The precise temporal control of gene expression is critical for specifying neuronal identity in the Drosophila central nervous system (CNS). A particularly interesting class of genes are those expressed at stereotyped times during the cell lineage of identified neural precursors (neuroblasts): these are termed ‘sublineage’ genes. Although sublineage gene function is vital for CNS development, the temporal regulation of this class of genes has not been studied. Here we show that four genes (ming, even-skipped, unplugged and achaete) are expressed in specific neuroblast sublin- eages. We show that these neuroblasts can be identified in embryos lacking both neuroblast cytokinesis and cell cycle progression (string mutants) and in embryos lacking only neuroblast cytokinesis (pebble mutants). We find that the unplugged and achaete genes are expressed normally in string and pebble mutant embryos, indicating that temporal control is independent of neuroblast cytokinesis or counting cell cycles. In contrast, neuroblasts require cytokinesis to activate sublineage ming expression, while a single, identified neuroblast requires cell cycle progression to activate even-skipped expression. These results suggest that neuroblasts have an intrinsic gene regulatory hierarchy controlling unplugged and achaete expression, but that cell cycle- or cytokinesis-dependent mechanisms are required for ming and eve CNS expression.

A central question of neurobiology is how neuronal diversity is generated. Drosophila neurogenesis is an ideal system for studying this issue because of its genetic and molecular acces- sibility. The CNS develops from a ventral neuroectoderm, as individual neuroectodermal cells enlarge and delaminate in a stereotyped spatiotemporal order to become neural precursors, called neuroblasts (NBs) (Campos-Ortega and Hartenstein, 1985). Each NB divides asymmetrically to bud off a series of ganglion mother cells (GMCs), with each GMC dividing to form a pair of post-mitotic neurons or glia. Work in both grasshopper and Drosophila has shown that an identified NB has a unique and invariant cell lineage producing multiple types of neurons and glia (Udolph et al., 1993; Condron and Zinn, 1994). In addition, several aspects of cell determination within NB lineages can occur normally following in vitro culture of isolated NBs (Huff et al., 1989).

Underlying the broad spectrum of cell identities generated from NB lineages is a precisely regulated pattern of gene expression. A number of genes are expressed at specific points during the lineage of identified NBs; these are termed ‘sublin- eage’ genes (Cui and Doe, 1992). Sublineage genes are often directly involved in specifying cell fates in the CNS (e.g. Doe et al., 1988a,b; Duffy et al., 1991; Yang et al., 1993; Bhat and Schedl, 1994; Condron et al., 1994; Condron and Zinn, 1995).

For example, the Drosophila gene pdm2 is expressed in the first GMC from NB 4-2; it is not expressed in the RP2/RP2sib neuronal progeny or later in the NB lineage (Yang et al., 1993; Bhat and Schedl, 1994). If pdm2 is not expressed in the GMC, then RP2 and RP2sib fail to develop normally (Bhat and Schedl, 1994; Yeo et al., 1995). Conversely, if pdm2 expression is not down-regulated after GMC division, both neuronal progeny maintain the GMC identity, resulting in duplication of the RP2/RP2sib fates (Yang et al, 1993; Bhat and Schedl, 1994). Another example of the importance of sublineage gene expression can be found in the grasshopper embryo. The median NB (MNB) produces both neurons and glia in tempo- rally ordered phases (Condron and Zinn, 1994). The transitions between neuronal/glial production phases are likely controlled by the precisely timed activity of the engrailed (en) gene and cAMP-dependent protein kinase (PKA). en is expressed early in the MNB lineage, where it is required to trigger glial pro- duction; loss of en function in the MNB lineage results in premature neuronal production with a loss of glia (Condron et al., 1994). Subsequently, the activation and nuclear localization of PKA is necessary and sufficient for switching the MNB from glial to neuronal production; inhibition of PKA leads to continued glial production and a loss of neurons, whereas premature activation of PKA results in supernumerary neurons at the expense of glia (Condron and Zinn, 1995).

Because sublineage-specific gene activity is fundamental for generating neuronal and glial diversity, we would like to identify the mechanisms controlling the timing of gene expression during NB cell lineages. In some invertebrate systems, where cell lineage appears to be important, a minimum number of cell cycles or the segregation of cyto- plasmic determinants are required to trigger gene expression (Satoh, 1979, 1982; Satoh and Ikegami, 1981a,b; Whittaker, 1987; Edgar and McGhee, 1988). In contrast, during Xenopus development, where cell lineage plays little or no role, cell cycle progression is not required for the correct temporal acti- vation of many neural differentiation markers (Harris and Hartenstein, 1991). A previous study in Drosophila shows that several general neuronal markers are expressed in cell cycle- arrested embryos (Hartenstein and Posakony, 1990), however, more specific sublineage-type markers were not examined. It is important to distinguish between general neuronal markers and the cell-specific, tightly regulated expression of NB sub- lineage genes. Regulation of the general program of neural differentiation may not accurately reflect the complexity of mechanisms controlling sublineage-specific gene expression in neural precursors during their unique, invariant cell lineages.

