The Drosophila snail (sna) gene is first expressed in cells giving rise to mesoderm and is required for mesoderm formation. sna is subsequently expressed in the developing nervous system. sna expression during neurogenesis evolves from segmentally repeated neuroectodermal domains to a pan-neural pattern. We have identified a 2.8kb regulatory region of the sna promoter that drives LacZ expression in a faithful neuronal pattern. Deletion analysis of this region indicates that the pan-neural element is composed of separable CNS and PNS components. This finding is unexpected since all known genes controlling early neurogenesis, including the proneural genes (i.e. da and AS-C), are expressed in both the CNS and PNS. We also show that expression of sna during neurogenesis is largely independent of the proneural genes da and AS-C. The separate control of CNS and PNS sna expression and independence of proneural gene regulation add to a growing body of evidence that current genetic models of neurogenesis are substantially incomplete.

Formation of the nervous system (neurogenesis) during Drosophila embryonic development is a multi-step process. During early neurogenesis, ectodermal cells make a choice between neural or epidermal fates. Genetic data suggest that groups of ectodermal cells must first gain competence to make the neural versus epidermal decision, then a subset of these competent cells are chosen to become primary neuronal precursor cells through a lateral inhibitory process (for reviews, see Ghysen and Dambly-Chaudiere, 1989; Jan and Jan, 1990; Artavanis-Tsakonas and Simpson, 1991; Campos-Ortega and Jan, 1991). Competence to form neural tissue depends on the activation of a group of genes called proneural genes, which include daughterless (da) and genes of the achaete-scute complex (AS-C) (Caudy et al., 1988a,b; Villares and Cabrera, 1987; Alonso and Cabrera, 1988; Gonzalez et al., 1989). These genes all encode bHLH proteins and are thought to function as transcriptional activators of neuronal genes (Caudy et al. 1988b; Alonso and Cabrera, 1988; Gonzalez et al., 1989; Murre et al., 1989a,b). Several of the AS-C proneural genes are expressed in overlapping patches of neuroectodermal cells (called proneural domains) that are believed to coincide with zones of neural competence (Cabrera et al., 1987; Romani et al., 1987, 1989). Lateral inhibition ultimately restricts peak expression of AS-C transcripts to cells fated to become primary neuronal precursor cells (Skeath and Carroll, 1991, 1992; Cubas et al., 1991). Enhanced AS-C expression in neuronal precursor cells is transient and is not maintained following segregation from the neuroectoderm, suggesting that these genes trigger neuronal precursor specification.

Once a neuronal precursor cell is selected, it carries out a stereotyped pattern of cell divisions to generate a specific type of neuron (and in some cases, associated non-neuronal support cells). As soon as all neuronal precursor cells form, they begin expressing a group of genes referred to as panneural genes (Bier et al., 1992). Although AS-C genes continue to be expressed for a short time in neuronal precursor cells, no single proneural gene is expressed in all neuronal precursor cells. The pan-neural genes are, therefore, the earliest genes known to be specifically expressed in all neuronal precursor cells, and may play a role in establishing neuronal tissue type identity.

In this study we examine the basis for expression of the snail (sna) gene in the developing nervous system following its initial expression in the presumptive mesoderm (Ip et al., 1992a; Alberga et al., 1991). The early neuroectodermal pattern of sna expression is similar to that of AS-C and then becomes restricted to all, or most, neuronal precursor cells in a pan-neural pattern. Expression of sna in the neuroectoderm and then in neuroblasts is independent of the known proneural genes da and AS-C, suggesting that an alternative pathway is capable of activating gene expression in neu-roectodermal patches and neuronal precursor cells. We have identified a segment of the sna promoter, which directs the full neuronal expression pattern when fused to the bacterial LacZ reporter gene. Deletion analysis of the sna promoter suggests that sequences directing expression in the central nervous system (CNS) can be separated from those required for expression in the peripheral nervous system (PNS). These data suggest that yet unidentified genetic pathways contribute to establishing the neuronal tissue type and to distinguishing major subdivisions of the nervous system.

Fly stocks

P-element transformed lines carrying various sna promoter fragments are described by Ip et al. (1992a). The Cyo-P[ftz-LacZ] balancer was kindly provided by Y. Hiromi. dakx136 and scB57 alleles were kindly provided by Y.N. Jan. Other genetic markers and chromosome balancers used are described in Lindsley and Grell (1968) and Lindsley and Zimm (1992).

Antibody staining of embryos

Embryos were dechorionated with bleach, rinsed with NaCl/Triton, and fixed with equal volumes of 4% formaldehyde in 0.1 M sodium phosphate (pH 7.2) and heptane. The embryos were then devitellinized with equal volumes of 90% methanol and heptane, rehydrated with 0.1 M sodium phosphate and 0.3% Triton X-100, and blocked with 2% bovine serum albumin. Antibody incubations and rinses were all done in the above buffer. The anti-Sna antiserum was used according to Kosman et al. (1991) and visualized with a rhodamine conjugated anti-guinea pig secondary antibody. In situ and antibody double labeling experiments were performed according to Sturtevant et al. (1993).

In situ hybridization to whole-mount embryos

In situ hybridization to whole-mount discs and embryos was performed using digoxigenin-abeled RNA probes (Boehringer-Mannheim, 1093 657) according to Tautz and Pfeiffle (1989) using 4 μg/ml Proteinase K instead of 40 μg/ml as required for digoxigenin-labeled DNA probes.

Other molecular techniques

RNA probe synthesis was performed according to Boehringer-Mannheim protocols and other cloning techniques followed standard procedures, as in Maniatis et al. (1982).

The sna gene is expressed early in the neuroectoderm and then continues to be expressed in neuronal precursor cells

During the early blastoderm stage of embryogenesis, the sna gene is expressed in a broad strip of ventral cells, which will give rise to mesoderm. sna function is required in these cells since mesoderm fails to form in sna mutant embryos (Boulay et al., 1987). At this stage sna functions, at least in part, as a transcriptional repressor of genes expressed in the mesectoderm such as singleminded, and genes expressed in the neuroectoderm such as AS-C transcripts and rhomboid (Kosman et al., 1991; Rao et al., 1991; Leptin, 1991; Ip et al., 1992a,b). After invagination of presumptive mesodermal cells, sna expression fades rapidly and disappears midway through germband extension. During the last period of sna expression in the presumptive mesoderm, transcription begins in the neuroectoderm initially in a pair-rule pattern (not shown), which rapidly evolves into a pattern of segmentally repeated stripes (Fig. 1A,B). This expression of sna occurs during the time that AS-C transcripts are expressed in neuroectodermal checkerboard patterns (Cabrera et al., 1987). sna is expressed in segmentally repeated blocks of cells and in longitudinal connecting rows of cells (r1-r3 in Fig. 1A). AS-C transcripts, in contrast, are expressed in disconnected patches of neuroectodermal cells at this time (Cabrera et al., 1987; Skeath and Carroll, 1992). The rows of cells strongly expressing sna correspond roughly to the locations where the three rows of neuroblasts will delaminate during the first (S1) wave of neuroblast segregation (Campos-Ortega and Hartenstein, 1985; Jimenez and Campos-Ortega, 1990; Bier et al., 1992). Since more cells express sna than will delaminate as neuroblasts, neuroectodermal cells that will ultimately become epidermal cells must also express sna mRNA at this stage.

