1 Institute of Zoology, Biocenter/Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
2 Department of Biology, Howard Hughes Medical Institute, Indiana University, Jordan Hall A507, Bloomington, IN 47405-6801, USA
*Author for correspondence (e-mail: frank.hirth{at}unibas.ch)
Accepted September 12, 2001
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SUMMARY |
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Key words: labial, Hox proteins, Brain development, Genetic rescue, Drosophila
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INTRODUCTION |
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Hox genes are expressed in the developing brain and ventral nerve cord of Drosophila in an ordered set of domains. In the embryonic brain, specific Hox genes are expressed in the posterior half of the tritocerebrum (and to a small extent in the deutocerebrum) as well as in the three subesophageal neuromeres. The tritocerebrum is the posterior neuromere of the supraesophageal ganglion and consists of two bilaterally symmetric hemiganglia that are bounded anteriorly by the deutocerebrum and are linked by the tritocerebral commissure that runs across the midline beneath the gut (Burrows, 1996; Reichert and Boyan, 1997). The tritocerebrum is connected to more posterior parts of the brain through longitudinal connectives, and forms projections to the frontal ganglion via the frontal connectives. The Hox gene that is specifically expressed in the posterior half of the tritocerebral neuromere is labial (lab). Loss-of-function lab mutations cause profound defects in the establishment of the tritocerebral neuromere (Hirth et al., 1998). In lab mutants, the tritocerebral commissure is missing and the longitudinal connectives are reduced or absent. Moreover, the cells in the lab mutant domain do not acquire a neuronal identity as exemplified by the lack of expression of neuronal markers indicating that lab is required for the specification of neuronal identity in the tritocerebrum. Comparable effects are seen in Deformed mutants, the only major difference being that these effects were observed in the mandibular and anterior maxillary brain neuromere, which is the expression domain of Deformed. None of the other Hox gene mutants show comparable brain defects (Hirth et al., 1998).
We have used genetic rescue experiments to investigate the functional equivalence of all of the Drosophila Hox genes in specifying the neuronal identity in the tritocerebral neuromere. For this we use the Gal4-UAS system (Brand and Perrimon, 1993) for targeted misexpression of Hox genes in the posterior tritocerebral domain (in which lab is normally expressed) of lab null mutants. As expected, we find that the lab mutant brain phenotype can be rescued by targeted expression of the Lab protein under the control of CNS-specific lab regulatory elements. We then demonstrated that under the control of these CNS-specific regulatory elements most of the other Drosophila Hox gene products are also able to replace the Lab protein in the specification of the tritocerebral neuromere. Only the Abdominal-B protein does not efficiently rescue the lab mutant phenotype in the brain. For the other Hox proteins, we observe a correlation between their efficiency of rescue the lab mutant brain phenotype and the chromosomal arrangement of their encoding loci. Our results indicate that, despite considerably diverged sequences, most Hox proteins are functionally equivalent in their ability to replace Labial in the specification of neuronal identity in the brain. This suggests that differences of Hox gene action in brain development rely mainly on cis-acting regulatory elements and not on Hox protein specificity.
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MATERIALS AND METHODS |
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For lab::Gal4-specific targeted misexpression of proboscipedia (pb), Deformed (Dfd), abdominal-A (abd-A) and Abdominal-B (Abd-B) in lab mutant embryos, the following UAS::Hox responder lines were used: p[UAS::pb 49.1] homozygous on chromosome II (Aplin and Kaufman, 1997); p[UAS::Dfd] homozygous on chromosome II (Brown et al., 1999); p[UAS::abd-A 21.6] homozygous on chromosome I (Greig and Akam, 1993), supplied by M. Akam; and p[UAS::Abd-Bm] homozygous on chromosome II (Castelli-Gair et al., 1994) driving the expression of the Abd-Bm form (Casanova et al., 1986; Zavortink and Sakonju, 1989), supplied by M. Akam.
