Skip to main content
NCBI home page
Genes & Development logoLink to Genes & Development
. 1998 Nov 15;12(22):3603–3612. doi: 10.1101/gad.12.22.3603

Dorsoventral patterning in the Drosophila central nervous system: the vnd homeobox gene specifies ventral column identity

Jocelyn A McDonald 1,2, Scott Holbrook 2, Takako Isshiki 1,2, Joseph Weiss 3, Chris Q Doe 1,2,5, Dervla M Mellerick 4,5
PMCID: PMC317246  PMID: 9832511

Abstract

The Drosophila CNS develops from three columns of neuroectodermal cells along the dorsoventral (DV) axis: ventral, intermediate, and dorsal. In this and the accompanying paper, we investigate the role of two homeobox genes, vnd and ind, in establishing ventral and intermediate cell fates within the Drosophila CNS. During early neurogenesis, Vnd protein is restricted to ventral column neuroectoderm and neuroblasts; later it is detected in a complex pattern of neurons. We use molecular markers that distinguish ventral, intermediate, and dorsal column neuroectoderm and neuroblasts, and a cell lineage marker for selected neuroblasts, to show that loss of vnd transforms ventral into intermediate column identity and that specific ventral neuroblasts fail to form. Conversely, ectopic vnd produces an intermediate to ventral column transformation. Thus, vnd is necessary and sufficient to induce ventral fates and repress intermediate fates within the Drosophila CNS. Vertebrate homologs of vnd (Nkx2.1 and 2.2) are similarly expressed in the ventral CNS, raising the possibility that DV patterning within the CNS is evolutionarily conserved.

Keywords: NK-2, ind, msh, neuroectoderm, neuroblast, neurogenesis, cell shape

The Drosophila embryonic central nervous system (CNS) develops from a bilateral neuroectoderm that forms adjacent to the specialized cells of the ventral midline. Neuroectoderm on each side of the ventral midline can be subdivided, on the basis of patterns of gene expression and neuroblast formation, into an orthogonal grid of four rows (1, 3, 5, 7) along the anteroposterior (AP) axis and three columns (ventral, intermediate, and dorsal) along the DV axis. The earliest neuroblast array has four neuroblasts in the ventral column, two in the intermediate column, and four in the dorsal column. Neuroblasts divide repeatedly to produce a series of smaller ganglion mother cells (GMCs), each of which produce two postmitotic neurons or glia. Every neuroblast is uniquely identifiable on the basis of its AP and DV position, and each generates a characteristic family of neurons and glia.

Neuroblast formation is regulated by the proneural genes achaete, scute, and lethal of scute (for review, see Campos-Ortega 1993). Each of these proneural genes is expressed in clusters of 4–6 cells at different positions within the neuroectoderm (e.g., achaete is expressed in four clusters, in the ventral and dorsal columns of rows 3 and 7). Proneural genes promote the formation of neuroblasts, whereas Delta–Notch signaling inhibits neuroblast formation; the balance of proneural and Notch activity results in the formation of a single neuroblast from each cluster (for review, see Campos-Ortega 1993).

What are the cues that specify correct neuroblast identity along the AP and DV axes? The segment polarity genes wingless, hedgehog, gooseberry, and engrailed are expressed in stripes in the neuroectoderm and specify the AP row identity of neuroblasts (Chu-LaGraff and Doe 1993; Zhang et al. 1994; Skeath et al. 1995; Bhat 1996; Matsuzaki and Saigo 1996; Bhat and Schedl 1997). Conditional inactivation (for wingless; Chu-LaGraff and Doe 1993) or misexpression (for gooseberry; Skeath et al. 1995) experiments show that segment polarity gene function is required in the neuroectoderm, prior to neuroblast delamination, for the proper specification of neuroblast identity.

Less is known about how neuroectoderm and neuroblast fates are established along the DV axis. ventral nervous system defective (vnd) is an NK-2 homeobox gene expressed in the ventral column of neuroectoderm and neuroblasts (Jiménez et al. 1995; Mellerick and Nirenberg 1995). vnd is required to activate proneural gene expression in a subset of the ventral column neuroectoderm and neuroblasts (Skeath et al. 1994), and for the formation of a subset of ventral and intermediate column neuroblasts (Jiménez and Campos-Ortega 1990; Skeath et al. 1994). Although vnd is expressed in the ventral column and is required for proper neuroblast formation, the role of vnd in specifying ventral column identity has not been investigated. Dorsal column neuroblast identity is specified, at least in part, by the muscle segment homeobox (msh) gene (Buescher and Chia 1997; Isshiki et al. 1997), which is expressed in the dorsal column neuroectoderm and neuroblasts (D’Alessio and Frasch 1996; Isshiki et al. 1997).

In this study, we show that Vnd protein is detected in the nuclei of all ventral column neuroectoderm and neuroblasts, and is maintained in a subset of neurons derived from these neuroblasts. We use three methods to assay DV column identity in wild-type and vnd mutant embryos; molecular markers specific for different DV columns, cell morphological differences between DV columns, and a cell lineage marker for ventral and intermediate column neuroblasts. We show that loss of vnd transforms ventral column into intermediate column identity; conversely, ectopic vnd transforms intermediate column into ventral column identity. We conclude that the vnd homeobox gene is necessary to specify ventral column cell fate and repress intermediate and dorsal column fate within the Drosophila CNS. In the accompanying paper (Weiss et al. 1998), we show that a newly identified homeobox gene, intermediate neuroblasts defective (ind), plays a similar role in specifying intermediate column fate.

Results

Vnd protein is detected in ventral column neuroectoderm and neuroblasts

Vnd protein is first detected at the blastoderm stage in bilateral stripes corresponding to the future ventral column of the CNS (Fig. 1A). Following gastrulation, stage 8 embryos have Vnd protein in the nuclei of the ventral midline cells, as well as in the bilateral ventral column neuroectoderm and neuroblasts (data not shown). By stage 9, there is little or no Vnd protein in the ventral midline cells, but Vnd levels remain high in the ventral column neuroectoderm and neuroblasts (Fig. 1C–F). During these stages, the Vnd protein pattern is identical to the vnd RNA pattern (Jiménez et al. 1995; Mellerick and Nirenberg 1995) except that there is a slight lag between the time of transcription and translation. At stage 11 and later, Vnd protein is detected in a complex pattern of neurons including some, but not all, of the neuronal progeny of the ventral column neuroblasts. For example, Vnd protein is detected transiently in the U neurons derived from the ventral column neuroblast, NB 7-1 (Fig. 1G). In contrast, Vnd protein is detected in the pCC interneuron, but not its sibling aCC motoneuron, derived from the ventral column neuroblast, NB 1-1 (Fig. 1H). Vnd is detected in additional neurons derived from ventral column neuroblasts and is also detected in dorsally located neurons that may derive from intermediate or dorsal column neuroblasts that do not express vnd (Fig. 1H).

