BIOLOGICAL OVERVIEW

Dichaete, also known by its contemporary preferred name Sox box protein 70D, is a classic gene of Drosophila. First described by Bridges, the dominant mutation results in wings extended at 45 degrees from the body axis and elevated 30 degrees above, alulae missing, dorcentrals and some other bristles reduced in number, head often deformed or split in the postverticle region, halteres turned down and homozygous lethal (Lindsley, 1992). Dichaete (Russell, 1996), or fishhook, as described by Nambu (1996), is a SOX domain protein implicated in the regulation of pair-rule genes. The SOX domain is a sequence-specific DNA binding domain found in those proteins of the High Mobility Group (HMG) superfamily, which are closely related to the mammalian sex determining factor SRY. A known Drosophila HMG protein, HmgD is involved in the transition from a transcriptionally silent to a transcriptionally active embryo early in development (Ner, 1994).

HMG domain proteins possess the ability to bind to and bend DNA. Moreover, two SOX domain proteins from the mouse, SOX2 and LEF-1, have been shown to be unable to activate transcription on their own; they must act in concert with other enhancer binding proteins (Travis, 1991 and Yan, 1995). This has lead to the suggestion that SOX domain proteins play architectural roles. For example, HMG proteins could be involved in assemblying higher-order nucleoprotein complexes, either by bending DNA to juxtapose nonadjacent sites for interaction with other transcription regulators, or by physically interacting with other regulatory proteins to provide a scaffold upon which other proteins are assembled (Grosschedl, 1994). One of the best examples of the interaction of HMG proteins with chromatin, is replacement of Hmg-D with Histone H1 in the Drosophila mid-blastula transition (Ner, 1994).

Dichaete directly regulates pair-rule genes even-skipped, fushi tarazu, hairy and runt and directly or indirectly affects the segment polarity gene wingless. even-skipped, hairy, and runtall show reductions in levels of expression in Dichaete mutants, with variable stripe specific effects on eve, fushi tarazu, hairy and runt. Since the stripes of pair rule genes generally occur in the correct anterior-posterior position in Dichaete mutants, the gene is unlikely to provide key positional information; it is more likely to be required in the maintainance or establishment of appropriate levels of pair-rule gene expression in the central region of the embryo (Nambu, 1996 and Russell, 1996).

Dichaete mutation also disrupts the central nervous system resulting in a disorganized axon scaffold with some ganglia exhibiting narrowed longitudinal connectives and partial fusion of commissural axon tracts. The effect on the CNS is likely to be due to loss of and/or fusion of midline and lateral engrailed-expressing CNS cells, such as midline cells including the median neuroblast. Dichaete is strongly expressed in the CNS (Nambu, 1996).

Dichaete/fish-hook is required for postembryonic development. Anti-Fish immunostaining reveals that Fish is expressed in several tissues, including the optic lobes, the central brain and ventral ganglia; the eye-antennal and leg imaginal discs; the imaginal ring and proximal cells of the salivary gland, and the hindgut. In the optic lobes, strong Fish expression is observed in the inner and outer proliferation centers. Fish expression is also detected in lateral cells in the thoracic ganglia and a subset of CNS midline cells in posterior abdominal ganglia. In the eye-antennal disc, Fish is expressed in a thin strip of cells along the ventral edge of the antennal disc that extends into the ventral/anterior region of the eye disc. This region corresponds to cells that give rise to ventral head cuticle, which separates the eyes and antennae. Interestingly, no Fish expression is detected in either wing or haltere discs, suggesting that the wing posture defects in dominant Dichaete alleles are likely the result of ectopic fish expression in the wing disc. In all leg discs, Fish is expressed in a few cell-wide teardrop-shaped patterns: the strongest expression is detected in the ventral/posterior and dorsal/anterior quadrants. In ventral quadrants, Fish expression runs along the tibial/tarsal boundary, while expression in the dorsal quadrants is in more proximal domains. Fish protein is also detected in the salivary gland imaginal ring, proximal salivary gland cells, and the hindgut. In all the larval tissues examined, Fish protein is predominantly localized in cell nuclei (Mukherjee, 2000).