Fig. 1.

Developmental profile of sna expression during neurogenesis in whole-mount embryos. sna transcripts were visualized by in situ hybridization of a digoxigenin-labeled sna cDNA probe to RNA in wild-type embryos. sna is first expressed at the cellular blastoderm stage in presumptive mesodermal cells, which form a wide sharply defined strip of cells along the ventral portion of the embryo (data not shown). As sna expression in the mesoderm begins to fade during early germband elongation (approx. 4.5-5 hours), transcripts appear in the overlying ectoderm (A,B). For a short period of time sna expression can be observed in both the mesoderm (open arrow) and the ectoderm, but then is soon restricted to the ectoderm (arrowhead). The ectodermal expression initially exhibits a pair-rule modulation and then evolves into a crude segmentally repeated set of cells connected by longitudinal rows of cells at roughly the locations from which neuroblasts will form (r1, r2, and r3). As neuroblasts begin to delaminate (C,D: approx. 5.5 hours), sna expression is observed in both the nascent neuroblasts (nb; solid arrow) and in ectodermal cells (bracket in C; arrowhead in D), but not in the mesoderm (open arrow). The pattern of sna transcription differs from the distribution of Sna protein in that detectable levels of protein are evident only in neuroblasts (see Fig. 2). As the first wave of neuroblast segregation nears completion (E,F: approx. 5.5-6 hours), sna transcripts become restricted to the neuroblast layer (labels as in previous panels). sna is expressed in all neuroblasts and in PNS precursors by 6.5-7 hours (G). The PNS expression is first evident in neuroectodermal patches (arrow in H) prior to sensory mother cell delamination (G), as is the case for neuroblasts in the CNS. At later stages sna is expressed in postmitotic cells of the PNS and CNS (I and J respectively: approx. 9–10 hours). In I the arrow points to PNS cells of the dorsal cluster and the arrowhead indicates the level of the lateral cluster. Two strong patches of sna expression become visible beginning at about 11 hours in thoracic segments (not shown). Embryos are all oriented with anterior to the left and dorsal at the top. The embryos in A-H are germband extended. The embryos in A and H are viewed with the focal plane at the ectodermal surface; in E,G and I the focus is subectodermal (i.e. at the level of a single cell layer under the ventral epithelial surface); in C the focal plane is partly epidermal (bracket) and partly subectodermal (arrow); and in B,D,F and J the focus is in a sagittal cross-sectional plane near the center of the embryo. Embryos in A,C,E, and G are viewed from a ventral perspective and all other embryos are viewed from the side. Abbreviations for this and subsequent figures: CNS, central nervous system; ect., ectoderm; ms or open arrow, mesoderm; nb, neuroblast; PNS, peripheral nervous system; r1-r3, three rows of first wave neuroblast where r1 denotes the row closest to the ventral midline; vml, ventral midline.

Fig. 1.

Developmental profile of sna expression during neurogenesis in whole-mount embryos. sna transcripts were visualized by in situ hybridization of a digoxigenin-labeled sna cDNA probe to RNA in wild-type embryos. sna is first expressed at the cellular blastoderm stage in presumptive mesodermal cells, which form a wide sharply defined strip of cells along the ventral portion of the embryo (data not shown). As sna expression in the mesoderm begins to fade during early germband elongation (approx. 4.5-5 hours), transcripts appear in the overlying ectoderm (A,B). For a short period of time sna expression can be observed in both the mesoderm (open arrow) and the ectoderm, but then is soon restricted to the ectoderm (arrowhead). The ectodermal expression initially exhibits a pair-rule modulation and then evolves into a crude segmentally repeated set of cells connected by longitudinal rows of cells at roughly the locations from which neuroblasts will form (r1, r2, and r3). As neuroblasts begin to delaminate (C,D: approx. 5.5 hours), sna expression is observed in both the nascent neuroblasts (nb; solid arrow) and in ectodermal cells (bracket in C; arrowhead in D), but not in the mesoderm (open arrow). The pattern of sna transcription differs from the distribution of Sna protein in that detectable levels of protein are evident only in neuroblasts (see Fig. 2). As the first wave of neuroblast segregation nears completion (E,F: approx. 5.5-6 hours), sna transcripts become restricted to the neuroblast layer (labels as in previous panels). sna is expressed in all neuroblasts and in PNS precursors by 6.5-7 hours (G). The PNS expression is first evident in neuroectodermal patches (arrow in H) prior to sensory mother cell delamination (G), as is the case for neuroblasts in the CNS. At later stages sna is expressed in postmitotic cells of the PNS and CNS (I and J respectively: approx. 9–10 hours). In I the arrow points to PNS cells of the dorsal cluster and the arrowhead indicates the level of the lateral cluster. Two strong patches of sna expression become visible beginning at about 11 hours in thoracic segments (not shown). Embryos are all oriented with anterior to the left and dorsal at the top. The embryos in A-H are germband extended. The embryos in A and H are viewed with the focal plane at the ectodermal surface; in E,G and I the focus is subectodermal (i.e. at the level of a single cell layer under the ventral epithelial surface); in C the focal plane is partly epidermal (bracket) and partly subectodermal (arrow); and in B,D,F and J the focus is in a sagittal cross-sectional plane near the center of the embryo. Embryos in A,C,E, and G are viewed from a ventral perspective and all other embryos are viewed from the side. Abbreviations for this and subsequent figures: CNS, central nervous system; ect., ectoderm; ms or open arrow, mesoderm; nb, neuroblast; PNS, peripheral nervous system; r1-r3, three rows of first wave neuroblast where r1 denotes the row closest to the ventral midline; vml, ventral midline.