Figure 1.

Figure 1

Open in a new tab

Vnd protein pattern in wild-type embryos. (A) Cellular blastoderm, ventro-lateral view. Vnd is detected in two stripes adjacent to the mesoderm anlagen and extending ∼0%–90% of egg length. (B) Early stage 9, ventro-lateral view. Vnd is detected in the ventral column of neuroectoderm. (C) Stage 9, ventral view. Vnd labels all ventral column neuroectodermal cells; the ventral midline CNS cells are unstained at this stage. (D–F) Vnd labels all ventral column neuroblasts at stage 9 (D), stage 10 (E), and stage 11 (F). (G,H) Vnd is detected in a complex pattern of GMCs and neurons at stage 11 (G) and stage 15 (H). Vnd, green; Eve, red; coexpression, yellow. (G) Vnd is detected in the progeny of the ventral column neuroblasts, including the U and aCC/pCC (CC) neurons (yellow, arrow) derived from NBs 7-1 and 1-1. Vnd is not detected in the RP2 neuron (arrowhead) derived from the intermediate column NB 4-2. (H) Vnd is observed in a subset of neurons derived from ventral neuroblasts. For example, it is detected in pCC but not the sibling aCC neuron (arrows) derived from NB 1-1. In addition, Vnd is detected in neurons lying near the lateral edge of the CNS (arrowhead), likely derived from intermediate and/or dorsal column neuroblasts. Anterior is up; (triangle) ventral midline; (v) ventral column; (i) intermediate column; (d) dorsal column.

Loss of vnd transforms ventral to intermediate column identity

To assay CNS cell fates in embryos lacking vnd function (vnd embryos), we use markers that specifically label ventral, intermediate, or dorsal column neuroectoderm and neuroblasts. We use three ventral column markers: Achaete, Odd-skipped (Odd), and Prospero (Doe 1992; Skeath and Carroll 1992; Broadus et al. 1995). Achaete labels ventral and dorsal column neuroectoderm and neuroblasts of rows 3 and 7 (Fig. 2A,D), Odd labels the ventral and dorsal column neuroblasts of row 1 (Fig. 2G), whereas Prospero labels the ventral column neuroblast in row 3 (MP2; Fig. 2J). We use two intermediate column markers: Ind and Huckebein (Chu-LaGraff et al. 1995; McDonald and Doe 1997; Weiss et al. 1998). Ind labels all intermediate column neuroectoderm and neuroblasts (Fig. 3A,D), whereas Huckebein labels the intermediate neuroectoderm and neuroblasts of row 3 (e.g., NB 4-2) and the ventral and intermediate neuroectoderm of rows 1 and 5 (Fig. 3G). Lastly, we use the dorsal marker Msh, which labels all dorsal column neuroectoderm and neuroblasts (Fig. 3J; D’Alessio and Frasch 1996; Isshiki et al. 1997). Using these markers, we investigate whether vnd is necessary or sufficient to specify ventral column identity within the developing CNS.

Figure 2.

Figure 2

Open in a new tab

Vnd is necessary and sufficient to activate ventral column gene expression. Expression of the ventral column markers Achaete (Ac), Odd, and Prospero (Pros) in wild-type, vnd−, and hs–vnd embryos. (Arrow) Ventral column identity; (*) intermediate column identity; (arrowhead) dorsal column identity. (NE) Neuroectoderm; (NB) neuroblast. For other symbols and orientation, see legend to Fig. 1. (A–F) Achaete staining in neuroectoderm (A–C, stage 8) or neuroblasts (D–F, early stage 9). (A,D) In wild-type embryos, Achaete is detected in ventral and dorsal neuroectoderm and neuroblasts of rows 3 and 7, and is completely repressed in the intermediate column. (B,E) In vnd− embryos, Achaete is not detected in ventral column neuroectoderm and neuroblasts; expression is normal in the dorsal column. (C,F) In hs–vnd embryos, Achaete is ectopically detected in the intermediate column of neuroectoderm and neuroblasts of rows 3 and 7; expression is normal in the ventral and dorsal columns. (G–I) Odd staining. (G) In wild-type embryos, Odd labels a ventral column neuroblast (NB 1-1; arrow) and a dorsal column neuroblast (NB 2-5; arrowhead), but not the adjacent intermediate column neuroblast (NB 3-2;*). (H) In vnd− embryos, Odd is not detected in the ventral column NB 1-1; dorsal column expression is normal. (I) In hs–vnd embryos, there is ectopic Odd in an intermediate column neuroblast (probably NB 3-2; middle arrow); ventral and dorsal column Odd expression is normal. (J–L) Prospero staining. (J) In wild-type embryos, Prospero labels the large ventral column MP2 nucleus (arrow) as well as all smaller GMC nuclei. (K) In vnd− embryos, Pros is not detected in MP2, because of a failure in MP2 formation. (L) In hs–vnd embryos, ectopic Prospero is detected in a large nucleus of an intermediate column neuroblast (right arrow) in addition to the ventral column MP2 nucleus (left arrow).

Figure 3.

Figure 3

Open in a new tab

Vnd represses intermediate and dorsal column gene expression. Expression of the intermediate column markers Ind and Huckebein (Hkb) and the dorsal column marker Msh in wild-type, vnd−, and hs–vnd embryos. (Arrow) Ventral column identity; (*) intermediate column identity; (arrowhead) dorsal column identity. For other symbols and orientation see legends to Figs. 1 and 2. (A–F) Ind staining in neuroectoderm (A–C, stage 8) or neuroblasts (D–F, stage 9). (A,D) In wild-type embryos, Ind expression is restricted to the intermediate column neuroectoderm and neuroblasts. (B,E) In vnd− embryos, Ind is ectopically expressed in the ventral column neuroectoderm and neuroblasts; note that many Ind+ intermediate column neuroblasts shift ventrally because of loss of ventral neuroblasts. (C,F) In hs–vnd embryos, Ind is repressed in the intermediate column neuroectoderm and neuroblasts. (G–I) Huckebein staining in the neuroectoderm, stage 10; row numbers are indicated. (G) In wild-type embryos, Huckebein labels the intermediate column of row 3 and the ventral and intermediate columns of rows 1 and 5. (H) In vnd− embryos, Huckebein is ectopically expressed in the ventral column of row 3. (I) In hs–vnd embryos, Huckebein is repressed in the intermediate column of row 3, and ectopically detected in the dorsal column of row 1. (J–L) Msh staining in the neuroectoderm, stage 9. (J) In wild-type embryos, Msh expression is restricted to the dorsal column. (K) In vnd− embryos, the pattern of Msh is identical to wild type. (L) In hs–vnd embryos, Msh is partially repressed, resulting in small patches of Msh+ dorsal column neuroectoderm (arrowhead) and neuroblasts (data not shown).

