 hours of embryonic development in the neuronal cells of flb embryos, provides an early marker for the aberrant differentiation of these cells. To extend these observations, we followed additional markers of neuronal differentiation in homozygous flblF26 embryos that were maintained at the restrictive temperature. An informative marker was the monoclonal antibody BP104. This antibody first detects the nervous-system-specific form of the neuroglian protein on a subset of neuronal cells of the CNS (stage 11), and later it detects the protein in all neurons of the CNS and PNS (Hortsch et al., 1990). The initial number of cells detected by BP104 in mutant flb embryos is comparable to that of wild type embryos. Fig. 6 shows wild type and mutant embryos at stage 11. In both cases, 2 –3 cells per hemisegment display strong staining, while weak expression is seen in additional neuronal cells. However, subtle early defects in the organization of the cells can be seen in the mutant embryos. The spacing between the cells in the longitudinal axis is not completely regular, and occasionaly the clusters of cells detected by this antibody on both sides of the midline are more closely spaced (Fig. 6B; Zak et al., 1990). These results demonstrate that DER activity early in the development of the nervous system is not required for determination of the correct number of delaminating neuroblasts, or the neuronal cells stemming from them. When the first neuronal markers are detected at stage 11, the defects in the organization of the CNS in flb embryos are only subtle. As development proceeds, the abnormalities become much more prominent. For example, at stage 12, the longitudinal tracts are fused in all segments (Fig. 6C). At later stages, extensive neural cell death ensues.

Fig. 6.

Early CNS development in flb embryos. Stage 11 wild-type (A) or homozygous flblF26 embryos at the restrictive temperature (B) were stained with mAb BP104. Note that while number of cells detected by this antibody in flb embryos is comparable to wild type, occasional abnormalities in the spacing between them are observed. At stage 12 (C), the defects in the organization of the CNS in the mutant embryos become much more pronounced.

Fig. 6.

Early CNS development in flb embryos. Stage 11 wild-type (A) or homozygous flblF26 embryos at the restrictive temperature (B) were stained with mAb BP104. Note that while number of cells detected by this antibody in flb embryos is comparable to wild type, occasional abnormalities in the spacing between them are observed. At stage 12 (C), the defects in the organization of the CNS in the mutant embryos become much more pronounced.

The results presented above establish the paradigm that early defects in the ectoderm of flb embryos at 4 hours lead to defects in the neuroblasts delaminating from it. Following this paradigm, we wanted to test whether the expression of DER in the ectoderm is also required for the development of other cell types originating from it. For example, the glial cells are generated from meso-ectodermal cells located, after the invagination of the mesoderm, as a ventral stripe (Crews et al., 1988). The expression of early markers for the population of delaminating midline cells was monitored. Only subtle abnormalities in these midline cells were found in flb embryos, while the overall pattern did not differ markedly from wild-type embryos (Fig. 7H; Zak et al., 1990). To follow the development of the midline cells in flb embryos at a higher resolution, the expression of enhancer trap lines marking specific subsets of these cells were utilized. The AA142 enhancer trap line labels the three pairs of MG cells in each segment (Klambt et al., 1991). flblF26; embryos were grown at the restrictive temperature and shifted down at 6 hours. These embryos were maintained at the permissive temperature and stained at 11-12 hours. In the mutant embryos, the enhancer trap is not expressed in the midline, while normal expression is observed in cells at the anterior and posterior end of the embryo (Fig. 7B). DER is first expressed in the MG cells only at late stages, after germ band retraction - 3 hours after the shift down in this experiment took place (Zak et al.,1990). Therefore, the absence of MG cells must be attributed to abnormalities that have occurred at the time when the progenitors of these glial cells were still part of the ectoderm where DER is expressed.

Fig. 7.

The fate of midline glial cells in flb embryos. Wild-type embryos (A) or homozygous flblF26 embryos (B) containing the AA142 enhancer trap, marking the MG cells, were grown at 29°C, shifted to 18°C at early stage 11 and stained with X-Gal and BP104 monoclonal antibodies at stage 15. In the mutant embryos, very few MG cells staining with X-Gal are observed. Wild-type (C, E) or flblF26 embryos (D, F) containing the AA142 enhancer trap were grown at 18°C, shifted to 29°C at stage 12 and stained with X-Gal and BP102 monoclonal antibody at stage 14 (C, D - ventral view) or at stage 15 (E, F - side view). Note that in the. flb embryos, no staining of the midline glial cells is observed, while the typical staining of this enhancer line at the head and posterior region is unaltered. To show that the early and late defects in the development of the pairs of MG cells in flb embryos are specific, wild-type (G) or flbJE1 (H) embryos containing the 242 enhancer trap line, marking all midline cells, were stained at stage 13 with anti-lacZ antibodies. While some abnormalities are observed in the severe flbJE1 embryos, most midline cells retain the normal expression pattern.

