EvoprintHD of Scr

BIOLOGICAL OVERVIEW

Sex combs reduced is required for labial and first thoracic segment development. Mutants show a tendency to transform labial into maxillary structures. The mutated labial segment appears to be transformed into something closely resembling the more anterior segment, resulting in duplication of maxillary structures. This simplistic, rather linear view of gene action is belied by more than five years research into the regulation of Sex combs reduced. Complex regulation implies a complex function. The biological tools currently employed are as yet too crude to elucidate what must be a high level of interactive complexity. Current understanding is partial and at times contradictory, like the divergent views of the fabled elephant examined by blind men.

From the vertebrate perspective, the labial segment produces what is perhaps the most ornate and alien of the fly's structures, the proboscus. The proboscus protrudes out and downward from the head, a flexible dipstick adapted to gather liquid nourishment. At its working end hang the labella, or labial palps. These furrowed bulbs gather liquid, and move it upward through a series of collecting channels until it reaches the hollow, straw-like labrum. From here, food continues its upward journey into the pharynx by means of muscular action.

Scr is subject to complex regulation. The gene is sandwiched between chromosomal segments that constitute its regulatory region, but even this area of the DNA is complex. It comprises a span of over 70 kb of DNA extending from Antennapedia at one end, sandwiching in the fushi tarazu gene on the way to doing the same with Scr, and continuing to a point beyond the 3' terminus of Deformed . Thus the regulation region of Scr includes the insertion of fushi tarazu, a critically important pair-rule gene.

The existence of pair-rule gene ftz in the center of a regulatory region for a homeotic gene of the Antennapedia complex has implications beyond current understanding. What is ftz doing there, and how is it co-regulated with Scr?

Regulation of Scr requires silencers as well and these are found in a large fragment between ftz and Antp located 40 kb upstream of the Scr promoter (Gorman, 1995).

The regulation of Scr in most tissues is controlled by multiple regulatory elements; the simple view of a proximal promoter and a structural gene proves to be an inadequate explanation. Expression in the prothorax (thoracic segment number one) is controlled by an element in the first intron and an enhancer on the other side of ftz. Expression in the labial lobe requires sequences in the first exon and intron, sequences on the far side of ftz, as well as other sequences, even further away in the direction of Antennapedia. Expression in the salivary gland requires these same regions and expression in the midgut requires sequences found between Scr and ftz. Expression in the CNS requires sequences in the second intron and the 3' end of ftz, as well as other regions. Given this level of complexity, no simple picture is possible (Gindhart, 1995b).

The complexity of Scr regulation, not at all unexpected considering the existence of site specific regulatory elements in such genes as achaete and even-skipped, nevertheless highlights an unbelieveable level of complexity in the evolution of gene regulation. Here are at least three different uses for the Scr gene: labial differentiation, CNS development and mesodermal development, each with independent gene regulation. It will be some time before the details at this level of complexity are understood in terms of the regulation and function of this gene.

Salivary gland formation in the Drosophila embryo is linked to the expression of Sex combs reduced. When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere, salivary glands form in new places. However, not every cell that expresses Scr is recruited to a salivary gland fate. Along the anterior-posterior axis, the posteriorly expressed proteins encoded by the teashirt (tsh) and Abdominal-B (Abd-B) genes block Scr activation of salivary gland genes. Along the dorsal-ventral axis, the secreted signaling molecule encoded by decapentaplegic prevents activation of salivary gland genes by Scr in dorsal regions of parasegment 2. Five downstream components in the Dpp signaling cascade required to block salivary gland gene activation have been identified: two known receptors (the type I receptor encoded by the thick veins gene and the type II receptor encoded by the punt gene); two of the four known Drosophila members of the Smad family of proteins which transduce signals from the receptors to the nucleus [Mothers against dpp (Mad) and Medea (Med)], and a large zinc-finger transcription factor encoded by the schnurri (shn) gene. The expression patterns of d-CrebA and Trachealess were examined in embryos missing zygotic function of schnurri. In embryos homozygous for shn, a dorsal expansion of salivary gland protein expression is observed. The presence of amnioserosa, an extreme dorsal cell type, suggests that embryos lacking zygotic shn function are not ventralized, as are embryos missing maternal and zygotic function of tkv, pt, Mad, or Med or missing zygotic function of dpp. These results reveal how anterior-posterior and dorsal-ventral patterning information is integrated at the level of organ-specific gene expression (Henderson, 1999). Scr is normally expressed in all the cells that form the salivary gland. However, as the salivary gland invaginates, SCR mRNA and protein disappear. Homeotic genes, such as Scr, specify tissue identity by regulating the expression of downstream target genes. For many homeotic proteins, target gene specificity is achieved by cooperatively binding DNA with cofactors. Therefore, it is likely that Scr also requires a cofactor(s) to specifically bind to DNA and regulate salivary gland target gene expression. Two homeodomain-containing proteins encoded by the extradenticle and homothorax genes are also required for salivary gland formation. exd and hth function at two levels: (1) exd and hth are required to maintain the expression of Scr in the salivary gland primordia prior to invagination and (2) exd and hth are required in parallel with Scr to regulate the expression of downstream salivary gland genes. Scr regulates the nuclear localization of Exd in the salivary gland primordia through repression of homothorax expression, linking the regulation of Scr activity to the disappearance of Scr expression in invaginating salivary glands (Henderson, 2000).