In vnd embryos, Achaete, Odd, and Prospero are not detected in the ventral column neuroectoderm or neuroblasts (Fig. 2; Table 1). This phenotype is the result of a lack of gene expression in the neuroectoderm and in some neuroblasts, as well as a failure in neuroblast formation (data not shown; Jiménez and Campos-Ortega 1990; Skeath et al. 1994). For example, the ventral column NB 1-1 forms >80% of the time, yet it is never Odd+ (Fig. 2H). However, absence of a Prospero+ MP2 is the result of a failure in MP2 formation (Fig. 2K; Skeath et al. 1994). We conclude that vnd is necessary to specify ventral column neuroectoderm and neuroblast identity, and to form specific ventral column neuroblasts. The ventral column in vnd embryos could be fully or partially transformed to a different columnar identity (intermediate or dorsal) or could assume a novel identity. To distinguish between these two possibilities, we examined intermediate and dorsal column markers in vnd embryos.

Table 1.

Vnd activates ventral markers and represses intermediate and dorsal markers

Markera
Wild type (%)
vnd− (%)
hs–vndb (%)
v
i
d
v
i
d
v
i
d
Ventral
Achaete NE 100 0 97 0c 0c 100c 100 94 100
Achaete NB 99 0 100 0c 0c 100c 100 33 100
Odd NB 100 0 100 0 0 100 100 23 100
Prospero MP2 100 0 0 0 0 0 100 12 0
Intermediate
Ind NE 0 100 0 100 100 0 0 d 0
Ind NBe 0 100 0 94 100 0 0 d 0
Huckebein NEf 0 100 0 100 100 0 0 0 0
Dorsal
Msh NE 0 0 100 0 0 100 0 0 d
Msh NB 0 0 100 0 0 100 0 0 d
Open in a new tab
a

A minimum of five embryos and 112 hemisegments were assayed for each phenotype. 

b

Only embryos with a phenotype in at least one hemisegment were scored. 

c

Results from Skeath et al. (1994). 

d

Ind and Msh expression in the neuroectoderm and neuroblasts of hs–vnd embryos is partially repressed; neither marker resembles the wild-type pattern. 

e

In vnd− embryos, only hemisegments containing one or more neuroblasts were scored. 

f

Huckebein expression in row 3; for row 1, see text. 

(NE) Neuroectoderm; (NB) neuroblast; (v) ventral column; (i) intermediate column; (d) dorsal column. 

We find that in vnd embryos, intermediate column markers are ectopically expressed in the ventral column. Ind is detected in both ventral and intermediate column neuroectoderm (Fig. 3B; Table 1) and neuroblasts (Fig. 3E; Table 1), although because some ventral neuroblasts do not form in vnd embryos, the intermediate column neuroblasts often shift to a more ventral position (Fig. 3E). The intermediate column marker Huckebein is also detected in both the ventral and intermediate columns of row 3 neuroectoderm (Fig. 3H; Table 1) and neuroblasts (13% in the ventral column; n = 311 hemisegments; data not shown). Expression of Msh in dorsal column neuroectoderm (Fig. 3K; Table 1) and neuroblasts (Table 1) is normal in vnd embryos. In addition, dorsal column expression of Achaete and Odd are unchanged (Fig. 2B,E,H; Table 1). Taken together, our data show that vnd is necessary for the specification of ventral column identity and the repression of intermediate column identity within the CNS.

During the course of our gene expression analysis, we noticed that the ventral column neuroectoderm has a distinctive cellular morphology. The Vnd+ ventral column cells are frequently elongated along the DV axis to give them an asymmetry ratio of equal or >1.5 (long axis divided by short axis), whereas the Ind+ intermediate column cells are more often round, with an asymmetry ratio closer to 1.0 (Fig. 4A). In vnd embryos, we find that most ventral column cells fail to assume an elongated morphology, instead showing a round morphology characteristic of intermediate column cells (Fig. 4B). Taken together, our molecular marker and cell morphology analyses show that vnd is required to establish ventral column gene expression profiles (perhaps by direct transcriptional activation and/or repression) as well as to induce a cell shape change characteristic of the ventral column neuroectoderm.

Figure 4.

Figure 4

Open in a new tab

vnd regulates ventral column cell morphology. Camera lucida tracings of neuroectoderm in wild-type and vnd− embryos. Cells with an asymmetry ratio (long axis divided by short axis) of 1.5 or greater are black and shown as a percentage below and the number of cells counted are indicated. Ind+ cells are shown in gray and their boundary is indicated by the black line. Ventral midline, dotted line. For other symbols and orientation see legend to Fig. 1. (A) In wild-type embryos at late stage 8, 64% of the ventral column neuroectodermal cells are elongated (black; asymmetry ratio of 1.5 or more), whereas only 25% of the intermediate column neuroectodermal cells are elongated (black), the remainder being round (asymmetry ratio of 1.0–1.5). (B) In vnd− embryos at late stage 8, both ventral and intermediate columns are Ind+ (gray) and both columns now have only 24% elongated cells (black).

Ectopic vnd transforms intermediate to ventral column identity

To determine whether vnd is sufficient to specify ventral column fate, we used an hsp70–vnd transgene to ectopically express vnd in the intermediate and dorsal columns of neuroectoderm (see Materials and Methods). In embryos carrying the hsp70–vnd transgene that are heat shocked to induce ubiquitous vnd expression (hs–vnd embryos), we find that ventral markers are ectopically expressed in the intermediate and dorsal columns of the neuroectoderm and neuroblasts, and that intermediate and dorsal markers are lost. However, the transformation of intermediate to ventral cell fate is more complete than that of dorsal to ventral cell fate, as described below.