In this paper, we investigate the role of NB cell cycle tran- sition and NB cytokinesis in controlling activation or repres- sion of four sublineage-specific genes: achaete (ac), ming- lacZ (ming), even-skipped (eve) and unplugged-lacZ (unp). Similar to pdm2 and en, three of these genes are required to specify cell fate within identified NB lineages. ac is a proneural gene that controls NB vs. ectodermal cell fates (reviewed by Campuzano and Modolell, 1992), ming is required to specify the identity of a subset of neurons from identified NB lineages (Cui and Doe, 1992), and eve controls the fate of a subset of identified GMCs and/or neurons (Doe et al., 1988b); unp has not been assayed for CNS function. While further investigation of sublineage-specific gene function is clearly necessary, it is equally important to under- stand the mechanisms regulating their precise temporal expression.

We used the string (stg) mutation, which blocks both NB cell cycle and cytokinesis (Edgar and O’Farrell, 1989, 1990), to test whether sublineage gene expression is dependent on either of these events. We used the pebble (pbl) mutation, which specifically blocks NB cytokinesis (Lehner, 1992; Hime and Saint, 1992), to determine which of the two processes is required. If gene expression is affected by stg, but not pbl, then it is cell cycle dependent; if gene expression is affected by both mutations, it is dependent on NB cytokinesis. Our results show that NBs form normally and can be individually identified in stg and pbl embryos. We find that unp and ac are expressed normally in both stg and pbl mutant embryos, thus their regu- lation is independent of cell cycle and cytokinesis. In contrast, NBs require cytokinesis (but not cell cycle progression) to activate sublineage ming expression, whereas eve expression in a single, identified NB lineage requires cell cycle progres- sion (but not cytokinesis).

Fly stocks

A yellow white (y w) stock was used as wild type because our enhancer trap markers are in this genetic background. We used a string null allele, stg7B, and the strongest available pebble allele, pbl11D. The 1530 stock contains an enhancer trap P-element insert in the ming locus at 83C (Cui and Doe, 1992); it was recombined with stg7B or pbl11D which are both located on the third chromo- some. We refer to the expression of this enhancer trap as ‘ming expression’ for simplicity. To rule out the possibility that the homozygous lethal 1530 insertion interacts with stg or pbl to cause a cell cycle, cytokinesis, or ming expression phenotype, we compared ming (1530) expression in stg mutant embryos homozy- gous for the enhancer trap insertion (1530, stg/1530, stg) and het- erozygous for the enhancer trap insertion (1530, stg/+, stg). No dif- ference in ming expression was observed. Similar results were obtained for homozygous and heterozygous expression of ming in pbl mutant embryos. All ming expression data in the paper are from embryos homozygous for the 1530 insertion. The 1912 stock contains an enhancer trap P-element insert in the unplugged (unp) locus at 44B on the second chromosome (Doe, 1992; P. Beachy, personal communication). The 1912 insertion was crossed into stg7B or pbl11D mutant backgrounds. We refer to the expression of the 1912 enhancer trap gene as ‘unp expression’ for simplicity. In all genotypes, the stg7B and pbl11D alleles are balanced with TM3 bearing a ftzlacZ construct (Hiromi and Gehring, 1987). Homozy- gous mutant embryos were distinguished by lack of ftzlacZ expression.

Immunostaining

Staged embryos were processed for antibody staining as previously described (Doe, 1992). Primary antibodies were mouse MR1a anti-prospero used at 1:5 (Spana and Doe, 1995); mouse anti- engrailed used at 1:1 (Patel et al., 1989); rabbit anti-engrailed used at 1:75 (DiNardo et al., 1985); mouse anti-achaete used at 1:5 (Skeath and Carroll, 1992); mouse anti-even-skipped used at 1:1 (Patel et al., 1994); mouse antibody BP106 used at 1:3 (Hortsch et al., 1990); mouse anti-snail (Mauhin et al., 1993) used at 1:5 according to Skeath et al. (1994); and rabbit anti-β-galactosidase (Cappel) used at 1:1200.

For histochemical staining, secondary anti-mouse or rabbit anti- bodies (Jackson ImmunoResearch) conjugated with horseradish per- oxidase (HRP) or alkaline phosphatase (AP) were used at 1:200. For achaete and snail staining, ABC kits (Vector) were used to intensify the signal. After appropriate antibody incubation, embryos were treated for color development. For HRP color reaction, embryos were incubated with 20 mg/ml DAB before addition of 0.003% H2O2 with or without 0.05% NiCl (for enhancement of signal). For AP color detection, either the BCIP/NBT reaction or the Alkaline Phosphatase Substrate Kit III (Vector) were used. The latter gave more bluish color. After satisfying signals were achieved, embryos were washed with buffer and stored in either methyl salicylate or 80% glycerol. Glycerol treated embryos were opened with a tungsten needle and placed ventral side up on slide, coverslipped, and further flattened by tapping a pencil tip gently on top of the coverslip. Embryos were visu- alized on a Zeiss Axioplan microscope.

For fluorescent staining, secondary anti-mouse or rabbit antibodies (Jackson ImmunoResearch) conjugated with LRSC or Cy5 were used at 1:200. Co-labeled embryos were mounted in 80% glycerol con- taining 4% n-propyl gallate (Sigma). DNA contents were labeled with the DNA fluorochrome phenylenediamine according to Lundell and Hirsh (1994). Embryos were visualized on a BioRad 1000 confocal microscope.

Cell tracing and neuroblast identification

Camera lucida was used to trace the NB array and their GMC progeny. All embryos were flat-mounted and viewed from the ventral surface on a Zeiss Axioplan microscope. Criteria for identifying and naming NBs were based on Doe (1992) and the revised NB map of Broadus et al., 1995.