Expression of sna in the neuroectoderm continues as the S1 wave of neuroblast segregation begins. During the early phase of neuroblast segregation, sna is expressed at approximately equal levels in both neuroectodermal cells and in delaminating neuroblasts (Fig. 1C,D). After the S1 wave of neuroblast segregation is complete, sna transcripts vanish from the neuroectoderm but continue to be expressed in most or all neuroblasts (Fig. 1E,F). Expression of sna in the PNS follows a similar course. sna transcripts are first observed in ectodermal patches (arrow in Fig. 1H) and then in sensory mother cells (Fig. 1G). The full CNS neuroblast pattern can be observed at the time that PNS precursors are first observed (Fig. 1G). The number of cells expressing sna diminishes considerably for a brief period between approximately 8-8.5 hours. Transcripts subsequently reappear in early postmitotic neurons and possibly in some CNS ganglion mother cells and PNS secondary precursor cells. Neuronal expression is maintained until shortly after germband retraction (Fig. 1I,J) and disappears by 11 hours. Strong expression is also evident in the precursors of the wing and haltere discs (Alberga et al., 1991), which persists well into the first larval instar.

The pan-neural expression of sna transcripts is consistent with the pattern of sna protein (Sna) expression as determined by immunofluorescence using a polyclonal anti-Sna antiserum (Fig. 2; also see Fig. 1D in Kosman et al., 1991). This antiserum (Kosman et al., 1991) detects Sna protein in postmitotic neurons, as well as in presumptive mesodermal cells during the blastoderm stage, but curiously does not detect protein in neuroectodermal cells that express sub-stantial levels of sna mRNA. Differences between sna mRNA and Sna protein distributions have also been reported by Alberga et al. (1991). Since sna transcripts are expressed for a prolonged period (>1 hour) in the neuroectoderm at levels comparable to that observed in neuroblasts, it is likely that sufficient time is available for translation of the sna message in the neuroectoderm (see Discussion).

Fig. 2.

Pan-neural distribution of Sna protein during neurogenesis. Sna protein was visualized with a polyclonal anti-Sna antiserum (Kosman et al., 1991) followed by a fluoresceine-labeled secondary antibody. The embryo shown is germband extended when all three rows of first wave neuroblasts have segregated (approx. 6 hours). At the blastoderm stage of development, the anti-Sna antiserum labels the presumptive mesoderm. This antiserum, however, does not stain the ectoderm prior to neuroblast segregation. Thus, during the early stages of neurogenesis when the location and spacing of neuroblasts is being determined, there is a discrepancy between the transcription and translation patterns. This cannot be solely attributed to antibody sensitivity since there are equivalent levels of sna RNA present during the period of ectodermal expression and subsequent neuroblast expression. At the very least, translation of sna transcripts must be less efficient in the ectoderm compared with neuroblasts. Similar observations have been made in the case of deadpan and genes from the AS-C.

Fig. 2.

Pan-neural distribution of Sna protein during neurogenesis. Sna protein was visualized with a polyclonal anti-Sna antiserum (Kosman et al., 1991) followed by a fluoresceine-labeled secondary antibody. The embryo shown is germband extended when all three rows of first wave neuroblasts have segregated (approx. 6 hours). At the blastoderm stage of development, the anti-Sna antiserum labels the presumptive mesoderm. This antiserum, however, does not stain the ectoderm prior to neuroblast segregation. Thus, during the early stages of neurogenesis when the location and spacing of neuroblasts is being determined, there is a discrepancy between the transcription and translation patterns. This cannot be solely attributed to antibody sensitivity since there are equivalent levels of sna RNA present during the period of ectodermal expression and subsequent neuroblast expression. At the very least, translation of sna transcripts must be less efficient in the ectoderm compared with neuroblasts. Similar observations have been made in the case of deadpan and genes from the AS-C.

Distinct sna promoter elements control PNS and CNS expression

To identify segments of the sna promoter required for expression in the nervous system, genomic fragments 5′ to the sna transcription unit were fused to the bacterial LacZ gene (Fig. 3) in vectors designed for P-element mediated transformation (Ip et al., 1992a). These sna promoter-LacZ constructs were introduced into the germline of flies (Rubin and Spradling, 1982). Transformed flies carrying either constructs with 6.0 or 2.8 kilobases (kb) of sna 5′ DNA sequences (Fig. 3) express LacZ RNA in a neuronal pattern virtually identical to that of the endogenous sna gene (Fig. 4A-D). These constructs also express sna in an approximately normal pattern in the presumptive mesoderm during the cellular blastoderm stage (Ip et al., 1992a).

Fig. 3.

LacZ reporter constructs. The upstream gene are represented as horizontal bars and the transcription start site (+1 bp) is indicated by the arrow. The numbers on top represent the distance in kb from the start site. The 5′ end of various truncations are indicated to the left of each construct. They all contain the native snail leader up to +100 bp (the initiation codon of snail is at +160 bp) and are inserted into the pCaSpeR-AUG-β-gal vector, which uses the heterologous translation start site of the alcohol dehydrogenase (Adh) gene. The last construct, NB, contains an upstream fragment from the NaeI site at −2.8 kb to the BamHI site at −0.25 kb attached to the basal promoter of the pWHL vector. Levels of LacZ expression in various tissues are summarized in the right column. Wild-type levels of expression are denoted by (+), low levels by (+/−), and no detectable expression by (−). CNS denotes the region of the snail enhancer required for expression in the central nervous system and includes expression in early neuroectodermal cells, neuroblasts and postmitotic neurons. PNS denotes the region of the snail enhancer sufficient for expression in PNS sensory mother precursor cells and in postmitotic neurons. The enhancer element required for mesoderm expression (Meso) has been described in detail in Ip et al., 1992a. AE is an augmentation element, which is required for elevated expression in all three tissues (Ip et al., 1992a). The restriction sites marked below the line are: A, AccI; B, BamHI; E, EcoRV; Na, NaeI; Nc, NcoI; R, RsaI.

Fig. 3.

LacZ reporter constructs. The upstream gene are represented as horizontal bars and the transcription start site (+1 bp) is indicated by the arrow. The numbers on top represent the distance in kb from the start site. The 5′ end of various truncations are indicated to the left of each construct. They all contain the native snail leader up to +100 bp (the initiation codon of snail is at +160 bp) and are inserted into the pCaSpeR-AUG-β-gal vector, which uses the heterologous translation start site of the alcohol dehydrogenase (Adh) gene. The last construct, NB, contains an upstream fragment from the NaeI site at −2.8 kb to the BamHI site at −0.25 kb attached to the basal promoter of the pWHL vector. Levels of LacZ expression in various tissues are summarized in the right column. Wild-type levels of expression are denoted by (+), low levels by (+/−), and no detectable expression by (−). CNS denotes the region of the snail enhancer required for expression in the central nervous system and includes expression in early neuroectodermal cells, neuroblasts and postmitotic neurons. PNS denotes the region of the snail enhancer sufficient for expression in PNS sensory mother precursor cells and in postmitotic neurons. The enhancer element required for mesoderm expression (Meso) has been described in detail in Ip et al., 1992a. AE is an augmentation element, which is required for elevated expression in all three tissues (Ip et al., 1992a). The restriction sites marked below the line are: A, AccI; B, BamHI; E, EcoRV; Na, NaeI; Nc, NcoI; R, RsaI.