In hs–vnd embryos, the ventral column marker Achaete expands into the intermediate column, leading to Achaete expression extending continuously across ventral, intermediate, and dorsal columns of neuroectoderm (Fig. 2C; Table 1) and neuroblasts (Fig. 2F; Table 1). Similarly, the ventral neuroblast markers Prospero and Odd also show ectopic expression in the intermediate column in hs–vnd embryos (Fig. 2I,L; Table 1). Conversely, hs–vnd embryos show a loss of intermediate column marker expression. The intermediate column marker Ind is strongly repressed or completely abolished in hs–vnd embryos (Fig. 3C,F; Table 1). The row 3 intermediate column marker Huckebein is also repressed in hs–vnd embryos (Fig. 3I; Table 1); Huckebein row 5 expression appears unaffected, which is not surprising because both the ventral and intermediate columns express Huckebein in wild-type row 5 neuroectoderm. These results show that ectopic vnd results in a transformation of intermediate column to ventral column identity within both neuroectoderm and neuroblast cell types.

The dorsal column is mis-specified in hs–vnd embryos, but is not fully transformed into ventral column identity. In hs–vnd embryos, the row 1 ventral column marker Huckebein is ectopically detected in the dorsal neuroectoderm (Fig. 3I; 98% of hemisegments, n = 114), and the dorsal column marker Msh is partially repressed (Fig. 3L; Table 1). However, the ventral column marker Prospero is ectopically expressed in the intermediate column but not in the dorsal column (Fig. 2L; Table 1). Thus, ectopic vnd is sufficient to partially transform dorsal column to ventral column identity.

vnd regulates ventral column neuroblast cell lineages

To determine the extent to which Vnd controls ventral column neuroblast identity, we use a neuroblast cell lineage marker, Even-skipped (Eve), to assay the development of specific ventral and intermediate column neuroblasts. Eve labels the progeny of two ventral column neuroblasts (aCC/pCC neurons from NB 1-1; U/CQ neurons from NB 7-1) and the progeny of one intermediate column neuroblast (RP2/RP2sib neurons from NB 4-2) (Fig. 5A; Broadus et al. 1995). The pattern of Eve is a sensitive indicator for normal cell fates within these neuroblast cell lineages (see Doe et al. 1988a; Duffy et al. 1991; Chu-LaGraff and Doe 1993; Skeath et al. 1995).

Figure 5.

Figure 5

Open in a new tab

Vnd regulates neuroblast cell lineages. (A) In wild-type embryos, Eve is detected in the aCC/pCC/U/CQ neurons derived from ventral column neuroblasts 1-1 and 7-1 (v) and in the RP2/RP2sib neurons derived from the intermediate column neuroblast 4-2 (i). (B) In vnd− embryos, the Eve+ RP2/RP2sib neurons are duplicated (i; the two large cells are RP2, the two small cells are RP2sib), whereas the Eve+ aCC/pCC/U/CQ neurons are not detected. (C) In hs–vnd embryos, the Eve+ aCC/pCC/U/CQ neurons are duplicated (v); the fate of the Eve+ RP2/RP2sib neurons was not scored in this experiment. For other symbols and orientation see legend to Fig. 1.

In vnd embryos, the Eve+ aCC/pCC and U/CQ neurons, derived from ventral column neuroblasts, are never detected (Fig. 5B; n = 162 hemisegments). NB 1-1 forms and produces Prospero+, Eve− GMCs (data not shown; see Fig. 2K); thus loss of Eve from the aCC/pCC neurons is caused by an alteration in NB 1-1 identity or cell lineage. In contrast, the absence of the Eve+ U/CQ neurons is the result of failure of their parental NB 7-1 to form (see section below). In addition, vnd embryos show a duplication of the Eve+ RP2/RP2sib neurons derived from the intermediate column NB 4-2 (Fig. 5B; 12%; n = 160 hemisegments). This phenotype is the result of a transformation of a Huckebein− ventral column neuroblast into a duplicate Huckebein+ intermediate column NB 4-2 (data not shown).

Is vnd sufficient to induce ventral neuroblast cell lineages in the intermediate or dorsal column neuroblasts? In hs–vnd embryos, an excess number of the ventral column Eve+ aCC/pCC and U/CQ neurons develop (Fig. 5C). Because hs–vnd produces intermediate to ventral transformations of neuroblast identity, we think it is likely that the excess aCC/pCC and U/CQ neurons develop from duplicated ventral column neuroblasts (NBs 1-1 and 7-1). However, we cannot rule out the possibility that ectopic vnd triggers duplication of GMC identities or excess rounds of cell division. We conclude that loss of vnd results in a transformation of ventral to intermediate column neuroblast identity that is maintained in the cell lineage of at least three neuroblasts (NBs 1-1, 7-1, and 4-2), and ectopic vnd results in the converse transformation of intermediate to ventral column neuroblast identity that is maintained in the cell lineage of at least two neuroblasts (NBs 1-1 and 7-1).

vnd regulates neuroblast formation

Defects in the formation of ventral and intermediate column neuroblasts in vnd embryos have been described (Jiménez and Campos-Ortega 1990; Skeath et al. 1994). Here, we confirm this phenotype, showing that there is a loss of ventral column neuroblasts, particularly MP2 and NB 7-1 (Fig. 2). In addition, we show that in hs–vnd embryos, there is precocious formation of neuroblasts in the intermediate column. In wild-type embryos, intermediate column neuroblasts form at mid-stage 9 (Doe 1992), but in hs–vnd embryos these neuroblasts form earlier, at early stage 9, about the time the adjacent ventral column neuroblasts are forming (Fig. 2F). These data provide additional evidence for a transformation of intermediate to ventral column identity.

vnd regulates dorsoventral patterning of the procephalic neuroectoderm

vnd, msh, and ind are each expressed in the procephalic ectoderm: Vnd in a ventral domain, Ind in three small clusters of cells (1, 2, 3) at intermediate positions, and Msh in a dorsal domain (Fig. 6). There are two differences in gene expression and regulation in the procephalic region compared with the thoracic and abdominal neuroectoderm. First, Vnd and Msh share an extensive border, only interrupted by two small islands of Ind+ cells (Fig. 6A). In vnd embryos, Msh expands into the ventral domain of the procephalic neuroectoderm (Fig. 6C), showing that Vnd is required to repress msh expression in the head. Consistent with this result, misexpression of vnd leads to repression of msh (Fig. 6D). Second, the Ind+ anterior cell cluster 1 appears to coexpress Vnd (Fig. 6 and data not shown); coexpression of Vnd and Ind is never observed in the thoracic and abdominal neuroectoderm. Surprisingly, vnd embryos show a loss of the Ind+ cluster 1 (Fig. 6F), and misexpression of vnd does not affect Ind expression in cluster 1 (Fig. 6G); thus, in this domain of the embryo, Vnd is required for the development of the Ind+ cluster 1. Because the Ind+ cells of cluster 1 are primarily restricted to neuroblasts, one possibility is that loss of vnd in the neuroectoderm leads to a failure of neuroblast formation and thus to a loss of Ind+ cells, rather than that Vnd directly activates ind transcription in this domain.