Sublineage-specific gene expression is correlated with number of neuroblast cell cycles

NB formation occurs in five waves within the neurogenic region starting at stage 8 of embryonic development (Doe, 1992). Individual NBs enlarge and delaminate from the ectoderm layer; within 10-15 minutes of delaminating, each NB enters its first post-blastoderm mitosis (i.e. mitosis 14).

Subsequent NB cell cycles are 40-50 minutes long (Hartenstein et al., 1987, 1994). Here we focus on four genes expressed in NB sublineages: ac, unp, ming, and eve (Fig. 1). To determine when each gene is expressed during identified NB lineages, we correlate the timing of gene expression with the number of GMCs produced by a NB (Figs 1B, 2). GMCs can be identi- fied by the presence of nuclear prospero protein, which is a robust, early marker for GMCs (Vaessin et al., 1991; Matsuzaki et al., 1992; Spana and Doe, 1995). We assume close association between a GMC and a NB indicates that the GMC was born from the NB; this is clearly a valid assumption for the earliest stages of neurogenesis where NB and GMC density is low (Fig. 2). We find that gene expression within NB lineages is correlated with number of NB cell cycles:

Fig. 1.

Pattern of NBs and sublineage expression of achaete (ac), unplugged-lacZ (unp), ming-lacZ (ming), and even-skipped (eve); the positional marker engrailed (en) is also shown. (A) Each panel represents the pattern of NBs between 3.5 hours (S1 panel) and 7 hours (S5 panel) of development (from Broadus et al., 1995; modified from Doe, 1992). The NB cell cycle is 40-50 minutes (Hartenstein et al., 1987), or approximately one cell cycle per stage. Heading: NB stage. Anterior, top; ventral midline, dotted line. (B) Summary of typical NB sublineage expression of the genes used in this study. ac is active in three newly formed NBs (NB 3-5, 7-1 and 7-4) but is repressed before the second NB mitosis. unp is activated after two or more mitoses in five NB lineages. ming has two modes of NB expression: continuously (e.g. NB 6-1) or after two or more mitoses in later sublineages (e.g. NB 7-4). eve is expressed only in the first born GMC of three NB lineages (NB 1-1, 4-2, and 7-1; Broadus et al., 1995). Large circle, NB; medium circle, GMC; small circle, neuron.

Fig. 1.

Pattern of NBs and sublineage expression of achaete (ac), unplugged-lacZ (unp), ming-lacZ (ming), and even-skipped (eve); the positional marker engrailed (en) is also shown. (A) Each panel represents the pattern of NBs between 3.5 hours (S1 panel) and 7 hours (S5 panel) of development (from Broadus et al., 1995; modified from Doe, 1992). The NB cell cycle is 40-50 minutes (Hartenstein et al., 1987), or approximately one cell cycle per stage. Heading: NB stage. Anterior, top; ventral midline, dotted line. (B) Summary of typical NB sublineage expression of the genes used in this study. ac is active in three newly formed NBs (NB 3-5, 7-1 and 7-4) but is repressed before the second NB mitosis. unp is activated after two or more mitoses in five NB lineages. ming has two modes of NB expression: continuously (e.g. NB 6-1) or after two or more mitoses in later sublineages (e.g. NB 7-4). eve is expressed only in the first born GMC of three NB lineages (NB 1-1, 4-2, and 7-1; Broadus et al., 1995). Large circle, NB; medium circle, GMC; small circle, neuron.

Fig. 2.

Activation or repression of sublineage gene expression is correlated with number of NB cell divisions. (A-C) ac expression is repressed after the first NB division, as shown by the presence of GMCs (A,B) at the same time as ac expression (C). (A) Camera lucida of an early stage 9 embryo showing all S1 NBs have produced the first GMC. NBs are identified by size and position (large circles); GMCs are identified by nuclear prospero (small brown circles). (B) Stage 9 embryo stained for the GMC marker prospero (brown) with the ac-positive NBs indicated: NB 3-5 (left arrowhead), MP2 (right arrowhead), NB 7-4 (left arrow), and NB 7-1 (right arrow). At this stage ac is still expressed in these NBs (see C). (C) Stage 9 embryo stained for ac protein, showing expression in NB 3-5 (left arrowhead), MP2 (right arrowhead), NB 7-4 (left arrow), and NB 7-1 (right arrow). Soon after this stage of development ac expression is repressed in the NB 3-5, NB 7-1 and NB 7-4 (see Figs 1, 5). (D-F) Two types of ming expression: sublineage expression is activated after two or more NB divisions (e.g. NB 7-4), whereas continuous ming expression occurs from the time of NB formation (e.g. NB 6-1). (D) Camera lucida of an early stage 10 embryo stained for ming (blue) and the GMC marker prospero (brown). Sublineage ming expression: at least two GMCs have been produced by NB 7-4, but it does not yet express ming. Continuous ming expression: the newly-formed NB 6-1 expresses ming but has not produced a GMC. (E) Early stage 10 embryo showing expression of ming (blue) in NB 6-1 (arrowhead) but not in NB 7-4 (arrow), even though NB 7-4 has produced several prospero-positive GMCs (brown). (F) Late stage 11 embryo showing sublineage expression of ming (blue) in NB 7-4 (arrow). Anterior, top; ventral midline, white triangle.