Fig. 4.

LacZ expression from sna promoter LacZ fusion genes. LacZ transcripts were detected by whole-mount in situ hybridization to antisense LacZ RNA probes labeled with digoxigenin. Expression of LacZ directed by 2.8 kb of the sna promoter is virtually indistinguishable from that of the endogenous sna gene. During neurogenesis LacZ is first expressed in the neuroectoderm (A; approx. 4.5–5 hours - compare with sna: Fig. 1A) and then in both the ectoderm and in neuroblasts (B; approx. 5.5–6 hours - compare with sna: Fig. 1B). The open arrow points to mesoderm, the arrowheads point to ectoderm, and the solid arrow indicates the intervening neuroblast layer. LacZ expression is restricted to the neuroblast layer (arrow) after the first wave of neuroblast segregation is complete (C; approx. 6 hours - compare with sna: Fig. 1F) as expression is excluded from the ectoderm (arrowhead). LacZ is also expressed in sensory mother cell precursors of the PNS (D; approx. 6.5-7 hours). Sequences necessary for PNS expression, but not CNS or mesoderm expression, are contained on a fragment containing only 1.6 kb of the sna promoter. Expression in all or most PNS cells is also directed by a 0.8 kb upstream fragment (E; approx. 7.5 hours, F; approx. 9–10 hours - compare with sna: Fig. 1I), while CNS expression is virtually eliminated in the neuroectoderm and in neuroblasts (G; approx. 6.5-7 hours - compare to Fig. 1G) as well as later in postmitotic neurons (H; approx. 10 hours - compare to CNS expression in Fig. 1J). No LacZ expression is driven from a 0.25 kb promoter fragment. A 2.8 kb fragment lacking the most proximal 0.25 kb also fails to direct LacZ expression, suggesting that a necessary enhancer element for both CNS and PNS expression lies within the proximal 0.25 kb fragment. These data suggest that the sna promoter has separate CNS and PNS elements. The PNS and CNS elements are also separable from the wing and haltere imaginal disc elements since these disc primordia are not labeled in any of promoter LacZ fusions tested (Ip et al., 1992b). Embryos are oriented and labeled as in Fig. 1. The embryos in A,G and H are viewed from a ventral perspective and all other embryos are viewed from the side. The focal plane in A is at the ectodermal surface; in B and C is in a mid-sagittal section; and in D-H is subectodermal.

Fig. 4.

LacZ expression from sna promoter LacZ fusion genes. LacZ transcripts were detected by whole-mount in situ hybridization to antisense LacZ RNA probes labeled with digoxigenin. Expression of LacZ directed by 2.8 kb of the sna promoter is virtually indistinguishable from that of the endogenous sna gene. During neurogenesis LacZ is first expressed in the neuroectoderm (A; approx. 4.5–5 hours - compare with sna: Fig. 1A) and then in both the ectoderm and in neuroblasts (B; approx. 5.5–6 hours - compare with sna: Fig. 1B). The open arrow points to mesoderm, the arrowheads point to ectoderm, and the solid arrow indicates the intervening neuroblast layer. LacZ expression is restricted to the neuroblast layer (arrow) after the first wave of neuroblast segregation is complete (C; approx. 6 hours - compare with sna: Fig. 1F) as expression is excluded from the ectoderm (arrowhead). LacZ is also expressed in sensory mother cell precursors of the PNS (D; approx. 6.5-7 hours). Sequences necessary for PNS expression, but not CNS or mesoderm expression, are contained on a fragment containing only 1.6 kb of the sna promoter. Expression in all or most PNS cells is also directed by a 0.8 kb upstream fragment (E; approx. 7.5 hours, F; approx. 9–10 hours - compare with sna: Fig. 1I), while CNS expression is virtually eliminated in the neuroectoderm and in neuroblasts (G; approx. 6.5-7 hours - compare to Fig. 1G) as well as later in postmitotic neurons (H; approx. 10 hours - compare to CNS expression in Fig. 1J). No LacZ expression is driven from a 0.25 kb promoter fragment. A 2.8 kb fragment lacking the most proximal 0.25 kb also fails to direct LacZ expression, suggesting that a necessary enhancer element for both CNS and PNS expression lies within the proximal 0.25 kb fragment. These data suggest that the sna promoter has separate CNS and PNS elements. The PNS and CNS elements are also separable from the wing and haltere imaginal disc elements since these disc primordia are not labeled in any of promoter LacZ fusions tested (Ip et al., 1992b). Embryos are oriented and labeled as in Fig. 1. The embryos in A,G and H are viewed from a ventral perspective and all other embryos are viewed from the side. The focal plane in A is at the ectodermal surface; in B and C is in a mid-sagittal section; and in D-H is subectodermal.

We dissected the neural element of the sna promoter by generating a series of truncations of promoter sequences (Fig. 3). Flies carrying LacZ fusion genes containing 2.2 kb, 1.6 kb, or 0.8 kb (Fig. 4E) of upstream sequences from the sna promoter express LacZ in all or most precursors and postmitotic neurons of the PNS, but not in cells of the CNS (Fig. 4F). Fusion genes containing only 0.25 kb of sna upstream sequences also express LacZ throughout the PNS but at significantly lower levels than observed with either the 1.6 or 0.8 kb constructs (data not shown). The finding that an 0.8 kb promoter fragment is sufficient for full expression in the PNS, but not in the CNS, suggests that separate promoter elements are required for PNS and CNS expression. Expression in the PNS and CNS also appears to be separable from a previously identified mesoderm element (Ip et al., 1992a), since LacZ fusion genes with 2.2 kb of upstream sna sequences are expressed in the mesoderm (and PNS) but not in the CNS, while 0.8 kb or 0.25 kb constructs are expressed in the PNS but not in the mesoderm. Deletions of the 2.8 kb construct, removing as little as 0.25 kb at the 3′ end of the promoter, abolish all expression in the CNS and PNS and substantially reduce expression in the mesoderm, suggesting that a general augmentation element is contained in this region (Ip et al., 1992a; data not shown). Thus, sna expression is independently regulated in the PNS, CNS and mesoderm (Fig. 3).