fish null mutants are embryonic lethal and exhibit severe defects in segmentation and CNS development. Thus, to directly examine fish postembryonic functions mitotic clones of fish were generated using the fish87 null mutant and the FLP/FRT system. Recombination was induced at different developmental stages and the resulting fish mutant clones were detected in adult animals by morphological inspection using the yellow (y) and white (w) mutations as markers, and in larval tissues by anti-Fish immunostaining. When recombination was induced during the first and second instar (24 -48 h AEL), out of over 400 adults of the appropriate genotype no animals with fish mutant clones were detected, suggesting either that mitotic recombination was not induced or that the effects of early induction were lethal. This issue was investigated via anti-Fish immunostaining, which clearly reveals the formation of mutant clones that lack Fish expression in third instar larval brains, as well as eye-antennal and leg discs. These findings strongly suggest that the loss of Fish function during early larval stages results in lethality and confirms that Fish is required for postembryonic development (Mukherjee, 2000).

Recombination was induced during later stages of development, specifically during the third instar (72-96 h AEL). This treatment results in adults that exhibit large patches of fish mutant cells both in the eye, as detected via a w mutant phenotype, and in bristles along the wing margins, as detected via the y mutant phenotype. These fish mutant clones appear morphologically normal, consistent with the lack of Fish expression in regions of the eye disc that give rise to ommatidia, or in the wing disc. Interestingly, large clones of fish mutant cells were never detected in the head capsule or legs. Instead small patches of black necrotic cells were detected in the first antennal segment, the ventral head cuticle surrounding the eye, and the leg tibial/tarsal boundary region. These are tissues derived from Fish-expressing regions of the eye-antennal and leg imaginal discs. The small size of these mutant patches suggests that fish is important for cell proliferation or survival (Mukherjee, 2000).

The effects of loss of fish function on gene expression in developing larval tissues were analyzed. In particular, the expression of two key developmental regulators, wingless (wg) and engrailed (en), were examined in fish mutant clones using a P[wg-lacZ] reporter and Mab 4D9. In leg discs, wg is normally expressed in a wedge in the anterior/ventral quadrant. In some individuals where fish mutant clones were induced, wg expression was absent in small patches of cells near the tibial/tarsal boundary. This corresponds to a region of prominent Fish expression in normal leg discs. Thus, fish function is required for wg expression during larval development. The loss of fish function is also associated with defects in en expression. When fish clones were induced, small patches of cells lacking en expression were detected in regions of the antennal disc corresponding to sites of Fish expression. Fish is also normally expressed in many cells in the larval brain, and the loss of fish is associated with an absence of en expression in discrete clusters of brain cells. Taken together, these loss-of-function studies indicate that fish plays an important role in regulating larval gene expression and cell differentiation (Mukherjee, 2000).

To complement these studies on fish loss-of-function phenotypes, targeted expression experiments using the Gal4/UAS system to mis-express Fish in developing imaginal discs were carried out. A P[UAS-fish] strain was created to express full-length Fish protein and it was crossed to different P[Gal4] driver strains in which gal4 is expressed prominently in eye-antennal and leg discs. While several of these combinations result in lethality, a few yield viable adults that exhibited specific appendage defects. The P[UAS-fish] strain was crossed to P[dpp-Gal4] flies, which prominently express Gal4 protein in dpp-expressing domains of the imaginal discs. At 25°C P[dpp-Gal4]/P[UAS-fish] animals survive to pharate adults but fail to eclose. These flies exhibit multiple morphological disruptions of eyes, antennae, wings, legs, and bristles. For example, in P[dpp-Gal4]/P[UAS-fish] animals the arista region of the antenna is generally lacking and replaced by a thick cuticular structure. Some animals exhibit an arista-to-tarsal transformation. More proximal regions of the antennae are normal, indicating that ectopic fish expression specifically disrupts the elaboration of distal structures. Defects in the elaboration of the aristae were also observed when P[UAS-fish] flies were crossed to the P[30A-Gal4] strain, which expresses Gal4 in distal regions of the antennal disc. P[30A-Gal4]/1; P[UAS-fish]/1 flies are viable, but exhibit reduced aristae that are compressed along the proximal/distal axis. The main aristal branch is severely shortened and the side branches are also stunted, though not reduced in number. P[dpp-Gal4]/P[UAS-fish] animals also exhibit severely truncated legs, as tarsal segments 2-4 are generally fused or missing. In addition, both tarsal and tibial regions often exhibit ventralization, and in some animals, a small ectopic distal leg segment is also detected (Mukherjee, 2000).