Fig. 7.

The fate of midline glial cells in flb embryos. Wild-type embryos (A) or homozygous flblF26 embryos (B) containing the AA142 enhancer trap, marking the MG cells, were grown at 29°C, shifted to 18°C at early stage 11 and stained with X-Gal and BP104 monoclonal antibodies at stage 15. In the mutant embryos, very few MG cells staining with X-Gal are observed. Wild-type (C, E) or flblF26 embryos (D, F) containing the AA142 enhancer trap were grown at 18°C, shifted to 29°C at stage 12 and stained with X-Gal and BP102 monoclonal antibody at stage 14 (C, D - ventral view) or at stage 15 (E, F - side view). Note that in the. flb embryos, no staining of the midline glial cells is observed, while the typical staining of this enhancer line at the head and posterior region is unaltered. To show that the early and late defects in the development of the pairs of MG cells in flb embryos are specific, wild-type (G) or flbJE1 (H) embryos containing the 242 enhancer trap line, marking all midline cells, were stained at stage 13 with anti-lacZ antibodies. While some abnormalities are observed in the severe flbJE1 embryos, most midline cells retain the normal expression pattern.

Late functions of DER in the CNS

Temperature-shift experiments at later stages of embryonic development have demonstrated that, in addition to its early function in CNS development, DER is also required at 9 –10.5 hours. In the absence of normal DER activity at this stage, the CNS displays non-separated commissures (Fig. 2D). This phenotype is similar to that seen in spitz, rhomboid and Star embryos (Meyer et al., 1988; Klämbt et al., 1991). These three mutations have been shown to cause specific defects in the MG cells, leading either to cell death or their failure to migrate posteriorly over the MGA cells and separate the two axon tracts in each commissure (Klâmbt et al.,1991)

At this stage of CNS development (stage 13), it has been shown that DER is expressed in cellular processes of the MG cells (Zak et al., 1990 and data not shown). We therefore wanted to test if the late CNS defects in flb embryos can be attributed to abnormalities in these cells. For this purpose, the expression of the AA142 enhancer trap line markingthe MGA, MGM and MGP cells was monitored in flblF26 embryos that were shifted to the restrictive temperature at 8 hours. It is important to emphasize that by shifting up the embryos after the DER functions required for the early CNS differentiation have already taken place, the late function of DER in the CNS can be specifically followed. While flblF26 embryos that were continuously grown at 18°C showed MG cells as followed by the AA142 enhancer line (not shown), the flblF26 embryos that were shifted up at 8 hours failed to show any blue staining of the MG cells (Fig. 7D and 7F). In the same embryos, other tissues marked by this enhancer trap line including a cluster of cells at the anterior and posterior end of the embryo, were unaffected. This result indicates that, in the absence of DER activity in the MG cells from 8 hours and onwards, these cells die or fail to differentiate. The same conclusion was reached when the MG cells were stained with anti-slit antibodies: no staining was observed m flblF26 embryos that were shifted up at 8 hours of development (not shown). This late CNS defect of flb appears to be specific to the MG cells. Additional markers used to follow glial cells in the severe flbJE1 mutant show that the other glial cells are intact. For example, the fas Ill-stained glial cells underlying the segmental grooves appear normal (Fig. 5B). Also, an enhancer trap fine that marks all midline cells, termed 242 (Nambu et al., 1990), shows that in severe flb embryos most midline cells continue to express the marker (Fig. 7H). Thus, the absence of functional DER at stage 13 specifically affects the three pairs of MG cells in each segment.