sna neural expression is largely independent of the AS-C and da proneural genes

We have examined sna expression in AS-C and da mutant embryos to determine whether the early or late components of the sna neuroectodermal expression pattern are under the control of the proneural genes or are independently regulated. The early neuroectodermal expression of sna is indistinguishable from the normal pattern in null dakx136 (Fig. 5A) and Df(1)scB57 mutant embryos. Subtle defects in the neuroectodermal pattern may have evaded our attention, however, since sna expression is dynamic during this period. The pan-neural expression of sna in early neuroblasts also appears normal in dakx136 (Fig. 5C), although at later stages some neuroblasts may prematurely stop expressing sna (data not shown). sna is also expressed in PNS preclusters (Fig. 5D) and early sensory mother cells indicating that neuronal precursor cells form in the PNS, as well as in the CNS of dakx136 mutants, but fail to generate mature neurons. These sna-expressing cells in dakx136 mutant embryos are tentatively identified as part of the PNS because an epidermal precluster is first observed, followed by brief expression in cells that are enlarged and subectodermal. Consistent with the view that these peripheral cells are PNS cells and not mesodermal precursors (derived from an early role of sna in mesoderm formation) is the observation that cells in similar positions are observed in sna mutants and sna twi double mutants, which entirely lack mesoderm (data not shown). As other known early neuronal markers are not expressed in da mutants, sna is a useful marker for this early stage of PNS neurogenesis. sna pan-neural expression is also normal in scB57 mutants (Fig. 5E) taking into account that a subset of neuroblasts fail to form in these embryos (Jimenez and Campos-Ortega, 1990).

Fig. 5.

sna expression in da and AS-C mutants. sna expression in early stages of neurogenesis is normal in dakx136 null embryos (A,C and D) and is expressed in most or all remaining neuroblasts in scB57 mutants (E). Homozygous dakx136 mutant embryos were identified by using a CyO ftz-LacZ balancer that expresses β-galacosidase protein (β-gal) in a pair-rule pattern during germband extension and in a subset of the nervous system at later stages (B). Embryos were double labeled (Sturtevant et al., 1993) for sna RNA expression using a digoxigenin-labeled RNA probe visualized by alkaline phosphatase and NBT as substrate (blue reaction precipitate) and for β-gal protein using an anti-β-gal antibody visualized by HRP activity using DAB as substrate (brown reaction product). Mutant embryos were identified as those only expressing sna transcripts (blue staining) without any brown labeling (ftz-LacZ from balancer). scB57 mutants were identified, based on the phenotype of missing neuroblasts (E). Embryos shown are (A) a dakx136 mutant embryo (ventral view of the ectoderm, 4-5 hours); (B) a wild-type embryo (saggital view, 5-6 hours); (C) a dakx136 mutant embryo (subectodermal view of S1 neuroblasts, 5.5-6 hours); (D) a dakx136 mutant embryo (ectodermal lateral view of PNS preclusters, 6 hours); and (E) a scB57 mutant embryo (subectodermal view of neuroblast phase when PNS sensory mother cells are formed, 6.5 hours). Stars indicate locations of missing r1 neuroblasts.

Fig. 5.

sna expression in da and AS-C mutants. sna expression in early stages of neurogenesis is normal in dakx136 null embryos (A,C and D) and is expressed in most or all remaining neuroblasts in scB57 mutants (E). Homozygous dakx136 mutant embryos were identified by using a CyO ftz-LacZ balancer that expresses β-galacosidase protein (β-gal) in a pair-rule pattern during germband extension and in a subset of the nervous system at later stages (B). Embryos were double labeled (Sturtevant et al., 1993) for sna RNA expression using a digoxigenin-labeled RNA probe visualized by alkaline phosphatase and NBT as substrate (blue reaction precipitate) and for β-gal protein using an anti-β-gal antibody visualized by HRP activity using DAB as substrate (brown reaction product). Mutant embryos were identified as those only expressing sna transcripts (blue staining) without any brown labeling (ftz-LacZ from balancer). scB57 mutants were identified, based on the phenotype of missing neuroblasts (E). Embryos shown are (A) a dakx136 mutant embryo (ventral view of the ectoderm, 4-5 hours); (B) a wild-type embryo (saggital view, 5-6 hours); (C) a dakx136 mutant embryo (subectodermal view of S1 neuroblasts, 5.5-6 hours); (D) a dakx136 mutant embryo (ectodermal lateral view of PNS preclusters, 6 hours); and (E) a scB57 mutant embryo (subectodermal view of neuroblast phase when PNS sensory mother cells are formed, 6.5 hours). Stars indicate locations of missing r1 neuroblasts.

We have analyzed the basis for nervous system specific expression of the sna gene during embryogenesis. We found that sna mRNA is first expressed in the neuroectoderm prior to neuronal precursor formation and then is maintained in most or all neuronal precursor cells. In the CNS neuroectoderm, sna is expressed in a pattern of broad segmentally repeated stripes modulated in longitudinal rows of cells that will subsequently contribute to the S1 wave of neuroblasts. This neuroectodermal expression differs from that of AS-C transcripts in several respects. AS-C genes T3, T4, and T5 are expressed earlier in the presumptive neuroectoderm than sna (i.e. during the late cellular blastoderm) and then are expressed in sharply defined patches of cells just prior to neuroblast segregation during late germband extension (Cabrera et al., 1987). Additionally, sna expression is maintained for a longer period than AS-C transcripts in neuroblasts following delamination.