To better understand how mis-expression of fish disrupts elaboration of distal leg segments, expression of the dpp, wg, Distal-less (Dll), and bric-a-brac (bab) genes was examined. These genes all play key roles in the differentiation of leg imaginal discs. Thus, mutually antagonistic interactions between dpp and wg define the dorsal and ventral regions of the leg, and both genes are required for activation of Dll. Dll is in turn essential for proximal/distal axis elaboration and activation of bab expression. In wild-type leg discs, dpp is expressed along the anterior/posterior compartment boundary. In P[dpp-Gal4]/P[UAS-fish] leg discs, the ventral expression of dpp is eliminated, and levels are reduced in the dorsal domain. fish mis-expression also disrupts wg expression. While wg expression is normally restricted to a ventral/anterior wedge in the leg disc, in P[dpp-Gal4]/ P[UAS-fish] leg discs wg expression is expanded into the ventral/posterior quadrant. These defects likely contribute to the ventralization phenotype detected in the legs of P[dpp-Gal4]/P[UAS-fish] flies. Because the proximal/distal leg defects in P[dpp-Gal4]/ P[UAS-fish] flies resemble those of Dll mutants, one possibility is that ectopic Fish expression disrupts Dll expression. In wild-type leg discs, Dll expression is observed as broad rings that span most of the disc. In P[dpp-Gal4]/P[UAS-fish] leg discs, this expression pattern is essentially unaltered. Thus, the proximal/distal patterning defects in P[dpp-Gal4]/P[UAS-fish] flies do not result from alterations in Dll expression. An analysis was therefore carried out to determine whether expression of bab, which is downstream of Dll, is affected by ectopic fish. In wild-type leg discs, bab expression is detected in a small circular pattern in the tarsal region. Strikingly, in P[dpp-Gal4]/P[UAS-fish] flies bab expression is completely eliminated. This result suggests that ectopic Fish directly represses bab expression and/or inhibits the ability of Dll to activate bab in the developing leg disc (Mukherjee, 2000).

The pattern of fish expression in the eye disc suggests that it could play a role in setting an anterior boundary of the eye field. Indeed, ectopic fish expression in the eye disc dramatically suppresses eye development. P[dpp-Gal4]/P[UAS-fish] flies exhibit moderate to severe loss of eye tissue, with many individuals exhibiting a complete absence of ommatidia. These defects are correlated with disruptions in dpp and wg expression in the developing eye imaginal disc. In wild-type animals, dpp is expressed within the morphogenetic furrow (MF) as it progresses from posterior to anterior; dpp is required for MF progression and eye formation. wg is normally expressed in lateral regions of the eye disc and suppresses inappropriate MF initiation. dpp and wg exhibit mutually antagonistic interactions, since loss of dpp function results in expanded wg expression, while increased dpp function leads to reduced wg expression. In P[dpp-Gal4]/P[UAS-fish] flies the eye discs are greatly reduced in size and no MF is apparent. Correspondingly, dpp expression is greatly reduced while wg expression is expanded into central regions of the eye disc. These results suggest that ectopic fish suppresses eye formation by altering dpp and wg expression patterns: this results in a blockade of MF formation (Mukherjee, 2000).

The Fish protein possesses distinct DNA binding and bending as well as transcriptional activation properties, suggesting that it can perform multiple transcriptional regulatory functions. In order to begin to determine which activities may be required for specific developmental functions, the properties of several truncated Fish proteins were examined. Six different P[UAS] strains were generated that express specific regions of Fish, including Fish-NH2 (amino acids 1-139), Fish-HMG (amino acids 140-218), Fish-COOH (amino acids 219-382), Fish-NH2/HMG, Fish-HMG/COOH, and Fish-NH2/COOH. Does Fish contain an intrinsic nuclear localization signal (NLS)? This is of interest because while Fish protein exhibits nuclear localization in larval tissues, during early embryonic stages Fish is present in both nuclear and cytoplasmic compartments. Antisera against the Fish NH2, HMG, or COOH regions were used to monitor the distribution of the truncated proteins ectopically expressed in larval salivary glands via the P[SaGa52a-Gal4] strain. The salivary glands were utilized because of the large cell sizes and absence of endogenous Fish expression in the main body of the gland. The HMG domain was found to be sufficient for nuclear targeting, as all versions containing the HMG domain exhibit extensive nuclear accumulation. In contrast, all versions of Fish lacking the HMG domain are largely cytoplasmic (Mukherjee, 2000).