Figure 6.

Figure 6

Open in a new tab

Vnd represses ind and msh expression in the procephalic neuroectoderm. (A) In wild-type embryos Vnd (brown) and Msh (blue) are detected in nonoverlapping domains within the procephalic neuroectoderm at stage 9. Ind is expressed in three domains (see E); domain 1 coexpresses Vnd, whereas domains 2 and 3 do not express either Vnd or Msh (data not shown). (B–D) Msh expression. In each panel, the approximate wild-type boundary is indicated by a broken line. (B) In wild-type embryos, Msh is detected in dorsal/posterior neuroectoderm. (C) In vnd− embryos, Msh expands into the ventral/anterior ectoderm. (D) In hs-vnd embryos, Msh is partially repressed, most frequently in the most ventral portion of the cluster. (E–G) Ind expression. (E) In wild-type embryos, Ind labels three small cell clusters (1, 2, 3). Cluster 1 coexpresses Vnd and the staining is primarily restricted to neuroblasts; clusters 2 and 3 do not express Vnd or Msh and the staining is equally strong in neuroectoderm and neuroblasts. Cluster 3 is an extension of the intermediate column expression in the germ band. (F) In vnd− embryos, cluster 1 is missing, cluster 2 is unaffected, and cluster 3 expands ventrally and anteriorly. (G) In hs–vnd embryos, Ind is repressed in cluster 2 and 3, whereas cluster 1 is unaffected. For other symbols and orientation see legend to Fig. 1.

The remaining two Ind+ cell clusters (2 and 3) are expressed and regulated in a manner consistent with the thoracic and abdominal neuroectoderm. Both Ind+ cell clusters 2 and 3 directly abut Vnd+ cells but do not express Vnd (Fig. 6E). In vnd embryos, the Ind+ cluster 3 expands ventrally into the domain normally expressing vnd, whereas Ind+ cluster 2 appears unaffected (Fig. 6F). Misexpression of vnd represses ind expression in clusters 2 and 3 (Fig. 6G). Thus, vnd can both activate ind (cluster 1) or repress ind (clusters 2 and 3) depending on the position within the procephalic neuroectoderm.

Discussion

The Vnd NK-2 type homeodomain protein is localized to the ventral column neuroectoderm and neuroblasts, and has a complex pattern in mature neurons. Analysis of molecular and morphological markers shows that loss of vnd produces a transformation of ventral to intermediate column identity, whereas ectopic vnd leads to a transformation of intermediate to ventral column identity. We conclude that Vnd plays a fundamental role in establishing ventral column identity within the Drosophila CNS.

Vnd expression during neurogenesis

Vnd is restricted to bilateral ventral columns of neuroectoderm, neuroblasts, and GMCs; it is transiently detected in the ventral midline cells. Later in neurogenesis, Vnd is detected in a subset of the neuronal progeny of ventral column neuroblasts (e.g., in pCC but not in the sibling aCC neuron derived from NB 1-1), and in additional neurons possibly derived from intermediate or dorsal column neuroblasts. This change in spatial pattern of expression is characteristic of many segmentation genes, which are utilized for patterning the neuroectoderm but then display a novel spatial distribution and function within neurons of the CNS (Carroll and Scott 1985; Frasch et al. 1987; Doe et al. 1988a,b; Jiménez and Campos-Ortega 1990; Duffy et al. 1991). It is likely that Vnd has at least two functions in the CNS: an early DV patterning function, and a later function in neuronal determination or differentiation.

vnd promotes neuroblast formation

MP2 and NB 7-1 are always missing in vnd embryos, yet other ventral column neuroblasts are much less affected (this paper; Jiménez and Campos-Ortega 1990; Skeath et al. 1994). One possible explanation is that vnd acts in combination with achaete and scute to promote the formation of MP2 and NB 7-1 (these are the only ventral column neuroblasts that express all three genes, all others express vnd and lethal of scute; Skeath et al. 1994). Embryos lacking achaete and scute can still form an MP2 neuroblast in a small percentage of cases, but if vnd is absent, MP2 never forms (this paper; Skeath et al. 1994). Thus, vnd must have an additional role in promoting neuroblast formation apart from activating achaete and scute expression.

In addition to the high frequency loss of MP2 and NB 7-1 in vnd embryos, there is a low frequency loss of other ventral and intermediate column neuroblasts (Jiménez and Campos-Ortega 1990; Skeath et al. 1994). Loss of ventral neuroblasts may be the result of an autonomous function of vnd, but the loss of intermediate column neuroblasts is harder to explain because vnd is not expressed in the intermediate column during neuroblast formation. We favor the hypothesis that vnd acts to create distinct proneural clusters (ventral vs. intermediate) within each row of neuroectoderm. In the absence of vnd, cells in each row behave as if they are in a single, enlarged proneural cluster spanning both ventral and intermediate columns, and thus may occasionally only generate a single neuroblast. This could lead to the observed low frequency, variable loss of ventral and intermediate neuroblasts. Alternate explanations are as follows: (1) vnd is transiently expressed in the intermediate column anlagen during blastoderm stage, in which it autonomously functions to promote neuroblast formation; and (2) that vnd is required in the ventral column to generate a nonautonomous signal promoting neuroblast formation in the intermediate column.

When vnd is misexpressed, the spatiotemporal pattern of neuroblast formation in the intermediate column is altered to a pattern closely resembling that of the ventral column. Intermediate column neuroblasts form earlier, at the same time as ventral column neuroblasts. There are also more than the normal number of intermediate column neuroblasts, closer to the number observed in the ventral column. These observations provide additional evidence for a transformation of intermediate to ventral column identity in hs–vnd embryos.

vnd establishes ventral column neuroectoderm and neuroblast identity

When does vnd specify ventral column identity? The earliest phenotypes observed in vnd loss- and gain-of-function embryos are alterations in neuroectoderm gene expression and cell morphology. Moreover, when vnd is ectopically expressed in the neuroectoderm, it is sufficient to induce ventral column neuroblast identity, but not when expressed after neuroblast formation (data not shown). Identical results were found for the specification of neuroblast identity along the AP axis by the segment polarity genes wingless and gooseberry (Chu-LaGraff and Doe 1993; Skeath et al. 1995). Thus, neuroblast specification along both the AP and DV axes is established in the neuroectoderm, just prior to neuroblast formation.