The distribution of Sna protein observed using the antiserum generated by Kosman et al. (1991) closely follows that of sna mRNA, with the one notable exception that it is not detectable in neuroectodermal cells. Since sna mRNA is expressed at comparable levels in the neuroectoderm and in neuronal precursor cells, it is likely that sna is regulated post-transcriptionally. Possible explanations for this difference between protein and RNA expression in the neuroectoderm include: (1) translation of the sna message is significantly less efficient in neuroectodermal cells than in neuroblasts, (2) Sna exists in a modified form in the neuroectoderm versus in neuroblasts, or (3) Sna protein is less stable in neuroectodermal cells than in neuroblasts. Similar observations of potential posttranscriptional regulation of genes in neuronal precursor cells versus proneural clusters have been described (Cabrera, 1990; Martin-Bermudo et al., 1991; Bier et al., 1992). In the case of the T3 AS-C gene, one study reports that T3 RNA, but not protein, is expressed in proneural preclusters (Cabrera, 1990), while results from another group using a different anti-T3 antiserum find both RNA and protein in the preclusters (Martin-Bermudo et al., 1991). The antiserum used by Cabrera et al. was raised against a short peptide containing a potential phosphorylation site, while the antiserum used by Martin-Bermudo et al. was generated to a larger region of the protein. Thus, one explanation for the different staining results could be due to the antiserum of Cabrera recognizing the unphosphorylated form of the T3 peptide (hypothesized to be in neuroblasts), but not the phosphorylated protein (the form present in proneural patches). Presumably, the Martin-Bermudo antiserum recognized both the phosphorylated and unphos-phorylated forms. The basis for the different staining in the neuroectoderm versus neuroblasts in the case of sna (and dpn) is more likely to be at the level of translational efficiency or protein stability since the antisera were generated against large fusion proteins, but the possibility of post-translational modifications cannot be ruled out. These data suggest that genes encoding at least two different classes of transcription factors are subject to different post-transcriptional processing in the neuroectoderm than in other cell types and that neuroectodermal cells may possess posttran-scriptional mechanisms that distinguish between various classes of transcripts or proteins.

We localized cis-acting sequences required for expression of sna in the nervous system to a 2.8 kb promoter fragment. This fragment drives LacZ expression in a pattern virtually identical to that of the endogenous sna gene. 2.2 kb,1.6 kb, or 0.8 kb upstream fragments are capable of directing expression in the PNS but not in the CNS. Whether these data reflect a requirement for distinct PNS versus CNS promoter elements, or alternatively, a requirement for more copies of a common iterated element in CNS cells versus PNS cells remains to be determined. We have recently analyzed a pan-neural promoter fragment controlling the expression of an unrelated pan-neural gene (deadpan) which also decomposes into PNS- and CNS-specific elements (J. Emery and E. Bier, unpublished data). Separate control of CNS and PNS expression in these two cases is unexpected since all known genes acting at the precursor formation stage of neurogenesis are expressed in both the CNS and PNS and affect the development of both components of nervous system. These results suggest that yet uncharacterized genetic pathways distinguish these two obvious morphological and functional subdivisions of the nervous system at a very early developmental stage.

Based on current models in which da and AS-C play key roles in establishing zones of proneural competence, it might have been anticipated that proneural genes acting through a single promoter element could regulate sna expression in both CNS and PNS precursor cells. Our data, however, indicate that if activation of sna transcription is mediated by promoter elements responding to various combinations of proneural genes, there must be more than one such element, one of which is sufficient for PNS but not for CNS expression. Furthermore, these promoter elements appear to be responsive to other activators, since sna expression is largely normal in da and AS-C mutants. It should be noted that normal expression of sna during neurogenesis in dakx136 and scB57 mutants is similar to the normal expression of Hunchback protein in neuroblasts in these mutants (Jimenez and Campos-Ortega, 1990), but differs dramatically from regulation of the pan-neural gene deadpan. deadpan is not expressed in any neuronal precursor cells in dakx136 mutants and is expressed only in a subset of the remaining neuroblasts in scB57 embryos (Bier et al., 1992). These data strongly suggest that there is a genetic pathway independent of da and AS-C that contributes to activating sna and hb expression prior to and during neuronal precursor selection. Alternatively, the neuronal enhancers of these genes may respond to the low levels of da provided by the maternal contribution of da whereas the deadpan enhancer may require higher levels, derived from zygotic da expression, to be active. The existence of an alternative genetic pathway specifying neuronal precursor cells is also consistent with the observation that the neuroectodermal pattern of sna differs significantly from that of AS-C. Evidence for parallel pathways in neuronal precursor selection has also been reported in the case of the R7 photoreceptor. An R7 enhancer trap marker is expressed independently of the sevenless signaling pathway (Mlodzik et al., 1992).

Although sna mutant embryos are contorted due to the lack of mesoderm, it is clear that development of the nervous system is essentially normal in these mutants, based on staining with neuronal markers such as deadpan, sna itself, mAb 22C10, anti-Eve, and anti-Engrailed (E.B., unpublished observations), indicating that sna alone is not required for morphological development of this tissue. We and others (Whitely et al., 1992) have identified additional genes encoding Zn finger proteins that are highly related to sna, at least one of which, called scratch, is expressed in a panneural pattern (E.B. unpublished data). Thus, in addition to the known helix-loop-helix proneural genes, it is possible that a family of related Zn finger proteins contributes to early nervous system development. An interplay between the sna Zn finger protein acting as a transcriptional repressor and helix-loop-helix transcriptional activators has been observed during mesoderm specification of ventral blastoderm cells (Kosman et al., 1991, Rao et al., 1991; Leptin, 1991; Alberga et al., 1991; Ip et al., 1992a,b). It will be interesting to determine whether Sna, possibly together with other Zn finger proteins, functions analogously during neurogenesis to repress non-neuronal fates.

The separate regulation of sna expression in the CNS versus PNS, the relatively normal expression of sna in da mutants, and the differences in the details of the sna and AS-C expression patterns all underscore the limitations of current models of neurogenesis in which da and AS-C play key roles in establishing the competence to form CNS neuroblasts and PNS sensory mother cells from which individual neuronal precursor cells are selected by a lateral inhibitory action of the neurogenic genes (Ghysen and Dambly-Chaudiere, 1989; Jan and Jan, 1990; Campos-Ortega and Jan, 1991). Thus, analysis of sna pan-neural expression has revealed that important components of the genetic hierarchy regulating neurogenesis remain to be discovered. This view is also supported by the relative scarcity of lethal mutations affecting nervous system formation (less than 1%) identified in screens of deficiency stocks (Jan et al., 1986) or in EMS screens (Seeger et al., 1993; H. Bellen, personal communication; E. Bier, unpublished data), relative to the high frequency (30-40%) of enhancer traps expressed in the nervous system (Bier et al., 1989). These data imply that a significant fraction of genes expressed in the nervous system may be individually dispensable. The instances of genetic ‘redundancy’ in neurogenesis (e.g. AS-C, E(spl), and deadpan) may therefore not be rare exceptions, but rather may be representative examples of parallel genetic control of nervous system formation.

