Institut für Genetik, Universität Mainz, Saarstrasse 21, D-55122 Mainz, Germany
*Author for correspondence (e-mail: jurban{at}mail.uni-mainz.de)
Accepted 16 November 2001
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SUMMARY |
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Key words: Drosophila, NB sublineage genes, hunchback, Central nervous system, Serotonin, Corazonin
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INTRODUCTION |
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The VNC of Drosophila develops from neural precursor cells, the so called neuroblasts (NBs), which delaminate in five successive waves. Each NB is characterized by its time of delamination, its position and gene expression (Broadus et al., 1995; Doe, 1992
). Most NBs divide asymmetrically several times and are believed to generate one ganglion mother cell (GMC) at each division, which subsequently produces two neurons and/or glia cells (Hartenstein et al., 1987
). At the end of embryogenesis, the VNC consists of
320 neurons and
30 glia cells per hemineuromere. Previous work indicates that NBs give rise to characteristic reproducible cell lineages (Bossing et al., 1996
; Schmidt et al., 1997
). As specific neurons have always been shown to derive from the first born GMC of the parental NB, it has been suggested that NB lineages are generated in a fixed temporal sequence (Brody and Odenwald, 2000
). This has highlighted Drosophila NB lineages as being a very good model system for studying sequential neural cell fate specification.
Recently, the involvement of a set of genes encoding transcription factors that are expressed sequentially within most, if not all, NB lineages, has been detected (Kambadur et al., 1998; Brody and Odenwald, 2000
). This gene cassette consists of the genes hunchback (hb), the redundantly acting POU domain protein 1 (pdm1; nub FlyBase) and POU domain protein 2 (pdm2), castor (cas), and grainyhead (grh) (Brody and Odenwald, 2000
), which are expressed in early-, middle- and late-born neurons of NB lineages. Furthermore, klumpfuss (klu) has to be added to this list, as it is also expressed in NB sublineages (Yang et al., 1997
). The successive time periods of hb, pdm1 and pdm2 and cas expression is partially established through the inhibition of pdm1 and pdm2 expression via hb and cas in early and late sublineages, respectively. The expression patterns of these genes clearly suggest a function in sequential determination of neural cell types. Indeed, pdm1 and pdm2 (Johnson and Hirsh, 1990
; Billin et al., 1991
; Dick et al., 1991
; Lloyd and Sakonju, 1991
; Prakash et al., 1992
; Yang et al., 1993
; Bhat and Schedl, 1994
; Ng et al., 1995
; Yeo et al., 1995
), cas (Mellerick et al., 1992
; Cui and Doe, 1992
; Cui and Doe, 1995
) and klu (Yang et al., 1997
) have a role in neural cell fate specification. However, a function of hb and grh in CNS development has not yet been shown.
In this study, we have analyzed the function of hb within NB lineages by focusing on the NB7-3. We chose this lineage for two reasons: first, it is small, consisting of only four neurons (Bossing et al., 1996; Schmid et al., 1999
); and second, all NB7-3 neurons can be distinguished by their characteristic position, expression of marker genes and/or their transmitter phenotype (Bossing et al., 1996
; Higashijima et al., 1996
; Dittrich et al., 1997
; Lundell and Hirsh, 1998
) (this paper). Using BrdU incorporation, we show that the NB7-3 neurons derive from three GMCs. We found that hb is expressed in the early NB7-3, its first GMC (GMC7-3a) and its progeny, and that this gene is indeed necessary for the specification and/or maintenance of the early-born neurons within this lineage. Ectopic expression of hb in the later born sublineage is sufficient to transform these cells into neurons with the same fate as the GMC7-3a progeny. Moreover, additional neurons of early GMC7-3a fate are observed. We also found that hb might play a similar role in at least one other NB lineage NB 7-1. Thus, our data support the hypothesis that hb is an important early cell fate determinant in the process of sequential cell fate specification within the CNS development.
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MATERIALS AND METHODS |
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BrdU experiments
BrdU injection was as described previously (Prokop and Technau, 1991) with the following changes: the embryos were chemically dechorionized with 7.5% bleach shortly before injection. After injection they were allowed to develop until stage 15 and subsequently fixed with heptane saturated with 37% formaldehyde for 20 minutes.