Vnd activates the expression of genes required for ventral neuroectodermal cell identity and represses the expression of genes characteristic of the intermediate and dorsal columns of the neuroectoderm (Fig. 7). For example, vnd is required to keep ind expression out of the ventral column neuroectoderm and neuroblasts. Vnd protein directly binds three target sequences in the promoter of ind (Weiss et al. 1998), consistent with direct repression of ind. We cannot distinguish whether Vnd directly represses other intermediate column genes (e.g., huckebein in row 3) or whether Vnd directly activates transcription of ventral column genes (e.g., achaete and odd). Alternatively, Vnd may act indirectly to modulate the expression of these genes, perhaps exclusively through repression of ind expression.

Figure 7.

Figure 7

Open in a new tab

Vnd is necessary and sufficient to establish ventral column identity in the CNS. Cartoon summary of gene expression patterns along the DV axis in wild-type, vnd− and hs–vnd embryos. (Rectangles) Neuroectoderm; (circles) neuroblasts; (vm) ventral midline cells; (d ecto) dorsal ectoderm. In wild-type embryos, the ventral column (black, v) expresses Vnd, and the Achaete, Odd, and Prospero markers; the intermediate column (gray, i) expresses Ind and Huckebein; and the dorsal column (white, d) expresses Msh, Achaete, and Odd. In vnd− embryos, the ventral column is transformed into an intermediate column identity; the dorsal column is unaffected. Neuroblasts in the ventral column occasionally do not form (broken line). In hs–vnd embryos, the intermediate column is transformed into a ventral column identity, and the dorsal column shows a mixture of dorsal and ventral markers (stipple) suggesting a partial dorsal to ventral column transformation.

Vnd regulates the expression of ind, achaete, odd, and huckebein within the ventral column, but Vnd also is required to establish the elongated cellular morphology characteristic of the ventral column neuroectoderm. The mechanism by which transcription factors regulate cell or tissue morphology is an important but difficult problem. To make progress, one approach would be to characterize Vnd transcriptional targets, which may uncover genes involved in cytoskeletal rearrangements or cell shape changes.

Parallels with vertebrate neural patterning

Recent work has revealed striking parallels between the development of the vertebrate and Drosophila CNS. The neuroectodermal territory is established by Short gastrulation/Chordin-mediated repression of Decapentaplegic/BMP-4 activity in both Drosophila and vertebrates (for review, see Bier 1997; Holley and Ferguson 1997). Within the Drosophila neuroectoderm, the ventral column is specified by vnd function (this paper; Skeath et al. 1994), the intermediate column is specified by ind function (Weiss et al. 1998), and the dorsal column appears to be partially or completely specified by msh function (Isshiki et al. 1997). Interestingly, the closest vertebrate homologs to each of these three genes is expressed in analogous DV domains during neurogenesis: The mouse Nkx2.1, Nkx2.2, and Nkx2.9 genes are vnd homologs expressed in ventral neuroectoderm domains adjacent to the floor plate (Guazzi et al. 1990; Price et al. 1992; Pabst et al. 1998); the mouse Gsh-1 and Gsh-2 genes are ind homologs expressed in intermediate neuroectodermal domains (Hsieh-Li et al. 1995; Valerius et al. 1995); and the mouse Msx genes are homologs of msh expressed in dorsal neuroectodermal domains (for review, see Davidson 1995). To determine whether the Nkx, Gsh, and Msx homeobox genes control DV patterning of the vertebrate CNS, similar to the function of the Drosophila vnd, ind, and msh genes, may require the generation of double or triple knockouts to overcome possible functional redundancy among the closely related genes within each family.

Materials and methods

Drosophila strains, transgenic flies, and heat shock conditions

The yellow white and white strains were used for wild-type analysis. The vnd6 allele (Jiménez and Campos-Ortega 1990) was used for vnd loss-of-function analysis. Standard molecular techniques were used to make the hs–vnd construct. A partial EcoRV restriction digest of the vnd cDNA generated a 2.44-kb fragment containing the vnd ORF plus 18 bp of 5′ and 257 bp of 3′ untranslated sequence. This was cloned into the pHSBJ-Casper vector (Jones and McGinnis 1993). The hs–vnd construct was coinjected with pPi25.7wc DNA into white embryos as described by Spradling (1986) and multiple independent insertions into the third chromosome were generated. Four hs–vnd lines were identified that expressed high levels of Vnd throughout the embryo. The following protocol was used to misexpress Vnd: 1 hr embryo collections were aged for 3 hr at room temperature, placed on a coverslip in a solution of PBS and 70% glycerol, and heat shocked for 7 min at 36°C; following a 1 hr recovery on grape juice agar, a second heat shock was performed by the same conditions. The embryos were aged 90 min (to obtain stage 9 embryos) or 2.5 hr (to obtain stage 11 embryos) and fixed by standard methods (Doe 1992). For some experiments, four heat shocks (with a 30 min recovery between heat shocks) were performed by the identical protocol; embryos were subsequently aged 2.5 hr before fixation. white embryos that were simultaneously subjected to identical heat-shock conditions did not exhibit defects. When embryos were heat shocked before 3 hr of development, white embryos developed normally, whereas hs–vnd embryos failed to develop.

Antibody production and staining

To generate the anti-Vnd antibody, a 1.68-kb fragment extending from the translation start site into the first helix of the homeodomain was cloned into the pRSET C vector (Invitrogen) and the fusion protein was purified under denaturing conditions. Rabbits were immunized five times with 200 μg of renatured antigen and bled out following the final boost. Following ammonium sulfate precipitation of IgG from the serum, Vnd-specific antibodies were affinity purified from the rabbit serum with renatured antigen cross-linked to AminoLink Plus Coupling gel (Pierce) as described in Mellerick et al. (1990).