The observation that the sna neuronal enhancer element does not behave according to expectation, based on prevalent models of early neurogenesis, is in striking contrast to the remarkable degree to which genetic models have predicted the relevant regulators of genes functioning during early anterior-posterior and dorsal-ventral patterning. For example, when enhancer elements directing expression of even-skipped stripe-2 along the anterior-posterior axis were dissected (Stanojevic et al., 1991; Small et al., 1992), or when enhancers controlling expression of sna (Ip et al., 1992a), twi (Jiang et al., 1991; Jiang and Levine, 1993) and rho (Ip et al., 1992b) in longitudinal domains along the dorsal-ventral axis were analyzed, the great majority of key regulators were already known from mutants affecting early patterning (e.g. bcd, hb, gt, and kr for eve stripe 2; and dl, sna, and twi for the dorsal ventral axis). If we wish to understand nervous system formation in the same detail as we do early pattern formation, the fragmentary genetic analysis of neurogenesis and recurring examples of parallel genetic pathways need to be addressed by further systematic classic mutant screens and alternative methods to reveal redundant pathways.

We thank Jason W. O’Neill for help with whole-mount in situ hybridization, Bill Harris and Kathryn S. Burton for critical comments and Kathryn S. Burton for preparing figures. This work was supported by an NIH Grant (RO1-NS29870-01), and Research Grant No. 5-FY92-1175 from the March of Dimes Birth Defects Foundation. Y. T. I. is a Hoffman LaRoch Fellow of the Life Sciences Foundation and E. B. was supported by funds from the McKnight Neuroscience Foundation, Sloan Foundation and an ACS Junior Faculty Award.