For lab::Gal4-specific targeted misexpression of labial (lab), Sex combs reduced (Scr), Antennapedia (Antp) and Ultrabithorax (Ubx) in lab mutant embryos, p[UAS::lab], p[UAS::Scr], p[UAS::Antp] and p[UAS::Ubx] responder lines were generated (Miller et al., 2001). The respective Hox cDNAs were cloned into a polylinker downstream from a minimal hsp70 promoter of the Gal4 responder plasmid pUAST (Brand and Perrimon, 1993), which contains a P-element with the white mini-gene as a marker. The hsp70 promoter is activated in the presence of Gal4 because of five upstream Gal4 binding sites (UAS). For generating p[UAS::lab], a 2.1 kb cDNA derived from a 2.4a minigene (including the second intron) (Chouinard and Kaufman, 1991) encompassing the entire lab-coding region, was digested with the SspI to generate the 2.1 kb cDNA that was inserted into pBlueScriptKS+ (Stratagene) at the EcoRV site. The cDNA was subsequently removed with EcoRI(5') and KpnI(3') for insertion into pUAST at the same sites. For generating p[UAS::Scr], the 1.2 kb BamHI(5') and MluI(3') truncated Scr L3 cDNA (Mahaffey and Kaufman, 1987) was inserted into pSE280 (Invitrogen) using the same sites. A partial Scr cDNA was then removed from pSE280 with NcoI(5'), blunted with Klenow and then released with XhoI. This modified cDNA was inserted into pUAST at the Klenow blunted EcoRI and XhoI sites. For generating p[UAS::Antp], the entire Antp G1100 cDNA (Scott et al., 1983) was inserted into pUAST at the EcoRI site. For generating p[UAS::Ubx], the previously reported Ubx NAB3 cDNA containing isoform 1S, which is the predominant embryonic cDNA (OConnor et al., 1988), was inserted into pUAST at the EcoRI site. All strains, as well as all experimental genotypes, were maintained in standard laboratory cultures at 25°C.
Control experiments verified that the P{w+ lab::Gal4}K5J2 driver is expressed in a spatial pattern, which corresponds to that of endogenous lab in the procephalon, and in the tritocerebral neuromere. UAS::transgene activation in the procephalon is delayed for 2.5 hours when compared with earliest presence of endogenous Lab protein (Kaufman et al., 1990), thus under the control of P{w+ lab::Gal4}K5J2, UAS::responder activation starts at late stage 10 (5-5.5 hours AEL) (Campos-Ortega and Hartenstein, 1997). Phenotypic penetrance of the lab mutant brain phenotype was 88.6% (n=209) when determined with the lab null allele labvd1 (Merrill et al., 1989; Hirth et al., 1998) using flies of the genotype labvd1/TM6B-UbxlacZ. The ability of the Hox proteins to rescue the lab mutant brain phenotype was determined by crossing P{w+ lab::Gal4}K5J2; labvd1/TM6B-UbxlacZ to either P{UAS::lab}, labvd1/TM3-AntplacZ or to flies of genotype P{UAS::Hox}, labvd1/ TM6B-UbxlacZ where Hox=pb, Dfd, Antp and Abd-B; or to flies of genotype P{UAS::Hox}/+; labvd1/+ for Hox=Scr, Ubx and Abd-A. All rescue experiments were carried out at 25°C; no significant differences in rescue efficiency were obtained when rescue experiments were carried out at 28°C. To identify rescued lab/ cells and their axonal projection pattern, UAS::tau-lacZ located on the X chromosome (Callahan and Thomas, 1994) was additionally crossed in.