Fig. 2.

Activation or repression of sublineage gene expression is correlated with number of NB cell divisions. (A-C) ac expression is repressed after the first NB division, as shown by the presence of GMCs (A,B) at the same time as ac expression (C). (A) Camera lucida of an early stage 9 embryo showing all S1 NBs have produced the first GMC. NBs are identified by size and position (large circles); GMCs are identified by nuclear prospero (small brown circles). (B) Stage 9 embryo stained for the GMC marker prospero (brown) with the ac-positive NBs indicated: NB 3-5 (left arrowhead), MP2 (right arrowhead), NB 7-4 (left arrow), and NB 7-1 (right arrow). At this stage ac is still expressed in these NBs (see C). (C) Stage 9 embryo stained for ac protein, showing expression in NB 3-5 (left arrowhead), MP2 (right arrowhead), NB 7-4 (left arrow), and NB 7-1 (right arrow). Soon after this stage of development ac expression is repressed in the NB 3-5, NB 7-1 and NB 7-4 (see Figs 1, 5). (D-F) Two types of ming expression: sublineage expression is activated after two or more NB divisions (e.g. NB 7-4), whereas continuous ming expression occurs from the time of NB formation (e.g. NB 6-1). (D) Camera lucida of an early stage 10 embryo stained for ming (blue) and the GMC marker prospero (brown). Sublineage ming expression: at least two GMCs have been produced by NB 7-4, but it does not yet express ming. Continuous ming expression: the newly-formed NB 6-1 expresses ming but has not produced a GMC. (E) Early stage 10 embryo showing expression of ming (blue) in NB 6-1 (arrowhead) but not in NB 7-4 (arrow), even though NB 7-4 has produced several prospero-positive GMCs (brown). (F) Late stage 11 embryo showing sublineage expression of ming (blue) in NB 7-4 (arrow). Anterior, top; ventral midline, white triangle.

Fig. 3.

NBs form normally in cell cycle-arrested or cytokinesis-arrested embryos. Stage 10 wild-type (A), stg (B), and pbl (C) embryos labeled for the pan-NB marker snail (brown) and the positional marker en (blue). All embryos have 19 NBs per hemisegment: four lateral NBs (arrows), five intermediate NBs (arrowheads); the nine medial NBs are mostly out of focus, except NB 6-1 (white arrow). Not all segments have all 19 NBs in the same focal plane. The lateral NB 6-4, GP and X are not indicated. Anterior, top; ventral midline, black triangle.

Fig. 3.

NBs form normally in cell cycle-arrested or cytokinesis-arrested embryos. Stage 10 wild-type (A), stg (B), and pbl (C) embryos labeled for the pan-NB marker snail (brown) and the positional marker en (blue). All embryos have 19 NBs per hemisegment: four lateral NBs (arrows), five intermediate NBs (arrowheads); the nine medial NBs are mostly out of focus, except NB 6-1 (white arrow). Not all segments have all 19 NBs in the same focal plane. The lateral NB 6-4, GP and X are not indicated. Anterior, top; ventral midline, black triangle.

(1) unp is expressed in the middle of NB lineages (Fig. 1B). It is first detected in a subset of NBs at late stage 11 (Fig. 4); all of these NBs form earlier and divide several times before expressing unp.

Fig. 4.

Sublineage activation of unplugged is independent of neuroblast cell cycle or cytokinesis. Wild type (A,D), stg (B,E), and pbl (C,F) embryos labeled for unp (blue) and the positional marker en (brown). (A-C) In mid-stage 11 embryos (S4 NB stage) unp is only expressed in midline cells. (D-F) In late stage 11 embryos (S5 NB stage), unp is detected in NB 4-1, NB 5-3, NB 6-2 and NB 7-2 (arrows from right to left); all of these NBs have produced at least one progeny by this stage (see Fig. 2). In stg and pbl embryos, some of the unp expressing NBs are out of the focal plane, partly due to the disorganized CNS morphology; nevertheless, we observe a relatively normal unp NB pattern in most hemisegments by camera lucida tracing (data not shown). Anterior, top; ventral midline, white triangle.

Fig. 4.

Sublineage activation of unplugged is independent of neuroblast cell cycle or cytokinesis. Wild type (A,D), stg (B,E), and pbl (C,F) embryos labeled for unp (blue) and the positional marker en (brown). (A-C) In mid-stage 11 embryos (S4 NB stage) unp is only expressed in midline cells. (D-F) In late stage 11 embryos (S5 NB stage), unp is detected in NB 4-1, NB 5-3, NB 6-2 and NB 7-2 (arrows from right to left); all of these NBs have produced at least one progeny by this stage (see Fig. 2). In stg and pbl embryos, some of the unp expressing NBs are out of the focal plane, partly due to the disorganized CNS morphology; nevertheless, we observe a relatively normal unp NB pattern in most hemisegments by camera lucida tracing (data not shown). Anterior, top; ventral midline, white triangle.

(2) ac is repressed in the middle of NB lineages (Fig. 1B). It is first expressed in four clusters of neuroectodermal cells and in the single NB that delaminates from each cluster (Skeath and Carroll, 1992), but it is turned off soon after the first GMC is born (Figs 2, 5).

Fig. 5.