Epidermal functions of DER at 6 hours

The temperature-shift experiments demonstrated that the temperature-sensitive period for the epidermal denticle belt defects occurs at 6 hours. To look for possible morphological manifestations of these defects in the epidermal cells, we examined cross-sections of 7-9 hour old flb1F26 embryos that were shifted up at 5-6 hours of development. The overall structure of the epidermis appears intact. However, pockets serving as sites for accumulation of darkly staining, picnotic cells can be identified between the epidermis and the CNS in the ventral region, or between the epidermis and the mesoderm in the lateral region (Fig. 8B). It is not clear whether the source of dead cells is immediately adjacent to these islands, or if these pockets serve to collect dead cells from more distant regions as well. It is thus not possible to conclude whether the increased cell death is restricted to a subset of epithelial or mesodermal cells, or whether it is a general defect resulting in the death of different cell types. The increased cell death appears to correlate temporally with the defects in the secretion of denticle belts and not with the earlier defects. Embryos that were shifted down at 5-6 hours, at a time point that results in a non-retracted germ band with normal denticle belts (as in Fig. 1E), showed only a marginal increase in cell death (Fig. 8C).

Fig. 8.

Cell death in flb1F26 embryos. CA) Cross-section of a wild-type embryo at stage 12; (B) embryo grown at 18°C, shifted up to 29°C at early stage 11 and sectioned at stage 12. Note the islands of small, darkly staining dead cells (de). (C) flb1F26 embryo grown at 29°C, shifted down to 18°C at early stage 11 and sectioned at stage 12. Note that there are very few dead cells in this embryo. Bar=10 μm.

Fig. 8.

Cell death in flb1F26 embryos. CA) Cross-section of a wild-type embryo at stage 12; (B) embryo grown at 18°C, shifted up to 29°C at early stage 11 and sectioned at stage 12. Note the islands of small, darkly staining dead cells (de). (C) flb1F26 embryo grown at 29°C, shifted down to 18°C at early stage 11 and sectioned at stage 12. Note that there are very few dead cells in this embryo. Bar=10 μm.

Temporal and spatial dissection of the flb phenotype

Although the DER protein is involved in multiple aspects of embryonic development, it has not been possible to dissect the separate components by genetic means. All embryonic lethal hypomorphic alleles otflb show a reduced severity of all aspects of the phenotype (Schejter and Shilo, 1989; Raz et al., 1991). This suggests that in spite of the diversity of functions in which DER/ffb is involved, the same signal transduction pathway is used in all cases. In this work, the complex embryonic phenotype of flb was resolved by temperature shifts of a temperature-sensitive allele. This analysis has demonstrated that there are at least five separate aspects constituting this phenotype. The first requirement tot flb appears to take place at 4 hours, and affects both germ band retraction and the structure of the CNS. At 6 hours, flb is required for differentiation of the epidermal cells secreting the denticle belts. At 9 hours, it is required once again for proper differentiation or survival of the MG cell pairs. In addition, the function of DER appears to be required continuously from 4 to 8 hours for proper differentiation of the head structures. Since the determination of the different temporal requirements for DER depends exclusively on the temperature shift analysis, one should be cautious about the precise extrapolation of these results. If the protein is not inactivated immediatly upon the shift to the restrictive temperature, or conversely, if a shift down to the permissive temperature relies on newly synthesized DER protein for biological activity, the apparent temperature-sensitive period would be shifted to an earlier time of embryogenesis. Table 1 summarizes the time points and tissues in which DER is required, and the phenotypic consequences of the absence of a functional DER in each case.

Table 1.

Temporal and spatial dissection of the embryonic DER/flb phenotype

Temporal and spatial dissection of the embryonic DER/flb phenotype
Temporal and spatial dissection of the embryonic DER/flb phenotype

We have determined the TSP for germ band retraction to be as early as 4 hours. However, the functional basis for this aspect of the phenotype remains an open question. Since germ band retraction is poorly understood, there are no available markers to follow it. One possibility is that the abnormal organization of the mesoderm interferes with germ band retraction. Alternatively, the epidermis may be involved in the process of retraction. Proper differentiation of the ectoderm at an early stage could be crucial for its ability to support the process of germ-band retraction later.