Immunocytochemistry and genetic rescue analysis
Whole-mount immunocytochemistry and laser confocal microscopy was performed as previously described (Hirth et al., 1998). In genetic rescue experiments, P{w+ lab::Gal4}K5J2 driven P{UAS::Hox} activity in homozygous lab null mutants (labvd1/labvd1) was confirmed by the absence of balancer-specific (TM6B-UbxlacZ; TM3-AntplacZ) ß-gal and/or Labial immunoreactivity, as well as by the presence of corresponding Hox immunoreactivity in the tritocerebral lab domain. The criteria used to judge lab/ embryos as fully rescued were: (1) the presence of the tritocerebral commissure linking the two tritocerebral hemiganglia; (2) the restoration of the longitudinal pathways between the supra- and subesophageal ganglia; and (3) the expression of neuron-specific molecular labels as assayed by anti-HRP and anti-Elav immunoreactivity (Hirth et al., 1998). Only when all three criteria were fulfilled was the tritocerebrum of a lab/ mutant embryo scored as rescued. Additionally, in embryos of the genotype UAS::tau-lacZ/+; lab::Gal4/UAS::Hox; lab/, the specificity of rescue was also determined by the presence of correct axonal projections of rescued lab/ cells along the rescued tritocerebral commissure.
Laser confocal microscopy
For laser confocal microscopy, a Leica TCS SP was used. Optical sections ranged from 0.4 to 2 µm recorded in line average mode with picture size of 512x512 pixels. Captured images from optical sections were arranged and processed using IMARIS (Bitplane). Figures were arranged and labeled using Adobe Photoshop.
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RESULTS |
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Next, we expressed each of the remaining seven UAS::Hox responders under the control of the lab::Gal4 driver in the lab mutant domain. We first investigated the Hox proteins of the Antennapedia-Complex, as in the wild type, all five proteins of this complex are expressed in specific domains of the developing brain (Hirth et al., 1998). Surprisingly, all of the Antennapedia-Complex Hox proteins were able to rescue the lab mutant brain defects in these experiments. Examples of the ability of these Hox proteins to rescue the labial mutant brain phenotype are shown for Sex combs reduced (Scr) and Antennapedia (Antp) (Fig. 4). In both cases, an efficient rescue of the tritocerebral defects in the lab mutants was obtained; the tritocerebral commissure was present, the longitudinal pathways were restored, and cells in the mutant domain showed correct neuron-specific molecular labels. In addition to the lab::Gal4 driven ectopic expression of Scr and Antp in the tritocerebral labial mutant domain, the large endogenous expression domains of these genes were observed unchanged in the subesophageal ganglion for Scr, and in the subesophageal ganglion and ventral nerve cord for Antp (Fig. 4C,F) (Hirth et al., 1998). A quantification of the rescue efficiency for all of the Antennapedia-Complex Hox proteins in these experiments is given in Table 1.
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DISCUSSION |
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There are several possible explanations for this discrepancy. First, the functional role of Lab in the specification of neuronal identity in the brain may differ from the role of other Hox proteins in other parts of the CNS. For example, Hirth et al. (Hirth et al., 1998) have found that the loss-of-function phenotype of lab (and Dfd) in the embryonic CNS differs from that of the remaining Hox genes. Moreover, in contrast to other domains in the embryonic CNS, there is an absence of overlapping expression with other Hox proteins, so that there is no genetic backup in the tritocerebrum. Similar observations on Lab have been made in epidermal structures (Kaufman et al., 1990; McGinnis and Krumlauf, 1992; Morata, 1993). Second, it is conceivable that all Hox proteins can specify neuronal identity and the generic formation of commissural and longitudinal connections in the CNS. However, this seems unlikely, as the morphology and innervation of the triocerebral neuromere is unique and highly specific, and unlike that of any other neuromere in the CNS (Burrows, 1996). Similarly, the morphology, mode of formation and gut-specific association of the developing tritocerebral commissure is clearly different from that of the other ganglionic commissures in the embryonic CNS (Wildemann et al., 1997). Third, Hox proteins may indeed be to a larger degree functionally interchangable in the CNS than hitherto expected. In this respect, two sets of recent functional complementation experiments carried out on mammalian Hoxa3/Hoxd3 genes and on mammalian Hox11a/Hox11d genes are noteworthy because they indicate that paralogous gene products can carry out identical biological functions if they are placed under the control of the appropriate cis-acting regulatory elements (Zakany et al., 1996; Greer et al., 2000). Our results extend this notion of functional equivalence of Hox genes from the level of paralogous genes to the level of the entire Hox gene cluster, excepting Abd-B. This, in turn, suggests that almost all of the Hox proteins can carry out identical biological functions in the Drosophila brain, if they are under the control of the same cis-acting regulatory elements.