Sublineage repression of achaete is independent of neuroblast cell cycle or cytokinesis. Wild-type (A,D), stg (B,E) and pbl (C,F) embryos labeled for ac protein. (A-C) In stage 9 embryos ac is expressed in three S1 NBs (NB 2-5, right arrowhead; NB 7-1, left arrow; and NB 7-4, right arrow) and the MP2 precursor (left arrowhead). In pbl embryos (C), some of these cytokinesis-arrested NBs show two nuclei containing ac protein (e.g. NB 7-1, left arrow), this is expected since ac repression normally occurs only after the first NB division. (D-F) In stage 10 embryos ac NB expression is repressed at the same time in all genotypes. In addition, activation of ac expression in cells outside the CNS occurs normally in all genotypes (ventral midline, arrow; lateral ectoderm, black arrowhead). Anterior, top; ventral midline, white triangle.

Fig. 5.

Sublineage repression of achaete is independent of neuroblast cell cycle or cytokinesis. Wild-type (A,D), stg (B,E) and pbl (C,F) embryos labeled for ac protein. (A-C) In stage 9 embryos ac is expressed in three S1 NBs (NB 2-5, right arrowhead; NB 7-1, left arrow; and NB 7-4, right arrow) and the MP2 precursor (left arrowhead). In pbl embryos (C), some of these cytokinesis-arrested NBs show two nuclei containing ac protein (e.g. NB 7-1, left arrow), this is expected since ac repression normally occurs only after the first NB division. (D-F) In stage 10 embryos ac NB expression is repressed at the same time in all genotypes. In addition, activation of ac expression in cells outside the CNS occurs normally in all genotypes (ventral midline, arrow; lateral ectoderm, black arrowhead). Anterior, top; ventral midline, white triangle.

(3)ming has two types of CNS expression (Fig. 1B): in some NBs it is expressed continuously from the time of NB formation, while in other NBs it is first expressed midway through the lineage (‘sublineage’ expression; Cui and Doe, 1992). In this paper we focus on one NB of each type: NB 6- 1, which expresses ming continuously throughout its lineage; and NB 7-4, which expresses ming only after two or more GMCs have been born (Figs 2, 6).

Fig. 6.

Sublineage expression of ming requires neuroblast cytokinesis. Wild-type (A,D,G), stg (B,E,H) and pbl (C,F,I) embryos stained for ming (blue) and the positional marker en (brown). (A-C) Stage 10 embryos (S3 NB stage). Newly formed NB 6-1 (arrowhead) expresses ming prior to its first cell division. NB 7-4 (arrow) forms at stage 9 and has divided at least twice by this stage (see Fig. 2) but does not yet express ming. NB 7-4 is unambiguously identified as the most lateral, posterior en-positive NB. (D-F) Late stage 11 embryos (S5 NB stage). (D) In wild-type embryos, some NBs express ming continuously throughout their lineage (NB 6-1, arrowhead; NB 3-4, asterisk); other NBs express ming only in late sublineages (e.g. NB 7-4, arrow). (E) In stg embryos, NBs showing continuous expression of ming are unaffected (NB 6-1, arrowhead; NB 3-4, asterisk), whereas sublineage ming expression is never observed (e.g. NB 7-4, arrow). (F) In pbl embryos, NBs expressing ming continuously are normal (NB 6-1, arrowhead; NB 3-4, asterisk); however, sublineage expression of ming is blocked (e.g. NB 7-4, arrow). We observe low frequency ming expression in NB 7-4 (10%, n = 105). (F′ and F′′) pbl embryo is triple labeled for ming (green), a DNA marker (white), and a membrane marker BP106 (red). (F′) Expression of ming and the membrane marker shows mononucleate NB 7-4 (arrow); NB 3-4, which expresses ming continuously, has two nuclei (asterisk). (F′′) Expression of DNA and membrane marker in the same embryo shows the single nucleus in NB 7-4 as well as a small GMC located nearby. We conclude that the low frequency ming expression in NB 7-4 only occurs after cytokinesis of NB 7-4 due to incomplete penetrance of the pbl mutation. In both stg and pbl embryos, the midline CNS has become internalized, leading to a general shift of the NB array towards the midline. (G-I) Camera lucida tracings of the NBs in late stage 11 embryos showing expression of ming (blue) and en (red). F and I are at lower magnification than other panels. Anterior, top; ventral midline, white triangle.

Fig. 6.