The TSP for the defects in ventral cuticle secretion was determined to be at a later point in time - around 6 hours of development (Fig. 3B). In sections of flb1F26 embryos shifted to the restrictive temperature at 5 hours, the only obvious morphological abnormality is extensive cell death. It is not clear whether this increased cell death is responsible for the failure of the epidermis to secrete the denticle belts. The observed pattern of cell death in the mutant embryos appears as an exaggeration of the normal pattern of cell death (Campos-Ortega and Hartenstein, 1985). This is based on the fact that it starts at the same time (stage 11) and that the remaining epidermal cells are able to maintain the epithelial structure and allow the dead cells to accumulate at the normal sites (Fig. 8B; Campos-Ortega and Hartenstein, 1985). The extensive cell death observed in flb mutant embryos may result from failure of the cells expressing DER to assume their proper identities. It is interesting to note that such an extensive but organized cell death is also found in embryos mutant for segmentation genes such as hunchback and fushi tarazu, which are required for specifying ectodermal cell identity (Lehmann and Nüsslein-Volhard, 1987; Magrassi and Lawrence, 1988).

The roles of DER in CNS development

One of the most informative results of the temperature-shift experiments was the elucidation of the basis for the CNS defects in flb embryos, which appear to be biphasic. Earlier analysis of this phenotype showed that, while the final structure of the CNS is completely disrupted, at early stages of its development the morphological CNS defects observed are only subtle (Zak et al., 1990). This observation, coupled with the fact that no DER expression was seen in the neuronal cells, has led to the suggestion that the basis for the flb defect is a collapse of the CNS after its initial proper assembly. Contradicting this notion, the temperature-shift experiments showed that the severe CNS defect of flb is determined as early as 4 hours. Thus, any explanation for the phenotype must postulate a function tor flb at this early stage, and take into account the observation that the protein is found in the ectoderm but not in the delaminating neuroblasts.

The analysis of the expression pattern of fasciclin III in flb embryos provides an explanation for the early CNS defects. In wild-type embryos, fas III is expressed at 6 hours in several tissues with a distinct pattern. In the ectoderm, it is expressed in a segmentally repeated pattern in the ectodermal cells lying between the segmental grooves. Strikingly, the same pattern of ectodermal expression is reiterated in the neuronal cells lying parallel to them. This alignment could be explained by a putative signal transduction mechanism in which spatial cues are transferred from the ectoderm to the neuronal cells. Such a mechanism was shown to be responsible for transmitting information between the mesoderm and the endoderm germ layers (Immerglück et al., 1990; Panganiban et al., 1990). An alternative explanation is that the basis for the alignment between the ectodermal and neuronal cells expressing fas III is their common origin, since the neuronal cells originate from neuroblasts delaminating from the ectoderm. An event occurring in the ventral ectoderm before the delamination of neuroblasts, may thus be responsible for triggering fas III expression two hours later, in both ectodermal and neuronal cells.

The defects in fas III expression observed in flb embryos suggest that the latter model is correct. We find that the expression of the segmentally repeated pattern in both ectodermal and neuronal cells is completely abolished, while all other sites of fas III expression remain unaltered. The TSP for this defect in fas III expression is indeed at 4 hours, at the time when the neuroblast precursors are still part of the ectoderm. Embryos that were shifted up at 5 hours or later, begin to show the typical fas III expression in both ectodermal and neuronal patches (not shown). These results suggest that DER is mediating an early event of communication between ectodermal cells. This interaction triggers separate differentiation paths that the two lineages will subsequently undertake. The absence of fas III expression in the flb embryos is likely to be a marker, reflecting an array of defects in both pathways. In support of this model, the patched (ptc) mutation known to affect differentiation of the epidermis at the central part of each segment, also lacks both the epidermal and neuronal staining of fas III at 6 hours (Patel et al., 1989). Like DER, ptc is also expressed in the ectoderm and in the mesoderm, but not in the neuroblasts (Nakano et al., 1989).

The identification of phenotypic defects in a diverse spectrum of tissues originating from a common lineage, has been previously reported by Meyer and Niisslein-Volhard (1988). When studying the terminal phenotype of the “spitz group” genes, they found that mutations in several independent loci lead to phenotypic defects in the same spectrum of tissues including the CNS and the lateral epidermis. Both tissues arise from the same region in the fate map of the embryo. Similarly, in this work, we followed fas III expression in flb embryos at 6 hours, and identified a common defect in two tissues sharing the same spatial origin. Furthermore, the ability to carry out temperature-shift experiments using a temperature-sensitive flb allele actually proves that the TSP for the common defect is indeed at the time when the ectodermal cells and the CNS progenitor cells were part of the same tissue.

