For clarification of a possible role of C15/clawless in the pretarsus/distal-tarsus establishment, the temporal and spatial expression patterns of Cll were compared with those of Al in leg discs collected at various stages. Pretarsus Al expression is first detected in early third instar in a domain slightly overlapping the Bar domain (Kojima, 2000). Similar to Al, pretarsus Cll expression first becomes discernible in early third instar, at which time, Cll expression largely overlaps that of Al. A close examination indicates that the Cll domain is slightly narrower than that of Al and that, in contrast to the Al domain, the Cll domain possesses no apparent peripheral gradation. Since both anti-Bar and anti-Cll antibodies were prepared from immunized rabbits, Bar expression could not be directly compared with Cll expression. However, it is believed that the Cll domain possesses almost no overlap with the Bar domain, because no appreciable overlap was recognized between cll mRNA and Bar expression domains (Kojima, 2005).
By mid third instar, the peripheral Al gradation disappears and the proximal extents of the Al and Cll domains became identical to each other. From this stage onward, the future distal leg region is virtually completely separated into two regions: Bar-positive future distal tarsus and Al/Cll-positive future pretarsus (Kojima, 2005).
An antibody raised against C15 revealed that it was expressed in exactly the same cells as Al and Lim1 in the center of leg discs, so that its expression domain abuts that of Bar, which is expressed in ta IV and V (there are actually two Bar genes, H1 and H2, which are co-expressed) (Campbell, 2005).
Signals from the BMP family member Decapentaplegic (Dpp) play a role in establishing a variety of positional cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although a reasonably clear picture is available of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. Three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, are described that are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. These genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, it has been demonstrated that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals (Reim, 2003).
The expression of a novel amnioserosa marker, which is encoded by the homeobox gene C15, was examined. In the normal situation, C15 is expressed in the amnioserosa from stage 7 until stage 17, when the amnioserosa undergoes apoptosis. In addition, from early stage 10 onwards there is a narrow domain of expression at the leading edge of the dorsal germ band, which later becomes segmental. In DocA mutant embryos, the level of C15 expression in early amnioserosa cells is unaltered; this allows use of C15 protein as a marker for the development of this tissue in the absence of Doc activity (Reim, 2003).
Until stage 9, the large majority of amnioserosa nuclei in DocA mutant embryos appear large and flattened as in wild-type embryos. Together with data from alpha-tubulin staining, this observation indicates that the amnioserosa cells begin to acquire the normal features of a squamous epithelium. However, the amnioserosa does not display a properly folded morphology during stages 8-10, and the posterior germ band is forced to bend towards the inside in DocA mutant embryos. In addition, some small nuclei become detectable within the amnioserosa during this stage. Altogether, these observations indicate that the amnioserosa initiates its differentiation process in the absence of Doc gene activity but fails to complete it, thus leading to morphological and functional abnormalities of this tissue towards the end of germ band elongation. Much stronger alterations can be observed during subsequent stages, when there are an increasing number of C15-stained amnioserosa nuclei with much smaller diameters than regular amnioserosa nuclei. At late stage 12, almost all amnioserosa cells feature small nuclei that are difficult to distinguish from dorsal epidermal cells. Co-staining for race indicates that it is predominantly the cells with the small nuclei that lose race expression, while most normally-sized nuclei are still surrounded by race signals. From this stage onwards, non-stained 'holes' appear in the amnioserosa and the number of C15-stained amnioserosa nuclei decreases prematurely. Hence, unlike wild-type embryos, stage 14 DocA mutant embryos are not covered dorsally by C15-stained amnioserosa cells. In addition to the observed alterations in the amnioserosa, the C15 expression domain at the leading edge of the epidermis appears significantly broadened (Reim, 2003).
Is the increasing number of smaller nuclei in the amnioserosa of DocA mutant embryos connected with abnormal cell divisions? The M-phase marker phospho-Histone H3 can be detected in numerous amnioserosa nuclei of DocA mutant embryos after stage 10; this increase is not seen in wild-type embryos. In addition, there is significant incorporation of BrdU in amnioserosa nuclei of DocA mutant embryos (particularly in the small nuclei, whereas no incorporation is observed in wild-type embryos. Mitotic spindles are also present in the amnioserosa of DocA mutants. These observations indicate that the normal G2 arrest of amnioserosa cells has been released and the cells re-enter the cell cycle. Whether the subsequent disappearance of small C15-stained amnioserosa nuclei in DocA mutant embryos is a result of premature apoptosis of cells in this tissue was also tested. This possibility was confirmed by the results of TUNEL labeling experiments, which produced signals in many amnioserosa nuclei from 12 onwards. Most of the TUNEL-labeled nuclei have reduced or are lacking C15 expression, which shows that wild-type amnioserosa nuclei at late stage 12 are not apoptotic). Altogether, these observations suggest that loss of Doc activity prevents the normal differentiation of the amnioserosa to a fully functional tissue, suspends the cell cycle block of amnioserosa cells, and causes premature apoptotic cell death in this tissue (Reim, 2003).
To uncover genes encoding for proteins that are required for Al activity or that lie upstream or downstream, a screen was devised to identify genes, which when mutated, could dominantly modify the phenotype of a weak al mutant. In null al mutants, the arista, the terminal portion of the antenna, is lost almost completely, although a vestige remains. However, in a weak allelic combination, alush/130, the arista is almost full length. Random mutagenesis yielded several mutants that dominantly reduced the size of the arista in alush/130 flies. One of these genetic enhancers was characterized as a mutation in the EGF-receptor (EgfrEal43), which has been shown to lie upstream of al, indicating that the screen could be successful in its goals. One more of these dominant enhancers corresponds to the C15 gene. In fact, three alleles of C15 were identified in the screen, C151, C152 and C153, and are the first mutations identified in this gene (Campbell, 2005).