Sublineage expression of ming requires neuroblast cytokinesis. Wild-type (A,D,G), stg (B,E,H) and pbl (C,F,I) embryos stained for ming (blue) and the positional marker en (brown). (A-C) Stage 10 embryos (S3 NB stage). Newly formed NB 6-1 (arrowhead) expresses ming prior to its first cell division. NB 7-4 (arrow) forms at stage 9 and has divided at least twice by this stage (see Fig. 2) but does not yet express ming. NB 7-4 is unambiguously identified as the most lateral, posterior en-positive NB. (D-F) Late stage 11 embryos (S5 NB stage). (D) In wild-type embryos, some NBs express ming continuously throughout their lineage (NB 6-1, arrowhead; NB 3-4, asterisk); other NBs express ming only in late sublineages (e.g. NB 7-4, arrow). (E) In stg embryos, NBs showing continuous expression of ming are unaffected (NB 6-1, arrowhead; NB 3-4, asterisk), whereas sublineage ming expression is never observed (e.g. NB 7-4, arrow). (F) In pbl embryos, NBs expressing ming continuously are normal (NB 6-1, arrowhead; NB 3-4, asterisk); however, sublineage expression of ming is blocked (e.g. NB 7-4, arrow). We observe low frequency ming expression in NB 7-4 (10%, n = 105). (F′ and F′′) pbl embryo is triple labeled for ming (green), a DNA marker (white), and a membrane marker BP106 (red). (F′) Expression of ming and the membrane marker shows mononucleate NB 7-4 (arrow); NB 3-4, which expresses ming continuously, has two nuclei (asterisk). (F′′) Expression of DNA and membrane marker in the same embryo shows the single nucleus in NB 7-4 as well as a small GMC located nearby. We conclude that the low frequency ming expression in NB 7-4 only occurs after cytokinesis of NB 7-4 due to incomplete penetrance of the pbl mutation. In both stg and pbl embryos, the midline CNS has become internalized, leading to a general shift of the NB array towards the midline. (G-I) Camera lucida tracings of the NBs in late stage 11 embryos showing expression of ming (blue) and en (red). F and I are at lower magnification than other panels. Anterior, top; ventral midline, white triangle.

(4)eve is expressed in the middle of NB lineages (Fig. 1B); it is not expressed in NBs, but rather in the first GMC of three NB lineages (Doe, 1992; Broadus et al., 1995; Fig. 7).

Fig. 7.

Sublineage even-skipped expression requires either cytokinesis or cell cycle progression. (A) Wild-type stage 11 embryo stained for eve (brown) and the positional marker en (blue stripe). Three different NB lineages produce eve-positive progeny: the en-positive NB 7-1, black arrow; NB 1-1, white arrow; and NB 4-2, arrowhead. (B) Stage 11 stg embryo labeled with anti-eve antibody; all three NB lineages fail to express eve. (C) Stage 11 pbl embryo stained for eve protein (brown) and en (blue stripe), showing one eve-positive NB located within the en stripe (NB 7-1; arrow); this NB can have two eve-positive nuclei (inset; eve is in red, the membrane marker BP106 is in green). Anterior, top; ventral midline, black triangle.

Fig. 7.

Sublineage even-skipped expression requires either cytokinesis or cell cycle progression. (A) Wild-type stage 11 embryo stained for eve (brown) and the positional marker en (blue stripe). Three different NB lineages produce eve-positive progeny: the en-positive NB 7-1, black arrow; NB 1-1, white arrow; and NB 4-2, arrowhead. (B) Stage 11 stg embryo labeled with anti-eve antibody; all three NB lineages fail to express eve. (C) Stage 11 pbl embryo stained for eve protein (brown) and en (blue stripe), showing one eve-positive NB located within the en stripe (NB 7-1; arrow); this NB can have two eve-positive nuclei (inset; eve is in red, the membrane marker BP106 is in green). Anterior, top; ventral midline, black triangle.

Neuroblast formation occurs normally in string and pebble mutant embryos

stg encodes a phosphatase that triggers mitosis by activating the Cdc2 kinase (Edgar and O’Farrell, 1989, 1990). In embryos homozygous for a stg null allele, all maternal stg transcript and protein are completely degraded after cycle 13, leading to G2- arrest at the cellular blastoderm stage (Edgar et al., 1994). This is over 30 minutes prior to the beginning of NB formation (Campos-Ortega and Hartenstein, 1985). Thus stg can be used to block all NBs in G2 of the cell cycle; this genetic approach circumvents the problems encountered when drugs are used to block the cell cycle, such as incomplete suppression of DNA replication or non-specific effects (Ikegami et al., 1978). The pbl gene is required for cytokinesis during the post-blastoderm cell divisions. In embryos homozygous for a strong pbl allele, nuclear cell cycles proceed normally but cytokinesis is blocked, resulting in multinucleate cells (Lehner, 1992; Hime and Saint, 1992).

A prerequisite for using the stg and pbl mutants to study the regulation of sublineage gene expression is that the NB pattern forms normally. We use the snail and en gene products to follow NB formation in stg or pbl embryos. snail protein marks all NBs (Alberga et al., 1991), whereas en provides a positional cue allowing individual NBs to be identified (Doe, 1992). In wild-type stage 10 embryos there are 19 NBs in each hemiseg- ment (Fig. 3A). In similarly aged stg embryos, although there is a dramatic reduction in the total number of embryonic cells, NBs delaminate normally and we observe approximately 19 NBs in each hemisegment (Fig. 3B). Furthermore, the spatial arrangement of NBs is relatively normal, so that by using a combination of snail and en expression we can identify indi- vidual NBs, e.g. NB 6-1 and NB 7-4 (Fig. 3B). In pbl embryos the NB array is more disorganized; nevertheless, we count approximately 19 NBs per hemisegment and can usually identify individual NBs (Fig. 3C).

To determine the extent of cell cycle and cytokinesis arrest in stg and pbl embryos, we assayed GMC formation by scoring for nuclear localization of prospero (Vaessin et al., 1991; Matsuzaki et al., 1992; Spana and Doe, 1995). We do not observe any GMCs in stg embryos, verifying that all post-blas- toderm divisions are blocked in G2 (data not shown). In contrast, a small number of GMCs can be observed in pbl embryos, showing that the pbl mutation is not a null (data not shown). Although a small fraction of NBs undergo cytokine- sis in pbl mutants, we can unambiguously identify the cleavage-inhibited NBs because they are multinucleate (see below).