Our findings agree with previous studies that have shown that the fate of neuronal cells is determined by cues generated within the ectoderm. For example, laser ablation of specific neuroblasts in the developing CNS of the grasshopper, leads to recruitment of neighboring neuroectodermal cells, which have assumed the fate of the ablated cells. This fate was shown to be determined by the position of the cells in the neuroectoderm (Doe and Goodman, 1985). The group of “neurogenic genes” is another example of the processes that occur in the neuro-ectoderm and determine the correct number of segregating neuroblasts [Reviewed in Artavanis-Tsakonas (1988); Campus-Ortega and Jan (1991)]. The putative role of flb in the ectoderm at 4 hours is different, however. In flb embryos, the segregation of neuroblasts and the expression of several neuronal markers is normal (e.g. those recognized by the anti-HRP, BP102 and BP104 antibodies). Yet, other specific markers of neuronal differentiation (such as fas III) fail to be expressed. DER is thus likely to be involved in a process crucial for determination of specific identities of neuronal cells, rather than in the establishment of their initial segregation pattern. The collapsed CNS pheno-type may develop from defects in neuronal pathfinding and migration, combined with the absence of several types of glial cells. Other examples of an early requirement for a gene product that is manifested only at late stages of neuronal differentiation are the fushi tarazu and prospero loci (Doe et al., 1988, 1991).

The function of DER in the ectoderm that is required for specification of neuroblast identity could either provide a permissive environment for subsequent differentiation pathways, or may actually transmit the inductive signal itself. Graded distribution of the DER ligand could provide individual cells with different levels of DER tyrosine kinase activity, initiating various developmental pathways. In support of the second alternative, the torso protein, which is also a receptor tyrosine kinase, has been shown to play a direct role in translating external positional information into specific cell fates (Casanova and Struhl, 1989; Sprenger et al., 1989).

Finally, the temperature-shift experiments have allowed us to attribute a role for the expression of DER in the MG cells at the stage of germ band retraction. Shift up of flb1V26 embryos after 8 hours, results in a phenotype resembling the spitz, rhomboid and Star mutants, in which the aberrant development or migration of the MGM pairs was shown to cause the failure to separate the axon tracts of the commissures (Klâmbt et al., 1991). Enhancer trap markers of the MG cells have indeed shown that in these flb embryos, the differentiation or viability of MG cell pairs is impaired. The elaborate process of migration carried out by the MG cells is likely to depend on the capacity of these cells to probe their environment and recognize neighboring cells by utilizing cell surface receptors. rhomboid was indeed shown to encode a putative transmembrane receptor protein (Bier et al., 1990). The crucial function of DER in the MG cells is a second example for the involvement of a cell surface receptor in the viability or differentiation of these cells. Future studies should give insights to the ligands that are likely to provide the spatial cues for the migration or differentiation of the MG cells.

In conclusion, this work has dissected and defined the complex temporal requirements for DER/fffi during embryonic development. The common theme between these diverse functions may be the regulation of cell fate by inductive signals transmitted through DER.

We would like to thank the following investigators for kindly providing strains or reagents used in this study: E. Wieschaus, C. Niisslein-Volhard, C. Goodman, H. Bellen, W. Gehring, Y. N. Jan, L. Jan, J. O’Donnel, J. Rothberg, S. Artavanis-Tsakonas, T. Volk and O. Leitner. In addition, we are grateful to all members of the Shilo lab for their support and helpful discussions, and to E. Schejter, T. Volk, R. Wides and N. Zak for critical reading of the manuscript.

This work was supported by NIH grant GM35998 and the Wolfson grant from the Israel academy of sciences to B. S.