Immunohistochemistry
Embryos and L1 larval brains were fixed, stained and mounted as described previously (Dittrich et al., 1997). Primary antibodies used were rat anti-Hunchback (1:100), rat anti-Sal (1:250), mouse anti-Zfh-1 (1:300), mouse anti-Zfh-2 (1:200), rabbit anti-Eagle (1:1000), mouse anti-Eagle (1:200), rabbit anti-Pdm-1 (1:500), rabbit anti-Serotonin (1:5000, Sigma), rabbit anti-Corazonin (1:2000), mouse anti-Eve (1:2, Developmental Studies Hybridoma Bank), rabbit anti-Eve (1:1000), rabbit anti-Castor (1:500) and anti-DIG-AP (1:1000, Roche). The secondary antibodies anti-rabbit-TRITC, anti-mouse-FITC and anti-rat-FITC from goat (Jackson Laboratories) were used at a 1:250 dilution. The mounted embryos were analyzed with a confocal laser scanning microscope (Leica TCS SPII). Scanning images were processed with Adobe Photoshop Macintosh Version 5.5.
Whole mount in situ hybridization
DIG-labeled RNA probe was synthesized with SP6 RNA Polymerase and pGEM-dSERT as a template according to the manufacturers protocol (Roche). The hybridization on embryos was performed as described previously (Tautz and Pfeifle, 1989; Plickert et al., 1997
).
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RESULTS |
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NB7-3 delamination takes place at about 6.5 hours after egg laying (AEL). Therefore BrdU was injected into embryos at two time points: between 6.5 and 7 hours AEL and between 7 and 7.5 hours AEL. After injection these embryos were allowed to develop until stage 15 and stained with anti-BrdU and anti-Eg antibody. At stage 15 all NB7-3 derived cells are postmitotic as they express the neuronal marker Elav (Robinow and White, 1991) (data not shown). Out of 111 abdominal NB7-3 lineages derived from injection between 6.5 and 7 hours AEL, 38% were BrdU positive in all four neurons, 55% in three neurons, 5% in two and 2% in one neuron. Injection at a later time point (7 to 7.5 hours AEL) resulted in an increase of NB7-3 lineages with less than three BrdU-positive neurons: 20% of hemisegments showed BrdU labeling in all four neurons, 28% in three neurons, 14% in two neurons and 39% in one neuron (n=153). Injection at around 8.5 hours AEL did not yield any BrdU staining in the NB7-3 cluster, indicating that after that time point there is no more S-phase in this lineage (Fig. 2E). By evaluating these results with respect to the position of the BrdU labeled neurons (see Fig. 2A-E,A'-E'), we found that GW and EW1 must be siblings as they are always labeled together (Fig. 2A,A',B,B'). This is in contrast to EW2 and EW3, which are very often separately labeled (63%, n=264; Fig. 2B,B',D,D'). Thus, according to our results, EW1 and GW are siblings, while EW2 and EW3 are generated separately.
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hb mutants exhibit a loss of the early NB7-3 sublineage
To test whether hb plays a role in the specification of GMC7-3a and its progeny we analyzed the NB7-3 lineage of embryos mutant for hb. To make sure that the obtained phenotypes are indeed related to Hb function we analyzed two independent mutations [the hypomorpic allele hb9 (Lehmann and Nüsslein-Volhard, 1987) and the null allele hb12 (Tautz et al., 1987
; Hulskamp et al., 1994
)] in parallel in most experiments. As similar results were obtained with both alleles, we will present the results found with hb12, unless stated otherwise. Because in hb mutant embryos the thoracic segments and the last abdominal segment are missing, and segments 7 and 8 are fused (Lehmann and Nüsslein-Volhard, 1987
), analyses were carried out only in the abdominal segments A1 to A6.