Sublineage unplugged and achaete expression is independent of neuroblast cell cycle or cytokinesis

unp is first expressed in NBs at late stage 11 (Fig. 4A,D). Most of the unp-positive NBs form earlier during neurogenesis and divide at least twice prior to expressing unp (Figs 1B, 4D). For example, NB 5-3 forms at early stage 9 but does not express unp until about 3 hours later, by which time it has probably generated at least four GMCs (assuming a 40 minute cell cycle; Hartenstein et al., 1987). In addition, NB 6-2 and NB 7-2 form at stage 9 but do not express unp until 2.5 hours later, by which time they should have divided three times. In both stg and pbl mutant embryos, we do not observe any significant change in the timing of unp NB expression (Fig. 4B,C,E,F).

The ac gene is first expressed in four clusters of neuroecto- dermal cells at stage 8 and in the single NB that delaminates from each cluster at early stage 9 (Skeath and Carroll, 1992). ac is maintained in the NBs until after they have divided to produce their first GMC at late stage 9 (Figs 1, 2). Thus ac is expressed in neuroectodermal cells at G2, maintained as the NB delaminates and divides, and is extinguished prior to the next NB mitosis. We observe a perfectly normal time course of ac expression in both stg and pbl mutant embryos (Fig. 5). Clearly the timing of both unp and ac sublineage expression is not dependent on passage through the NB cell cycle or on NB cytokinesis.

Sublineage ming expression requires neuroblast cytokinesis

The ming gene has two fundamentally different modes of expression during neurogenesis. Some NBs, such as NB 6-1 and 3-4, express ming continuously throughout their lineage. In both stg and pbl embryos, NBs with continuous ming expression are unaffected (Fig. 6A-C). More interesting are NBs with sublineage-specific ming expression (i.e. ming is activated midway through the NB lineage). The clearest example of sublineage ming expression is in NB 7-4. In wild- type embryos, NB 7-4 forms at early stage 9, yet it does not express ming until stage 11, after it has divided at least twice (Fig. 6D,G). In stg embryos, NB 7-4 forms normally but never expresses ming (Fig. 6E,H). This shows that either cell cycle progression or cytokinesis is required to activate sublineage expression of ming in NB 7-4.

To distinguish between these possibilities, we assayed ming expression in pbl mutant embryos, in which the NB cell cycle occurs normally, but NB cytokinesis is blocked. We find that NB 7-4 forms normally, but in over 90% of cases does not express ming (Fig. 6F,I). The 10% of NB 7-4s that express ming appear to have gone through cytokinesis: embryos triple labelled for ming, DNA, and a cell membrane marker (BP106) show that the ming-positive NB 7-4s are not multinucleate, and GMCs can be seen at nearby positions (Fig. 6F′,F′′). We have never observed a ming-positive multinucleate NB 7-4, although neighboring NBs are frequently multinucleate (e.g. NB 3-4; Fig. 6F′,F′′). We conclude that the low frequency ming expression in NB 7-4 only occurs after cytokinesis of NB 7-4 due to incomplete penetrance of the pbl mutation. These data suggest that NB cytokinesis is specifically required to activate sublineage expression of ming in NB 7-4.

Sublineage even-skipped expression requires either cytokinesis or cell cycle progression

When a NB divides, one sibling becomes the new NB and the other becomes a GMC. In addition to examining markers for gene expression in NBs, we have looked at one gene, eve, that is expressed in the first GMC of three NB lineages: NB 1-1, NB 4-2 and NB 7-1 (Fig. 7A; Broadus et al., 1995). In stg embryos, eve expression is lost in all three NB lineages (Fig. 7B). Thus, either cell cycle- or cytokinesis-arrest prevents eve expression in these lineages. To distinguish between these pos- sibilities, we examined eve expression in pbl mutant embryos, where only NB cytokinesis is blocked.

In pbl embryos, eve expression is observed only in NB 7-1 (Fig. 7C). This suggests that in this NB, eve expression requires cell cycle progression, not cytokinesis. To rule out the possi- bility that eve expression in NB 7-1 is due to an incomplete block of NB cytokinesis, we use confocal microscopy to double label for eve and the cell membrane marker BP106; we find that two eve-positive nuclei are frequently observed in NB 7-1 (Fig. 7C, inset). Interestingly, both nuclei in NB 7-1 contain eve protein, not just the ‘GMC’ nucleus. In contrast, eve is not expressed in NB 1-1 or NB 4-2 in stg or pbl embryos. This suggests that in these lineages, eve expression requires NB cytokinesis, not cell cycle progression, similar to the sub- lineage expression of ming.

The importance of coordinating gene expression and number of cell cycles is particularly acute in the Drosophila CNS: each NB has both a stereotyped cell lineage (e.g. Udolph et al., 1993; Condron and Zinn, 1994) and a complex yet repro- ducible pattern of gene expression (e.g. Doe, 1992; Cui and Doe, 1992). The precise modulation of gene expression during NB cell lineages results in each GMC inheriting different gene products; many of these sublineage-expressed genes are known to be essential for specifying cell fate in the developing CNS (Doe et al., 1988a,b; Cui and Doe, 1992; Bhat and Schedl, 1994; Yeo et al., 1995). It is critical to understand how sub- lineage gene expression is regulated; a combined analysis of gene function and gene regulation will be necessary to under- stand fully how neuronal diversity is generated.