Embryo fixation, antibody staining, and identification of homozygous mutant embryos were performed as in McDonald and Doe (1997). Primary antibodies were used at the following dilutions: mouse anti-Achaete monoclonal (990E5F1; 1:10; Skeath and Carroll 1992); rabbit anti-β-galactosidase serum (1:3000; Cappel); rat anti-β-galactosidase monoclonal (Srinivasan et al. 1998); mouse anti-Eve (2B8; 1:10; Patel et al. 1994); rat anti-Huckebein serum (1:50; McDonald and Doe 1997); rabbit anti-Msh serum (1:500; T. Isshiki and A. Nose, unpubl.); rat anti-Ind serum (1:500; Weiss et al. 1998); rabbit anti-Odd (1:2000; E. Ward and D. Coulter, unpubl.); mouse anti-Prospero monoclonal (MR2A; 1:4; Spana and Doe 1995); and rabbit anti-Vnd serum (1:10). Secondary antibodies of the appropriate species conjugated to biotin (Vector Labs), alkaline phosphatase (Southern Biotechnology Associates), DTAF and Cy5 (Jackson Immunoresearch) were used at a dilution of 1:400. Histochemical detection was done with the Vectastain Elite kit (Vector Labs) in conjunction with the Renaissance TSA Indirect kit (NEN). Embryos were dissected, mounted in 85% glycerol, examined on a Zeiss Axioplan microscope, and images were captured with a Sony DKC-5000 digital camera or traced with a camera lucida drawing tube. Fluorescent stainings were visualized on a Biorad MRC 1024 confocal microscope. Figures were assembled in Adobe Photoshop.

Acknowledgments

We thank Tonia Von Ohlen for providing the embryo in Figure 5A; Denise Montell for comments on the manuscript; Barbara Weiler and Jim Epperson for excellent technical assistance; E. Ward, D. Coulter, J. Skeath, and N.H. Patel for generously providing antibodies; and the University of Illinois Immunological Resource Center and Artist Services. This work was supported by grants from the National Institutes of Health (C.Q.D) and National Science Foundation (D.M.).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked ‘advertisement’ in accordance with 18 USC section 1734 solely to indicate this fact.

Note added in proof

Results similar to ours have been obtained independently by Chu et al. (1998). Chu, H., C. Parras, K. White, and F. Jiménez. 1998. Formation and specification of neutral neuroblasts is controlled by vnd in Drosophila melanogaster. Genes & Dev. (this issue).

Footnotes

E-MAIL cdoe@uoneuro.uoregon.edu; FAX (541) 346-4736; E-MAIL dervla_mellerick.pediatrics@mailgw.surg.med.umich.edu; FAX (734) 7644279.