hb12 mutant NB7-3 lineages consisted of two (20%) or three (80%) Eg-positive neurons at stage 13 (Fig. 4E) (n=66) but only two remaining Eg-positive cells at stage 15 (98%, n=50; Fig. 4B,E''). To test which of the cells are missing, we performed double staining with antibodies against cell specific markers. Double staining against Zfh-1 and Eg at stage 13 showed that one posterior lying cell is mostly Zfh-1 positive (98%, n=53; Fig. 4E') suggesting that the GW neuron is initially made, while at the later stage 15 only two Zfh-1-negative cells can be detected (n=49; Fig. 4E''). Thus, the GW neuron either does not survive or does not maintain zfh1 expression at this later time point. The two remaining cells always express zfh-2 (n=72; Fig. 4H), the marker for the late sublineage neurons (Fig. 1E, Fig. 4G). This strongly suggests that in hb12 mutant embryos, the neurons of the late NB7-3 sublineage (EW2 and EW3) are still present. These data were supported by RNA in situ hybridization against dSerT mRNA in hb mutant embryos, showing that only one neuron per hemisegment is labeled (n=56; Fig. 4K), which is probably the Zfh2-positive EW2 neuron. In addition, we tested for the presence of the neurotransmitters Serotonin and Corazonin in hb9 mutant early larval VNCs (Fig. 5). In accordance with the decrease in dSerT-positive neurons, a significant reduction of Serotonin levels in the VNC was detected, with about 20% of hemisegments showing only one serotonergic cell (n=66; Fig. 5E). However, although the EW3 neuron seemed to be correctly specified as judged by zfh-2 expression, the Corazonin staining is missing in 39% of the hemineuromeres (n=112; Fig. 5B). This suggests that, although present, the EW3 neuron is affected with respect to its transmitter phenotype.
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Hb has a similar function in other NB lineages
Having found that Hb is an important determinant of early cell fate within the NB 7-3 lineage, we next tested whether this might also be true for other lineages of the Drosophila VNC. To do this we chose the eve (even skipped)-expressing cells, because a known subset of these are early-born progeny of identified NBs (see Fig. 6). Indeed, using antibodies against Eve and Hb in stage 15 embryos, we found co-expression in a dorsal subset of eve-expressing cells that consists of aCC, pCC (from GMC1-1a), RP2 (from GMC4-2a) (Fig. 6A'), fpCC and the dorsalmost CQ neuron (most likely the first born neurons from NB7-1; Fig. 6A'') (Bossing et al., 1996). Hb was not found in the NB 3-3-derived EL cells. We subsequently analyzed the function of Hb within these lineages using spalt (sal) as an additional marker for aCC, pCC and RP2 (Fig. 6B'). One of the EL neurons is also Sal positive (Fig. 6B'') but this cell can be reliably identified by its much more ventral position. We could show that Hb affected the NB7-1-derived fpCC and CQ neurons in a similar way as the GW and EW1 neurons in the NB7-3 lineage. In hb12 mutant embryos, the dorsal subset of the NB 7-1 derived neurons was missing in 97% of hemineuromeres (n=72): 80% showing a loss of two, 10% of one and 7% of three cells (Fig. 6C''). The missing cells are probably fpCC and/or the dorsalmost CQ neuron, which normally express hb. We assume that the missing third cell might be shifted into the EL cluster where it can no longer be identified (Fig. 6C''). Conversely, we observed an increase of the number of Eve-positive neurons in CQ position when we ectopically expressed hb via scaGAL4 or engrailedGAL4 (Fig. 6D'',E'') (Tabata et al., 1995
). enGAL4 drives hb expression in all row 6 and 7 NBs. Among these neuroblasts, only NB7-1 gives rise to Eve-positive neurons (Broadus et al., 1995
; Bossing et al., 1996
). Therefore, we conclude that these additional neurons are most likely additional fpCCs and/or dorsal CQ neurons. However, owing to the lack of markers for the Eve-negative cells of the NB7-1 lineage, we could not check whether these additional neurons appear at the cost of other NB7-1-derived neurons.
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DISCUSSION |
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NB7-3 generates three GMCs with the first GMC giving rise to the EW1 and GW neurons
By injecting BrdU at different time windows during development, we determined the sibling relationships of the progeny of NB7-3, which consists of two serotonergic interneurons (EW1 and EW2), one interneuron that expresses the neuropeptide Corazonin (EW3) and one motorneuron (GW) (Fig. 1) (Higashijima et al., 1996; Lundell et al., 1996
; Dittrich et al., 1997
; Lundell and Hirsh, 1998
) (this work). According to our results, EW1 and GW are siblings, while EW2 and EW3 are generated sequentially from the NB7-3.