In our experiments, all of the Hox responders were expressed in the lab mutant under the control of the identical, lab-specific regulatory elements. Under these circumstances, the Lab responder achieved the best rescue efficiency, while the other Hox responders (with the exception of Abd-B) had somewhat lower rescue efficiencies that ranged from 86-59% of the rescue values achieved by Lab (see Fig. 7). Interestingly, the relative rescue efficiency of the Hox gene products Lab, Pb, Dfd, Scr, Antp, Ubx and Abd-A reflect their proximal-to-distal arrangement of their encoding loci on the chromosome. It is conceivable that this co-linear correlation of rescue efficiency among theses Hox gene products is due to the variability in the Gal4-UAS system, to positional effects of transgene insertions, or to differences in transgene expression levels. However, a more reasonable explanation is that the decline in relative rescue efficiency among these Hox proteins, as well as the qualitative difference between Abd-B and the other Hox proteins in their ability to rescue the lab mutant brain phenotype, is due primarily to Hox protein sequence differences. Hox proteins do indeed show sequence differences, the most notable of which reside in the homeodomain, the hexapeptide motif (lacking in Abd-B), and the linker lengths between the homeodomain and the hexapeptide motif (Gehring et al., 1994; Duboule, 1994; Mann, 1995; Chan et al., 1996; Mann and Chan, 1996; Piper et al., 1999; Passner et al., 1999).
We posit that the findings reported here have implications for understanding Hox gene function and evolution. The functional equivalence of almost all of the Hox proteins in brain neuromere specification implies that the specificity of Hox gene action is achieved mainly through regulatory elements that control position, timing and level of Hox gene expression and only to a lesser degree through Hox protein sequence differences. Similar findings have been obtained in studies on Pax gene interchangeability in Drosophila (Li and Noll, 1994). Thus, the genes paired and gooseberry, which have distinct developmental roles in embryogenesis and have considerably diverged coding sequences, can exert the same conserved function in genetic rescue experiments. Comparable findings have recently been reported in mammals (Bouchard et al., 2000), corroborating the idea put forward by Noll that the essential difference among these developmental regulatory genes of the same family may reside in their cis-regulatory regions.
The fact that the expression of different Hox genes in the lab mutant domain does not cause homeotic transformation of tritocerebral identity, suggests that Hox proteins act as mediators rather than as selectors within the developmental pathway that specifies segmental neuronal identity in the Drosophila brain. Recent experiments using both loss- and gain-of-function mutations suggest that this also applies to the specification of other structures along the anteroposterior body axis of Drosophila. For example, in haltere development, abd-A and to some extent Abd-B can substitute for Ubx gene action (Casares et al., 1996). Moreover, a comparable lack of Hox gene specificity has been observed in gonad development (Greig and Akam, 1995).
Finally, the high degree of functional interchangeability of Lab and all of the other Drosophila Hox proteins, with the exception of Abd-B, is consistent with evolutionary studies that propose a common origin of all of the Hox genes from a single ancestral progenitor and an early singularity of Abd-B-like genes in the ancestral Hox gene cluster (Schubert et al., 1993). Given the striking evolutionary conservation of structure, expression and brain-specific function of lab and its mammalian Hox1 orthologs (Hirth and Reichert, 1999; Reichert and Simeone, 1999), it will now be important to determine whether functional equivalence among non-paralogous Hox gene products is also valid for vertebrate hindbrain development.
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ACKNOWLEDGMENTS |
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