References

  1. Bhat KM. The patched signaling pathway mediates repression of gooseberry allowing neuroblast specification by wingless during Drosophila neurogenesis. Development. 1996;122:921–932. doi: 10.1242/dev.122.9.2921. [DOI] [PubMed] [Google Scholar]
  2. Bhat KM, Schedl P. Requirement for engrailed and invected genes reveals novel regulatory interactions between engrailed/invected, patched, gooseberry and wingless during Drosophila neurogenesis. Development. 1997;124:1675–1688. doi: 10.1242/dev.124.9.1675. [DOI] [PubMed] [Google Scholar]
  3. Bier E. Anti-neural inhibition: A conserved mechanism for neural induction. Cell. 1997;89:681–684. doi: 10.1016/s0092-8674(00)80250-0. [DOI] [PubMed] [Google Scholar]
  4. Broadus J, Skeath JB, Spana EP, Bossing T, Technau G, Doe CQ. New neuroblast markers and the origin of the aCC/pCC neurons in the Drosophila central nervous system. Mech Dev. 1995;53:393–402. doi: 10.1016/0925-4773(95)00454-8. [DOI] [PubMed] [Google Scholar]
  5. Buescher M, Chia W. Mutations in lottchen cause cell fate transformations in both neuroblast and glioblast lineages in the Drosophila embryonic central nervous system. Development. 1997;124:673–681. doi: 10.1242/dev.124.3.673. [DOI] [PubMed] [Google Scholar]
  6. Campos-Ortega JA. Early neurogenesis in Drosophila melanogaster. In: Bate M, Martinez-Arias A, editors. Development of Drosophila melanogaster. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1993. pp. 1091–1130. [Google Scholar]
  7. Carroll SB, Scott MP. Localization of the fushi tarazu protein during Drosophila embryogenesis. Cell. 1985;43:47–57. doi: 10.1016/0092-8674(85)90011-x. [DOI] [PubMed] [Google Scholar]
  8. Chu-LaGraff Q, Doe CQ. Neuroblast specification and formation is regulated by wingless in the Drosophila CNS. Science. 1993;261:1594–1597. doi: 10.1126/science.8372355. [DOI] [PubMed] [Google Scholar]
  9. Chu-LaGraff Q, Schmid A, Leidel J, Brönner G, Jäckle H, Doe CQ. huckebein specifies aspects of CNS precursor identity required for motoneuron axon pathfinding. Neuron. 1995;15:1041–1051. doi: 10.1016/0896-6273(95)90093-4. [DOI] [PubMed] [Google Scholar]
  10. D’Alessio M, Frasch M. msh may play a conserved role in dorsoventral patterning of the neuroectoderm and mesoderm. Mech Dev. 1996;58:217–231. doi: 10.1016/s0925-4773(96)00583-7. [DOI] [PubMed] [Google Scholar]
  11. Davidson D. The function and evolution of Msx genes: Pointers and paradoxes. Trends Genet. 1995;11:405–411. doi: 10.1016/s0168-9525(00)89124-6. [DOI] [PubMed] [Google Scholar]
  12. Doe CQ. Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system. Development. 1992;116:855–863. doi: 10.1242/dev.116.4.855. [DOI] [PubMed] [Google Scholar]
  13. Doe CQ, Hiromi Y, Gehring WJ, Goodman CS. Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis. Science. 1988a;239:170–175. doi: 10.1126/science.2892267. [DOI] [PubMed] [Google Scholar]
  14. Doe CQ, Smouse D, Goodman CS. Control of neuronal fate by the Drosophila segmentation gene even-skipped. Nature. 1988b;333:376–378. doi: 10.1038/333376a0. [DOI] [PubMed] [Google Scholar]
  15. Duffy JB, Kania MA, Gergen JP. Expression and function of the Drosophila gene runt in early stages of neural development. Development. 1991;113:1223–1230. doi: 10.1242/dev.113.4.1223. [DOI] [PubMed] [Google Scholar]
  16. Frasch M, Hoey T, Rushlow CA, Doyle H, Levine M. Characterization and localization of the even-skipped protein of Drosophila. EMBO J. 1987;6:749–759. doi: 10.1002/j.1460-2075.1987.tb04817.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guazzi S, Price M, De Felice M, Damante G, Mattei MG, Di Lauro R. Thyroid nuclear factor (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J. 1990;9:3631–3639. doi: 10.1002/j.1460-2075.1990.tb07574.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Holley SA, Ferguson SA. Fish are like flies are like frogs: Conservation of dorsal-ventral patterning mechanisms. BioEssays. 1997;19:281–284. doi: 10.1002/bies.950190404. [DOI] [PubMed] [Google Scholar]
  19. Hsieh-Li HM, Witte DP, Szucsik JC, Weinstein M, Li H, Potter SS. Gsh-2, a murine homeobox gene expressed in the developing brain. Mech Dev. 1995;50:177–186. doi: 10.1016/0925-4773(94)00334-j. [DOI] [PubMed] [Google Scholar]
  20. Isshiki T, Takeichi M, Nose A. The role of the msh homeobox gene during Drosophila neurogenesis: implication for the dorsoventral specification of the neuroectoderm. Development. 1997;124:3099–3109. doi: 10.1242/dev.124.16.3099. [DOI] [PubMed] [Google Scholar]
  21. Jiménez F, Campos-Ortega J. Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of D. melanogaster. Neuron. 1990;5:81–89. doi: 10.1016/0896-6273(90)90036-f. [DOI] [PubMed] [Google Scholar]
  22. Jiménez F, Martin-Morris LE, Velasco L, Chu H, Sierra J, Rosen DR, White K. vnd, a gene required for early neurogenesis of Drosophila, encodes a homeodomain protein. EMBO J. 1995;14:3487–3495. doi: 10.1002/j.1460-2075.1995.tb07355.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones B, McGinnis W. The regulation of empty spiracles by Abdominal-B mediates an abdominal segment identity function. Genes & Dev. 1993;7:229–240. doi: 10.1101/gad.7.2.229. [DOI] [PubMed] [Google Scholar]
  24. Matsuzaki M, Saigo K. hedgehog signaling independent of engrailed and wingless required for post-S1 neuroblast formation in Drosophila CNS. Development. 1996;122:3567–3575. doi: 10.1242/dev.122.11.3567. [DOI] [PubMed] [Google Scholar]
  25. McDonald JA, Doe CQ. Establishing neuroblast-specific gene expression in the Drosophila CNS: huckebein is activated by Wingless and Hedgehog and repressed by Engrailed and Gooseberry. Development. 1997;124:1079–1087. doi: 10.1242/dev.124.5.1079. [DOI] [PubMed] [Google Scholar]
  26. Mellerick DM, Nirenberg M. Dorsal-ventral patterning genes restrict NK-2 homeobox gene expression to the ventral half of the central nervous system of Drosophila embryos. Dev Biol. 1995;171:306–316. doi: 10.1006/dbio.1995.1283. [DOI] [PubMed] [Google Scholar]
  27. Mellerick DM, Osborn M, Weber K. On the nature of serological tissue polypeptide antigen (TPA); monoclonal keratin 8, 18, and 19 antibodies react differently with TPA prepared from human cultured carcinoma cells and TPA in human serum. Oncogene. 1990;5:1007–1017. [PubMed] [Google Scholar]
  28. Pabst O, Herbrand H, Hans-Henning A. Nkx2-9 is a novel homeobox transcription factor which demarcates ventral domains in the developing mouse CNS. Mech Dev. 1998;73:85–93. doi: 10.1016/s0925-4773(98)00035-5. [DOI] [PubMed] [Google Scholar]
  29. Patel NH, Condron BG, Zinn K. Pair-rule expression patterns of even-skipped are found in both short- and long-germ beetles. Nature. 1994;367:429–434. doi: 10.1038/367429a0. [DOI] [PubMed] [Google Scholar]
  30. Price M, Lazzaro D, Pohl T, Mattei M-G, Rüther U, Olivo J-C, Duboule D, Di Lauro R. Regional expression of the homeobox gene Nkx 2.2 in the developing mammalian forebrain. Neuron. 1992;8:241–255. doi: 10.1016/0896-6273(92)90291-k. [DOI] [PubMed] [Google Scholar]
  31. Skeath JB, Carroll SB. Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo. Development. 1992;114:939–946. doi: 10.1242/dev.114.4.939. [DOI] [PubMed] [Google Scholar]
  32. Skeath JB, Panganiban GF, Carroll SB. The ventral nervous system defective gene controls proneural gene expression at two distinct steps during neuroblast formation in Drosophila. Development. 1994;120:1517–1524. doi: 10.1242/dev.120.6.1517. [DOI] [PubMed] [Google Scholar]
  33. Skeath JB, Zhang Y, Holmgren R, Carroll SB, Doe CQ. Specification of neuroblast identity in the Drosophila embryonic central nervous system by gooseberry-distal. Nature. 1995;376:427–430. doi: 10.1038/376427a0. [DOI] [PubMed] [Google Scholar]
  34. Spana EP, Doe CQ. The prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila. Development. 1995;121:3187–3195. doi: 10.1242/dev.121.10.3187. [DOI] [PubMed] [Google Scholar]
  35. Spradling AC. P-element-mediated transformation. In: Roberts DB, editor. Drosophila: A practical approach. Oxford, UK: IRL Press; 1986. pp. 175–197. [Google Scholar]
  36. Srinivasan, S., C.-Y. Peng, S. Nair, J.B. Skeath, E.P. Spana, and C.Q. Doe. 1998. Biochemical analysis of Prospero protein during aymmetric cell division: Cortical Prospero is highly phosphorylated relative to nuclear Prospero. Dev. Biol. (in press). [DOI] [PubMed]
  37. Valerius MT, Li H, Stock JL, Weinstein M, Kaur S, Singh G, Potter SS. Gsh-1: A novel murine homeobox gene expressed in the central nervous system. Dev Dyn. 1995;203:337–351. doi: 10.1002/aja.1002030306. [DOI] [PubMed] [Google Scholar]
  38. Weiss, J.B., T. Von Ohlen, D. Mellerick, G. Dressler, C.Q. Doe, and M.P. Scott. 1998. Dorsoventral patterning in the Drosophila CNS: The intermediate neuroblasts defective homeobox gene specifies intermediate column identity. Genes & Dev. (this issue). [DOI] [PMC free article] [PubMed]
  39. Zhang Y, Ungar A, Fresquez C, Holmgren R. Ectopic expression of either the Drosophila gooseberry-distal or proximal gene causes alterations of cell fate in the epidermis and central nervous system. Development. 1994;120:1151–1161. doi: 10.1242/dev.120.5.1151. [DOI] [PubMed] [Google Scholar]

Articles from Genes & Development are provided here courtesy of Cold Spring Harbor Laboratory Press

RESOURCES