We have strong evidence that EW1 and GW are produced by the first GMC of NB7-3 (GMC7-3a). As EW3 is nearly always the last neuron labeled by BrdU, we hypothesize that this neuron is most likely to be generated by the third GMC, while GMC7-3b gives rise to EW2. The fact that we do not find any siblings for the later-born neurons can be explained in two ways: either the missing sibling is dying by PCD or the GMCs are able to differentiate directly without further cell division. We currently cannot conclude which of these possibilities is true but investigations in apoptosis-deficient mutant embryos clearly show that PCD occurs within the NB7-3 lineage. The frequent occurrence of more than six NB7-3 derived cells in these mutant embryos might be due to additional cell divisions of the surviving cells.
Hb is an important determinant for the specification of GMC7-3a
The restricted expression of hb in the early NB7-3 (before and shortly after its first division) and the first-born GMC, including its progeny suggest that hb is involved in the specification of these cells. Our analysis strongly supports this assumption: in stage 13 hb loss-of-function mutant embryos, we found either a complete loss of the GMC7-3a-derived EW1 and GW neurons or of the EW1 neuron alone. At stage 15 the GW neuron could also no longer be detected. Therefore, we conclude that the loss of Hb cannot transform GMC7-3a into GMC7-3b or GMC7-3c (Fig. 7B). This is different from the findings of Isshiki et al. (Isshiki et al., 2001); they found a duplication of EW2 neurons in a low number of cases. We do not know why this difference occurs, but it might be due to the use of different hb alleles. Our results suggest that either additional factors might be necessary for determining the fates of GMC7-3b and/or c, which are not present in the Hb positive GMC7-3a, or that GMC7-3a harbors additional factor(s) that suppress other GMC fates. However, ectopic expression of hb in all cells of the lineage leads to additional neurons of the EW1 and GW type at the cost of neurons normally generated by the later-born GMCs (Fig. 7C). This shows clearly that hb is sufficient to induce fates of the first GMC and to suppress those of the later-born GMCs. This can be interpreted in two ways. One possibility is that overexpression of hb in the progeny of GMC7-3a is sufficient to reiteratively cause both of these cells to adopt the fate of the parental GMC (GMC7-3a). But as hb is strongly expressed in the differentiating progeny of the first GMC anyway, it is very unlikely that a higher dose of Hb has such an effect. Therefore we favor the idea that ectopic Hb in the other GMCs and/or the late NB7-3 (after the first division) is able to transform the fate of these GMCs into the fate of the normally Hb-positive GMC (GMC7-3a). However, the fact that we found a high number of two to three additional EW1-like neurons (as judged by the presence of dSerT and absence of zfh2 expression), but mostly only one additional Zfh1-positive GW neuron suggests that only one additional GMC is fully transformed in most cases. This hints at an additional factor being important for the generation of GW, which is present mainly in one additional GMC beside GMC-1, most probably in the second GMC (Fig. 7C). A good candidate for such a factor appears to be Krüppel (Kr) (Isshiki et al., 2001
).
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Loss of Hb function affects the Hb negative corazonergic neuron
Our analysis of the NB7-3 lineage revealed that, in hb loss-of-function mutants, the neurons that are normally Hb positive are affected. However, using the hypomorphic allele hb9, which develops into L1 larval stage we could additionally show that the Hb negative EW3 is also affected and fails to express Corazonin in a significant number of cases. Because not only this cell but also the immediate precursor cell(s) must have had a reduced Hb function, we can imagine two scenarios. It is possible that Corazonin expression can be switched on only in the last generated neuron when the NB receives Hb activity during the early phase of lineage development. This would favor the idea that at least some aspects of neuronal fate are dependent on transient gene activity within the stem cell long before the neuron is generated. Alternatively, initiation and/or maintenance of Corazonin expression might depend on cellular interactions such as synaptic inputs or the presence of extrinsic factors that could be altered in the hb mutant CNS. This could also explain the difference in our results and those of Isshiki et al. (Isshiki et al., 2001), who report no change in Crz expression in hb mutant embryos at a much earlier stage than we have analyzed. That extrinsic factors indeed can play a prominent role in specifying and maintaining neurons, including the transmitter phenotype, has been extensively shown in vertebrates (for a review, see Edlund and Jessell, 1999
).