When al mutant alleles are separated from C15 alleles, homozygotes of all three C15 alleles survive to adulthood, although two, C152 and C153, die soon after emerging. Examination of these adults revealed that their legs and antennae have identical phenotypes to that of al, i.e., the aristae and the claws are either reduced or completely lost. In the weakest mutant, C151, the aristae are reduced but the claws are normal; this is similar to weak al mutants such as al1. In the stronger two mutants, C152 and C153, the arista is almost completely eliminated apart from a very small vestige and the structures found at the tip of the leg (claws, pulvilli, and empodium) are completely eliminated. In addition, although there are still five tarsal segments, ta IV and V are reduced to about half their normal size. All these phenotypes are identical to those of null or very strong alleles of al, but much stronger than that of null lim1 mutants, which often possess a claw (Campbell, 2005).
These mutants were shown to correspond to C15 as follows. (1) They were placed in the interval 93C3-93F by deficiency mapping: this region includes C15. All other available mutations in this region complement the C15 alleles. (2) In situ hybridization showed that C15 is expressed in the center of the leg and antennal discs, i.e., in cells giving rise to the regions affected in the mutant adults. Finally, sequencing of each of the mutants identified a single base change in C15 that, in C151, results in substitution of a conserved residue N-terminal to the homeodomain (H175Q), and, in both C152 and C153, results in a stop codon truncating the protein at residues 170 and 137, respectively. Both truncations occur before the homeodomain, suggesting that these two are probably null alleles (Campbell, 2005).
The subdivision of the developing field by region-specific expression of genes encoding transcription factors is an essential step during appendage development in arthropod and vertebrates. In Drosophila leg development, the distal-most region (pretarsus) is specified by the expression of homeobox genes, aristaless and Lim1, and its immediate neighbor (distal tarsus) is specified by the expression of a pair of Bar homeobox genes. One additional gene, clawless (cll), a homolog of vertebrate Hox11/tlx homeobox gene family and formerly known as C15, is specifically expressed in the pretarsus and cooperatively acts with aristaless to repress Bar and possibly to activate Lim1. Similar to aristaless, the maximal expression of clawless requires Lim1 and its co-factor, Chip. Bar attenuates aristaless and clawless expression through Lim1 repression. Aristaless and Clawless proteins form a complex capable of binding to specific DNA targets, which cannot be well recognized solely by Aristaless or Clawless (Kojima, 2005).
al involvement in the repression of Bar expression in the pretarsus has been demonstrated based on the observation that Bar is de-repressed in alex/alice leg discs (Tsuji, 2000). Bar repression in the pretarsus may include at least one additional factor expressed in the future pretarsus and functioning with al to repress Bar, since no appreciable reduction in Bar expression is detected (Kojima, 2000) upon al misexpression (Kojima, 2005).
While searching for a possible candidate gene functioning with al to repress Bar, a mutant with phenotype quite similar to that of alex/alice flies was found and named clawlessdl9-2 (clldl9-2) after the leg phenotype. Most clldl9-2 homozygotes developed into pharate adults and some eclosed. As with alex/alice flies, clldl9-2 flies lack pretarsus structures such as claws, the arista in the antenna and medial notum bristles (Kojima, 2005).
After meiotic recombination mapping and complementation tests using several deletion chromosomes, clldl9-2 was mapped to 93E1-93F8. In this interval, about 20 genes have been annotated by the Drosophila genome project. In situ hybridization of late third instar imaginal discs indicated that C15, previously identified as a Drosophila homolog of Hox11-type homeobox genes (Dear, 1994), is expressed specifically in the regions with morphological defects in clldl9-2 flies: the pretarsus and the medial region of the notum. The C15 expression domain in the late third instar leg disc completely overlaps the Aristaless domain. al mutant but not clldl9-2 flies display apparent defects in sternoplural bristles and the first wing vein. Postvertical bristles are absent from clldl9-2 but not alex/alice flies. In agreement with phenotype differences, al is expressed in the proximal region of the leg disc, where the sternoplural bristles develop, and in the anterior wing pouch with formation of the first vein. No al expression could be found in the future oceller region, where postvertical bristles are produced. C15 was expressed in the future oceller region but not in the proximal region of the leg disc, along with the wing pouch (Kojima, 2005).
The nucleotide sequence analysis of C15 cDNA prepared from mRNA extracted from clldl9-2 homozygous larvae shows a base substitution at nucleotide position 821, that alters the codon for tryptophan 240 to a termination codon. This mutation causes a 68-amino-acid-long C-terminal deletion, including five C-terminal amino acids of the homeodomain. A basic residue in this region of the homeodomain is implicated in DNA binding, and consequently, clldl9-2 may be concluded to be a mutant allele of C15. The phenotypes of hemizygotes are almost identical to those of homozygotes, indicating that clldl9-2 may be a functional null or a very strong hypomorphic allele. Since no C15 mutant has been reported, and Drosophila gene name is usually given after the mutant phenotype, C15 is referred to as clawless (cll) (Kojima, 2005).
Reference names in red indicate recommended papers.
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date revised: 20 February 2007
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