1-2

In this paper, we correlate the number of NB divisions with the activation or repression of four sublineage-specific genes. We show that NBs delaminate normally and can be individu- ally identified in stg and pbl embryos. We find that the unp and ac genes are expressed normally in both stg and pbl mutant embryos; their regulation is independent of cell cycle and cytokinesis. In contrast, NBs require cytokinesis (but not cell cycle progression) to activate sublineage ming expression, whereas a single, identified NB requires cell cycle progression (but not cytokinesis) to activate eve expression.

Both unp and ac are expressed normally in stg and pbl embryos. This indicates that the accurate activation of unp or repression of ac is not regulated by passage through the nuclear cell cycle, or by NB cytokinesis. While previous studies using general neural markers have shown that NBs can exhibit differ- entiation without cell division (Hartenstein and Posakony, 1990), our results suggest for the first time that cell cycle- arrested NBs can also maintain accurate sublineage-specific gene expression. What mechanisms could be used to precisely time unp and ac expression? One possibility is that cell inter- actions control NB gene expression. The NB pattern is approx- imately normal in both stg and pbl embryos, which would allow interactions between adjacent NBs, or between a NB and the ventral ectoderm or dorsal mesoderm. This mechanism seems improbable because some aspects of NB cell lineage can occur normally in NBs isolated in vitro (Huff et al., 1989). In addition, because NBs do not undergo cytokinesis in stg or pbl embryos, interactions between NBs and GMCs are clearly not required to regulate unp or ac gene expression. A more likely possibility is that sublineage expression of unp and ac is con- trolled by a transcriptional regulatory cascade which is estab- lished at the time of the NB formation. This regulatory hierarchy could control both specific gene expression and the timing of the NB cell cycle, which would ensure close coordi- nation between gene expression and GMC birth order. Moreover, it is known that the timing of cell cycle gene expression is regulated by developmental cues, rather than cell cycle progression itself (Knoblich et al., 1994; Edgar et al., 1994; Duronio and O’Farrell, 1994); this is consistent with a single regulatory hierarchy controlling both sublineage gene expression and the cell cycle.

Sublineage expression of ming is lost in both stg and pbl embryos. Because NBs in pbl embryos maintain their nuclear cell cycle, these data suggest that sublineage expression of ming requires NB cytokinesis. There are several possible explanations for this phenotype. Lack of cytokinesis may result in incorrect segregation of cytoplasmic factors; for example, repressors of ming transcription could be trapped in cytokinesis-arrested NBs. At least two proteins, numb and prospero, are asymmetrically localized into newly formed GMCs as they are budded off from the parental NB (Rhyu et al., 1994; Spana and Doe, 1995), showing that there exists a mechanism for asymmetrically localizing proteins into newborn GMCs. Alternatively, the absence of cytokinesis may prevent cell interactions between a NB and its GMC progeny. ‘Feedback’ signals from a newborn GMC to its parental NB could trigger changes in ming expression. These alternatives can be tested by in vitro isolation or ablation experiments.

In wild-type embryos, eve is expressed in the first GMC of three NB lineages (1-1, 4-2, and 7-1). Expression of eve is completely abolished in stg embryos, while in pbl embryos eve is expressed in NB 7-1, but not in NB 1-1 or NB 4-2. eve expression in NB 7-1 in pbl embryos is not due to occasional cytokinesis of this NB, since multiple eve-positive nuclei can be detected in the NB. Thus, eve expression in this lineage requires activity of the stg phosphatase and/or progression through the cell cycle. Other methods of blocking cell cycle progression can be used to determine if the stg protein is directly involved in activating eve expression. In contrast, eve expression requires NB cytokinesis in the NB 1-1 and NB 4-2 lineages. This suggests that, just like ming sublineage expression, cytoplasmic segregation or intra-lineage cell inter- actions may be required for eve expression in the NB 1-1 and NB 4-2 lineages.

The results described in this paper indicate that multiple mechanisms are used to regulate the timing of gene expression during NB cell lineages in the developing CNS. These include passage through the nuclear cell cycle (eve-expression in the NB 7-1 lineage), NB cytokinesis (sublineage ming expression and eve expression in NBs 1-1 and 4-2), and a mechanism independent of both the nuclear cell cycle and cytokinesis (unp and ac). Interestingly, within a single NB lineage the expression of two genes can be modulated by distinct mech- anisms. For example, in NB 7-4, mid-lineage repression of ac is independent of nuclear cell cycle or cytokinesis, whereas mid-lineage activation of ming requires cytokinesis. It is not surprising that multiple mechanisms control CNS gene expression, due to the vast array of cell types and complex patterns of gene expression found in the CNS. The new challenge will be to investigate the molecular basis of each of these mechanisms.

We thank A. Alberga, S. DiNardo, and N. H. Patel for providing antibodies. We also thank Jim Skeath and the anonymous reviewers for invaluable comments on the manuscript. Special thanks to Bruce Edgar for sharing his thoughts and unpublished results, and to Jim Skeath for assistance on achaete and snail stainings. This work was supported by the NIH (HD27056) and an NSF Presidential Young Investigator Award. C. Q. D. is an Assistant Investigator of the Howard Hughes Medical Institute.

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