Hb is also needed for early sublineage fates in other lineages
hb-positive cells of other lineages are also affected in the hb mutant background: the Eve-expressing neurons RP2 (derived from GMC4-2a) (Doe, 1992) and aCC and/or pCC (derived from GMC1-1a) (Doe, 1992
) are frequently undetectable. Additionally, fpCC and/or the dorsalmost of the four CQ neurons (derived from NB7-1) (Bossing et al., 1996
) are not found in 97% of the hemineuromeres. These cells are most probably identical to the U1 and U2 neurons described by Isshiki et al. (Isshiki et al., 2001
). The lower penetrance of the phenotype in RP2 and aCC/pCC (67% and 50%, respectively) could be due to other cell or lineage specific factors that are co-expressed with hb and are able to act redundantly in these cases. In particular, RP2 might be rescued by the presence of Pdm1 and Pdm2 activity in its parental GMC, because it has been shown that these two proteins are necessary for acquiring a GMC4-2a fate (Yang et al., 1993
; Yeo et al., 1995
). Maternally contributed hb (Lehmann and Nüsslein-Volhard, 1987
) might also be able to partially rescue the phenotype in the lineages of the early delaminating NB4-2 and NB1-1.
In any case, the phenotype of the loss-of-function mutant embryos suggests that hb is, as in NB7-3, involved in the specification or at least maintenance of certain NB progeny. In particular, the role of hb with respect to the NB7-1 derived fpCC and dorsal CQ neuron seems to be comparable with what we found in the NB7-3 lineage, as pan-neural hb overexpression leads to additional Eve-positive fpCCs and/or CQ neurons. Also, additional Sal-positive neurons were observed in the position of aCC/pCC. This is in agreement with the results of Isshiki et al. (Isshiki et al., 2001) who found a duplication of aCC/pCC.
hb in comparison to other sublineage genes
Our results indicate that hb must be added as a functional member of the group of CNS sublineage genes that are involved in the specification of GMCs. The other three genes of this group, which have been investigated on the NB lineage level are the redundantly acting pdm1 and pdm2 genes and klumpfuss (klu). As mentioned above, pdm1 and pdm2 have been shown to be involved in the specification of the first GMC (GMC-1) in the NB4-2 lineage (Yang et al., 1993; Yeo et al., 1995
). However, ectopic pdm2 expression did not lead to a transformation of the later-born GMCs as we postulate for hb in the NB7-3 lineage, but transformed the progeny of GMC4-2a into the fate of the parental cell (Yang et al., 1993
). Thus, although pdm1 and pdm2 are able to induce a NB4-2 specific first GMC fate, they can do this only in cells that derive from the first born GMC. This suggests that the quality of being GMC-1 (of NB4-2) is independent of Pdm1 and Pdm2 function.
However, in klu loss-of-function mutant embryos, the normally Klu-positive GMC-4-2b transforms into the fate of GMC-4-2a (Yang et al., 1997). Thus, in the absence of Klu, GMC4-2b has also a GMC-1 quality and Klu is necessary for the switch to a GMC-2 fate. What determines this GMC-1 quality in the NB4-2 lineage is not known, but our results imply that Hb might have such a function during NB7-3 lineage development, as it is able to induce ectopic GMC-1 fate(s) at the cost of the other GMC fates. Preliminary experiments show that, unlike in the NB4-2 lineage, Klu is not necessary for the fate of the second GMC of NB7-3 (data not shown). In accordance with this, Isshiki et al. (Isshiki et al., 2001
) have shown that Kr is the factor that specifies the GMC 7-3b fate.
Conclusions
Taken together, our data suggest that there are principle mechanisms leading to the sequential cell specification in NB lineages, but different lineages seem to use either different genes to determine specific sublineages (e.g. klu) or use the same genes with different contributions (e.g. hb).
In any case, it is clear that an exact temporal regulation of their expression is crucial for the correct specification of neurons along the time axis. Therefore, an important aspect in understanding the sequential sublineage specification within NB lineages, is to know how sublineage genes like hb are switched on and off at the right time during lineage development. There are strong hints that the cell cycle plays an important role in this respect (Cui and Doe, 1995; Weigmann and Lehner, 1995
). Owing to its accessibility, the NB7-3 lineage provides a powerful tool to investigate this and other related questions in the future.
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ACKNOWLEDGMENTS |
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