It was then asked whether activities of the truncated Fish proteins induce developmental defects when mis-expressed in developing imaginal discs. Surprisingly, when expressed via P[dpp-Gal4], none of the truncated proteins cause any phenotypic abnormalities; the resulting flies were all viable and morphologically normal. This indicates that at least when expressed via P[dpp-Gal4], all three regions of Fish protein are required to induce detectable phenotypes. Next to be tested was whether the truncated proteins might produce phenotypic effects when expressed in different patterns in the eye-antennal and leg imaginal discs. This was pursued by crossing each P[UAS] strain to P[GMR-Gal4] and P[Dll-Gal4]. When full-length Fish was expressed via P[GMR-Gal4] in the developing eye disc, the P[GMR-Gal4[/1;P[UAS-fish]/1 flies exhibit a reduction and disorganization of ommatidia and mechanosensory bristles. The defects are dosage-dependent, as P[GMR-Gal4[/1;P[UAS-fish] flies exhibit a total absence of ommatidia and mechanosensory bristles, and instead contain necrotic cell masses. These data further demonstrate that ectopic fish can repress eye formation. For the truncated Fish proteins, only ectopic expression of Fish-HMG/COOH results in eye defects. P[GMR-Gal4]/1;P[UAS-fish-HMG/ COOH]/1 flies exhibit a moderate eye roughening and disorganization of ommatidia. The phenotype is more severe when two copies of P[UAS-fish-HMG/COOH] are present; however, it is still reduced compared to the effects of full-length Fish. P[GMR-Gal4]-driven expression of each of the other Fish proteins yields flies with normal eyes (Mukherjee, 2000).

The Fish-HMG/COOH is also the only truncated version that produces detectable phenotypes when expressed in the leg and antennal imaginal discs via P[Dll-Gal4]. When raised at 18°C, P[Dll-Gal4] flies are viable and exhibit normal appendage morphology. However, when raised at 25°C P[Dll-Gal4] flies exhibit deleted tarsal segments and truncated aristae. At both temperatures P[Dll-Gal4]-targeted expression of full-length Fish results in complete prepupal lethality, and so effects on appendage development could not be examined. In contrast, P[Dll-Gal4]/P[UAS-HMG-COOH] flies are viable and exhibit specific antennal defects. At 18°C the aristae are shortened and thickened while at 25°C no aristae are formed; instead there are abnormal cuticular structures protruding from the third antennal segment. Taken together, these data suggest that the COOH region of Fish possesses significant, albeit unknown functional properties, and that the NH2-terminal transcriptional activation domain is not absolutely essential for Fish gain-of-function phenotypes (Mukherjee, 2000).

While the precise developmental functions of Sox genes are still being defined, Sox proteins do not appear to directly specify cell or tissue identities. Many Sox genes are expressed during early stages of tissue development and may enhance the ability of cells to respond to differentiation signals. In this regard they can be considered as developmental modulators that promote the efficient progression of specific processes by influencing the activity of other developmental regulators (Mukherjee, 2000).

GENE STRUCTURE

Exons - 1

PROTEIN STRUCTURE

Amino Acids - 382

Structural Domains

Dichaete contains a 76 amino acid stretch with 88% identity to the DNA-binding domain of SOX2 proteins from human, mouse and chicken. Outside the DNA-binding domain there is no similarity to other proteins, although there is a 30 amino acid stretch at the C-terminus end with limited similarity to a potential SOX2 activation domain. The HMG domain of Dichaete shares 83% identity with human Sox3 protein (Nambu, 1996 and Russell, 1996).

Based on homology within the HMG domains of SRY and SOX proteins, 6 distinct subgroups (A-F) have been proposed. The sequence of its HMG domain places Dichaete in the B group, which includes SOX1, SOX2, SOX3, SOX11, SOX 14 and SOX19. There are 25 positions at which an invariant residue is present in the HMG domains of SRY, Dichaete and the 13 SOX proteins that have been analyzed. Interestingly, the only position where Dichaete differs from an otherwise invariant residue is at postion 18, which, except for the most variant F subgroup, is a lysine, but in the Dichaete HMG domain it is a glutamine. Outside the HMG domain Dichaete possesses several short alanine-, glutamine-, and serine-rich stretches that may serve as transcriptional activation domains, as well as 11 copies of a repeat pentapeptide sequence (Nambu, 1996).

date revised:  28 February 2000 

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