Artavanis-Tsakonas
,
S.
(
1988
).
The molecular biology of the Notch locus and the fine tuning of differentiation in Drosophila
.
Trends Genet
.
4
,
95
100
.
Baker
,
N. E.
and
Robin
,
G. M.
(
1989
).
Effect on eye development of dominant mutations in Drosophila homologue of the EGF receptor
.
Nature
340
,
150
153
.
Bier
,
E.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1990
).
rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophila melanogaster
.
Genes Dev
.
4
,
190
203
.
Campos-Ortega
,
J. A.
and
Hartenstein
,
V.
(
1985
).
The Embryonic Development of Drosophila melanogaster. Berlin, Heidelberg, New York, and Tokyo: Springer-Verlag
.
Campos-Ortega
,
J. A.
and
Jan
,
Y. N.
(
1991
).
Genetic and molecular basis of neurogenesis in Drosophila
.
Annu. Rev. Neurosci
.
14
, in press.
Casanova
,
J.
and
Struhl
,
G.
(
1989
).
Localized surface activity of torso, a receptor tyrosine kinase, specifies terminal body pattern in Drosophila
.
Genes Dev
.
3
,
2025
2038
.
Clifford
,
R. J.
and
Schüpbach
,
T.
(
1990
).
Coordinately and differentially mutable activities of torpedo, the Drosophila melanogaster homolog of the vertebrate EGF receptor gene
.
Genetics
123
,
771
787
.
Crews
,
S. T.
,
Thomas
,
J. B.
and
Goodman
,
C. S.
(
1988
).
The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to per gene product
.
Cell
52
,
143
151
.
Doe
,
C. Q.
,
Chu-Lagraff
,
Q.
,
Wright
,
D. M.
and
Scott
,
M. P.
(
1991
).
The prospero gene specifies cell fates in the Drosophila central nervous system
.
Cell
65
,
451
464.
Doe
,
C. Q.
and
Goodman
,
C. S.
(
1985
).
Early events in insect neurogenesis: II. The role of cell interactions and cell lineage in the determination of neuronal precursor cells
.
Devi Biol. Ill, 206-219
.
Doe
,
C. Q.
,
Hiromi
,
Y.
,
Gehring
,
W. J.
and
Goodman
,
C. S.
(
1988
).
Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis
.
Science
239
,
170
175
.
Hortsch
,
M.
,
Bieber
,
A. J.
,
Patel
,
N. H.
and
Goodman
,
C. S.
(
1990
).
Differential splicing generates a nervous system-specific form of Drosophila neuroglian
.
Neuron
4
,
697
709
.
Immerglück
,
K.
,
Lawrence
,
P. A.
and
Bienz
,
M.
(
1990
).
Induction across germ layers in Drosophila mediated by a genetic cascade
.
Cell
62
,
262
268
.
Klâmbt
,
C.
,
Jacobs
,
R.
and
Goodman
,
C. S.
(
1991
).
The midline of the Drosophila central nervous system: A model for the genetic analysis of cell fate, cell migration, and growth cone guidance
.
Cell
64
,
801
815
.
Lehmann
,
R.
and
Nüsslein-volhard
,
C.
(
1987
).
hunchback, a gene required for segmentation of the anterior and posterior region of the Drosophila embryo
.
Devi Biol
.
119
,
402
417
.
Leptin
,
M.
(
1991
).
twist and snail as positive and negative regulators during Drosophila mesoderm development
.
Genes Dev
.
5
,
1568
1576
.
Livneh
,
E.
,
Glazer
,
L.
,
Segal
,
D.
,
Schlessinger
,
J.
and
Shilo
,
B.-Z.
(
1985
).
The Drosophila EGF receptor homolog: conservation of both hormone binding and kinase domains
.
Cell
40
,
599
607
.
Magrassi
,
L.
and
Lawrence
,
P. A.
(
1988
).
The pattern of cell death in fushi tarazu, a segmentation gene of Drosophila
.
Development
104
,
447
451
.
Meyer
,
U.
and
Nüsslein-volhard
,
C.
(
1988
).
A group of genes required for pattern formation in the ventral ectoderm of the Drosophila embryo
.
Genes Dev
.
2
,
1496
1511
.
Nakano
,
Y.
,
Guerrero
,
L
,
Hidalgo
,
A.
,
Taylor
,
A.
,
Whittle
,
J. R.
and
Ingham
,
P. W.
(
1989
).
A protein with several possible membrane-spanning domains encoded by the Drosophila segment polarity gene patched
.
Nature