Alberga
,
A.
,
Boulay
,
J.-L.
,
Kempe
,
E.
,
Dennefeld
,
C.
, and
Haenlin
,
M.
(
1991
).
The snail gene required for mesoderm formation in Drosophila is expressed dynamically in derivatives of all three germ layers
.
Development
111
,
983
992
.
Alonso
,
M. C.
and
Cabrera
,
C. V.
(
1988
).
The achaete-scute complex of Drosophila melanogaster comprises four homologous genes
.
EMBO J
.
7
,
2585
2591
.
Artavanis-Tsakonas
,
S.
(
1988
).
The molecular biology of the Notch locus and the fine tuning of differentiation in Drosophila
.
Trends Genet
.
4
,
95
100
.
Artavanis-Tsakonas
,
S.
and
Simpson
P.
(
1991
).
Choosing a cell fate: a view from the Notch locus
.
Trends Genet
.
7
,
403
407
.
Bier
,
E.
,
Vaessin
,
H.
,
Shepherd
,
S.
,
Lee
,
K.
,
McCall
,
K.
,
Barbel
,
S.
,
Ackerman
,
L.
,
Carretto
,
R.
,
Uemura
,
T.
,
Grell
,
E.
,
Jan
L. Y.
, and
Jan
,
Y. N.
. (
1989
).
Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector
.
Genes Dev
.
3
,
1273
1287
.
Bier
,
E.
,
Vaessin
,
H.
,
Younger-Shepherd
,
S.
,
Jan
,
L. Y.
and
Jan
Y. N.
(
1992
).
deadpan, an essential pan-neural gene in Drosophila, encodes a helix-loop-helix protein similar to the hairy gene product
.
Genes Dev
.
6
,
2137
2151
.
Boulay
,
J. L.
,
Dennefeld
,
C.
and
Alberga
,
A.
(
1987
).
The Drosophila developmental gene snail encodes a protein with nucleic acid binding fingers
.
Nature
,
330
,
395
398
.
Cabrera
,
C. V.
,
Martinez-Arias
,
A.
and
Bate
,
M.
(
1987
).
The expression of three members of the achaete-scute gene complex correlates with neuroblast segregation in Drosophila
.
Cell
50
,
425
433
.
Cabrera
,
C. V.
(
1990
).
Lateral inhibition and cell fate during neurogenesis in Drosophila: the interactions between scute, Notch, and Delta
.
Development
109
,
733
742
.
Campos-Ortega
,
J. A.
and
Hartenstein
,
V.
(
1985
).
The Embryonic Development of Drosophila melanogaster
.
New York
:
Springer-Verlag
.
Campos-Ortega
,
J. A.
and
Jan
,
Y. N.
(
1991
).
Genetic and molecular basis of neurogenesis in Drosophila melanogaster
.
Annu. Rev. Neurosci
.
14
,
399
420
.
Caudy
,
M.
,
Grell
,
E. H.
,
Dambly-Chaudiere
,
C.
,
Ghysen
,
A.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1988a
).
The maternal sex determination gene daughterless has zygotic activity necessary for the formation of peripheral neurons in Drosophila
.
Genes Dev
.
2
,
843
852
.
Caudy
,
M.
,
Vaessin
,
H.
,
Brand
,
M.
,
Tuma
,
R.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1988b
).
daughterless, a gene essential for both neurogenesis and sex determination in Drosophila has sequence similarities to myc and the achaete-scute complex
.
Cell
55
,
1061
1067
.
Dambly-Chaudiere
,
C.
and
Ghysen
,
A.
(
1987
).
Independent subpatterns of sense organs require independent genes of the achaete-scute complex in Drosophila larvae
.
Genes Dev
.
1
,
297
306
.
Ghysen
,
A.
and
Dambly-Chaudiere
,
C.
(
1989
).
Genesis of the Drosophila peripheral nervous system
.
Trends Genet
.
5
,
251
255
.
Gonzalez
,
F.
,
Romani
,
S.
,
Cubas
,
C.
,
Modolell
,
J.
and
Campuzano
,
S.
(
1989
).
Molecular analysis of the asense gene, a member of the achaete-scute complex of Drosophila melanogaster, and its novel role in optic lobe development
.
EMBO J
.
8
,
3553
3562
.
Hartenstein
,
V.
(
1988
).
Development of Drosophila larval sensory organs: Spatiotemporal pattern of sensory neurons, peripheral axonal pathways and sensilla differentiation
.
Development
102
,
869
886
.
Ip
,
Y. T.
,
Park
,
R. E.
,
Kosman
,
D.
,
Yazdanbakhsh
,
K.
and
Levine
,
M.
(
1992a
).
dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo
.
Genes Dev
.
6
,
1518
1530
.
Ip
,
Y. T.
,
Park
,
R. E.
,
Kosman
,
D.
,
Bier
,
E.
and
Levine
,
M.
(
1992b
).
The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo
.
Genes Dev
.
6
,
1728
1739
.
Jan
,
Y. N
,
Bodmer
,
R.
,
Ghysen
,
A.
,
Dambly-Chaudiere
,
C.
and
Jan
,
L. Y.
(
1986
).
Mutations affecting the peripheral nervous sytem in Drosophila embryos. Proceedings of the UCLA Symposium for Molecular Entomology
.
J. Cell Biochem
.
45
56
.
Jan
,
Y. N.
and
Jan
,
L. Y.
(
1990
).
Genes required for specifying cell fates in Drosophila embryonic nervous system
.
Trends Neurosci
.
13
,
493
498
.
Jiang
,
J.
,
Kosman
,
D.
,
Ip
,
Y. T.
and
Levine
,
M.
(
1991
).
The Dorsal morphogen gradient regulates the mesoderm determinant twist in early Drosophila embryos
.
Genes Dev
.
5
,
1881
1891
.
Jiang
,
J.
and
Levine
,
M.
(
1993
).
Binding affinities and cooperative interactions with bHLH activators delimit threshold responses to the Dorsal gradient morphogen
.
Cell
72
,
741
742
.
Jimenez
,
F.
and
Campos-Ortega
,
J. A.
(
1990
).
Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of D. melanogaster
.
Neuron
5
,
81
89
.
Klämbt
,
C.
,
Knust
,
E.
,
Tietze
,
K.
and
Campos-Ortega
,
J. A.
(
1989
).
Closely related transcripts encoded by the neurogenic gene complex Enhancer of split of Drosophila melanogaster
.
EMBO J
.
8
,
203
210
.
Kosman
,
D.
,
Ip
,
Y. T.
,
Levine
,
M.
and
Arora
,
K.
(
1991
).
Establishment of the mesoderm neuroectoderm boundary in the Drosophila embryo
.
Science
254
,
118
122
.
Leptin
,
M.
(
1991
).
twist and snail as positive and negative regulators during Drosophila mesoderm development
.
Genes Dev
.
5
,
1568
1576
.
Lindsley
,
D. L.
and
Grell
,
E. H.
(
1968
).
Genetic variations in Drosophila melanogaster
.
Carnegie Institute of Washington
,
Washington, D. C
.
Lindsley
,
D. L.
and
Zimm
,
G. G.
(
1992
).
The Genome of Drosophila melanogaster
.
San Diego, CA
:
Academic Press Inc
.
Maniatis
,
T.
,
Fritsch
,
E. F.
and
Sambrook
,
J.
(
1982
).
Molecular Coning: A Laboratory Manual. Cold Spring Harbor
,
New York
:
Cold Spring Harbor Laboratory
.
Martin-Bermudo
,
M. D.
,
Martinez
,
C.
,
Rodriguez
,
A.
and
Jimenez
,
F.
(
1991
).
Distribution and function of the lethal of scute gene product during early neurogenesis of Drosophila
.
Development
113
,
445
454
.
Mlodzik
,
M.
,
Hiromi
,
Y.
,
Goodman
,
C. S.
and
Rubin
,
G. M.
(
1992
).
The presumptive R7 cell of the developing Drosophila eye receives positional information independent of sevenless, boss, and sina
.
Mech. Dev
.
37
,
37
42
.
Murre
,
C.
,
Schonoleber-McCaw
,
P.
and
Baltimore
,
D.
(
1989a
).
A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, Myo D and myc proteins
.
Cell
56
,
777
783
.
Murre
,
C.
,
Schonoleber-McCaw
,
P.
,
Vaessin
,
H.
,
Caudy
,
M.
,
Jan
,
L. Y.
,
Jan
,
Y. N.
,
Cabrera
,
C. V.
,
Buskin
,
J. N.
,
Hauschka
,
S. D.
,
Lassar
,
A. B.
,
Weintraub
,
H.
and
Baltimore
,
D.
(
1989b
).
Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence
.
Cell
58
,
537
544
.
Rao
,
Y.
,
Vaessin
,
H.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1991
).
Neuroectoderm in Drosophila embryos is dependent on the mesoderm for positioning but not for formation
.
Genes Dev
.
5
,
1577
1588
.
Romani
,
S.
,
Campuzano
,
S.
and
Modolell
J.
(
1987
).
The achaete-scute complex is expressed in neurogenic regions of Drosophila embryos
.
EMBO J
.
6
,
2085
2092
.
Romani
,
S.
,
Campuzano
,
S.
,
Macagno
,
E. R.
and
Modolell
,
J.
(
1989
).
Expression of achaete and scute genes in Drosophila imaginal discs and their function in sensory organ development
.
Genes Dev
.
3
,
997
1007
.
Rubin
,
G. M.
and
Spradling
,
A.
(
1982
).
Genetic transformation of Drosophila with transposable vectors
.
Science
218
,
348
353
.
Seeger
,
M.
,
Tear
,
G.
,
Ferres-Marco
,
D.
and
Goodman
,
C. S.
(
1993
).
Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline
.
Neuron
10
,
409
426
.
Skeath
,
J. B.
and
Carroll
,
S.B.
(
1991
).
Regulation of achete-scute gene expression and sensory organ pattern formation in the Drosophila wing
.
Genes Dev
.
5
,
984
995
.
Skeath
,
J. B.
and
Carroll
,
S. B.
(
1992
).
Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo
.
Development
114
,
939
946
.
Small
,
S.
,
Blair
,
A.
and
Levine
,
M.
(
1992
).
Regulation of even-skipped stripe 2 in the Drosophila embryo
.
EMBO J
.
11
,
4047
4057
.
Spradling
,
A.
(
1986
).
P-element-mediated transformation
.
In Drosophila: A Practical Approach
(ed.
D. B.
Roberts
), pp.
175
197
.
Oxford, Washington, D. C
:
IRL Press
.
Stanojevic
,
D.
,
Small
,
S.
and
Levine
,
M.
(
1991
).
Regulation of a segmentation stripe by overlapping activators and repressors in the Drosophila embryo
.
Science
254
,
1385
1387
.
Sturtevant
,
M.
,
Roark
,
M.
and
Bier
,
E.
(
1993
).
The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signaling pathway
.
Genes Dev
.
7
,
961
973
.
Tautz
,
D.
and
Pfeifle
,
C.
(
1989
).
A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals a translational control of segmentation gene hunchback
.
Chromosma
98
,
81
85
.
Villares
,
R.
and
Cabrera
,
C. V.
(
1987
).
The achaete-scute gene complex of D. melanogaster: conserved domains in a subset of genes required for neurogenesis and their homology to myc
.
Cell
50
,
415
424
.
Whitely
,
M.
,
Noguchi
,
P. D.
,
Sensabaugh
,
S. M.
,
Odenwald
,
W. F.
and
Kassis
,
J. A.
(
1992
).
The Drosophila gene escargot encodes a zinc-finger motif found in snail-related genes
.
Mech. Dev
.
36
,
117
127
.