341
,
508
513
.
Nambu
,
J. R.
,
Franks
,
R. G.
,
Hu
,
S.
and
Crews
,
S. T.
(
1990
).
The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells
.
Cell
63
,
63
75
.
Nüsslein-volhard
,
C.
,
Wieschaus
,
E.
and
Kludlng
,
H.
(
1984
).
Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome
.
Wilhelm Rouxs Arch. devl. Biol
.
193
,
267
282
.
Panganiban
,
G. E. F.
,
Reuter
,
R.
,
Scott
,
M. P.
and
Hofhnann
,
M.
(
1990
).
A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis
.
Development
110
,
1041
1050
.
Patel
,
N. H.
,
Schafer
,
B.
,
Goodman
,
C. S.
and
Holmgren
,
R.
(
1989
).
The role of segment polarity genes during Drosophila neurogenesis
.
Genes Dev
.
3
,
890
904
.
Patel
,
N. H.
,
Snow
,
P. M.
and
Goodman
,
C. S.
(
1987
).
Characterization and cloning of fasciclin III: a glycoprotein expressed on subset of neurons and axon pathways in Drosophila
.
Cell
48
,
975
988
.
Price
,
J. V.
,
Clifford
,
R. J.
and
Schüpbach
,
T.
(
1989
).
The maternal ventralizing locus torpedo is allelic to faint little ball, an embryonic lethal, and encodes the Drosophila EGF receptor homolog
.
Cell
56
,
1085
1092
.
Rao
,
Y.
,
Vaessin
,
H.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1991
).
Neuroectoderm in Drosophila embryo is dependent on mesodenn for positioning but not for formation
.
Genes Dev
.
5
,
1577
1588
.
Raz
,
E.
,
Schejter
,
E. D.
and
Shilo
,
B.-Z.
(
1991
).
Inter-allelic complementation among DER 6 alleles: Implications for the mechanism of signal transduction by receptor-tyrosine kinases
.
Genetics
129
,
191
201
.
Schejter
,
E. D.
,
Segal
,
D.
,
Glazer
,
L.
and
Shilo
,
B.-Z.
(
1986
).
Alternative 5’ exons and tissue-specific expression of the Drosophila EGF receptor homolog transcripts
.
Cell
46
,
1091
1101
.
Schejter
,
E. D.
and
Shilo
,
B.-Z.
(
1989
).
The Drosophila EGF receptor homolog (DER) gene is allelic to faint little ball, a locus essential for embryonic development
.
Cell
56
,
1093
1104
.
Schüpbach
,
T.
(
1987
).
Germ line and soma cooperate during oogenesis to establish the dorsoventral pattern of egg shell and embryo in Drosophila melanogaster
.
Cell
49
,
699
707
.
Shilo
,
B.
and
Raz
,
E.
(
1991
).
Developmental control by the Drosophila EGF receptor homolog DER
.
Trends Genet
.
7
,
388
392
.
Sprenger
,
F.
,
Stevens
,
L. M.
and
Nüsslein-volhard
,
C.
(
1989
).
The Drosophila gene torso encodes a putative receptor tyrosine kinase
.
Nature
338
,
478
483
.
Thomas
,
J. B.
,
Crews
,
S. T.
and
Goodman
,
C. S.
(
1988
).
Molecular genetics of the single-minded locus: A gene involved in the development of the Drosophila nervous system
.
Cell
52
,
33
141
.
Torrence
,
S. A.
,
Law
,
M. I.
and
Stuart
,
D. K.
(
1989
).
Leech neurogenesis: II. Mesodermal control of neuronal patterns
.
Devi Biol
.
136
,
40
60
.
Wides
,
R. J.
,
Zak
,
N. B.
and
Shilo
,
B. Z.
(
1990
).
Enhancement of Tyrosine Kinase Activity of the Drosophila Epidermal Growth Factor Receptor Homolog by Alterations of the Transmembrane Domain
.
Eur. J. Biochem
.
189
,
637
645
.
Wieschaus
,
E.
and
Nüsslein-volhard
,
C.
(
1986
).
Looking at embryos
.
In Drosophila, a Practical Approach
, (ed.
D. B.
Roberts
), pp.
199
-
227
. Oxford, Washington, D.C.: IRL Press.
Zak
,
N. B.
and
Shilo
,
B.-Z.
(
1990
).
Biochemical properties of the Drosophila EGF receptor homolog (DER) protein
.
Oncogene
5
,
1589
1593
.
Zak
,
N. B.
,
Wides
,
R. J.
,
Schejter
,
E. D.
,
Raz
,
E.
and
Shilo
,
B.-Z.
(
1990
).
Localization of the DER/flb protein in embryos: implication on the faint little ball lethal phenotype
.
Development
109
,
865
874
.