To determine if Exd cooperates with Scr to control salivary gland gene expression, the accumulation of two early salivary gland proteins, CrebA and Trachealess (Trh), was examined in embryos lacking exd function. Zygotic loss of exd function results in a reduction in the number of salivary gland cells expressing CrebA and Trh, as well as a decrease in the level of protein made in these cells. This reduced level of salivary gland protein expression is not as severe as the one seen in Scr mutant embryos. Unlike SCR, EXD mRNA is supplied maternally and, thus, the maternal contribution may partially compensate for the loss of zygotic function. To test this possibility, the maternal contribution of exd was removed using the FLP-FRT system. In embryos lacking maternal exd function, salivary gland expression of CrebA and Trh is at wild-type levels. However, salivary gland expression of CrebA and Trh is completely absent in embryos lacking both the maternal and the zygotic contributions of exd. Thus, exd is required for embryonic salivary gland gene expression. Moreover, zygotically provided exd can rescue the loss of maternally provided exd and maternally provided exd can partially compensate for zygotic loss of exd (Henderson, 2000).

Since Scr, exd, and hth are required for salivary gland formation, the mRNA and/or protein expression patterns of these genes during normal salivary gland formation were examined. During stages 9 and 10, when salivary gland gene expression is established, Scr and hth are expressed in the salivary gland primordia, as well as other tissues, and Hth and Exd are nuclear. During stage 11, after the establishment of early salivary gland gene expression, the salivary glands begin to invaginate. At this stage, there are several changes in the expression and/or localization of these genes and/or proteins in the salivary gland cells: Scr and hth transcripts disappear, Hth protein disappears, and Exd protein becomes cytoplasmic (Henderson, 2000).

Hth is known to regulate the nuclear entry of Exd in other cells of the embryo and in the imaginal discs. In embryos lacking hth function, Exd is cytoplasmic throughout the embryo, including the cells of the salivary gland primordia. Since Hth is required for the activity of Exd, and Exd is required to maintain Scr expression in the salivary gland primordia, the expression of Scr in embryos lacking hth function was also examined. As observed in exd minus embryos, neither SCR mRNA nor Scr protein expression is maintained in the ventral cells of PS2 in hth mutants. Exd is required for the stability of Hth throughout the embryo and, correspondingly, a loss in Hth protein stability, but not HTH transcript accumulation is observed in exd mutant embryos. Thus, hth maintains the expression of Scr in the salivary gland primordia and regulates the nuclear localization of Exd throughout the embryo, including the cells of the salivary gland primordia. Furthermore, exd is required for accumulation of Hth (Henderson, 2000).

Scr, Exd, and Hth are expressed in the salivary gland primordia when salivary gland gene expression is established. Once the salivary gland cells begin to invaginate, the expression and/or localization of all three proteins changes in the salivary gland primordia. This result indicates that a gene(s) expressed in the salivary gland primordia during stage 11 is required for these changes in gene expression and protein localization. Since hth expression is specifically lost in the salivary gland primordia, a test was performed to see if Scr regulates hth expression in the salivary gland primordia. In embryos lacking Scr function, HTH mRNA and Hth protein are maintained in the cells that would normally form the salivary gland beyond stage 11. Correspondingly, Exd remains nuclear in the ventral cells of PS2 in embryos lacking Scr function. Therefore Scr is required to repress hth expression in the cells of the salivary gland primordia during stage 11. The loss of hth expression consequently leads to the loss of nuclear Exd accumulation (Henderson, 2000).

Since Scr may act either directly or indirectly to shut off hth expression, the expression of hth was examined in embryos mutant for two early salivary gland genes that encode transcription factors: fkh, which encodes a winged-helix transcription factor related to mammalian HNF-3beta, and Creb-A, which encodes a beta-Zip protein that binds cyclic-AMP-response elements. No change in hth expression is seen in embryos lacking either fkh or Creb-A function. Therefore, Scr does not act through fkh or Creb-A to shut off hth transcription. These findings suggest that prior to stage 11, Hth regulates the nuclear entry of Exd in the salivary gland primordia. During stage 11, Hth is no longer expressed in these cells and, as a consequence, Exd localizes to the cytoplasm. Without Exd and Hth in the nucleus, Scr expression is not maintained. To test this idea, hth expression was maintained in cells of the salivary gland primordia using the GAL4-UAS system. This expression pattern was achieved using a fkh-GAL4 construct to drive expression of UAS-hth in the salivary gland secretory cell primordia from stage 10 onward. When hth was expressed under the control of the fkh-GAL4 driver, Hth protein could be detected in the salivary gland nuclei throughout embryogenesis. Correspondingly, Exd and Scr were detected in the salivary gland until late stages of embryogenesis. These results indicate that Hth is not only necessary, but is also sufficient, to maintain Scr expression in the salivary gland (Henderson, 2000).

GENE STRUCTURE

Molecular mapping of Scr breakpoint lesions has defined a segment of greater than 70 kb of DNA necessary for proper Scr gene function. This region is split by the fushi tarazu (ftz) gene, with lesions affecting embryonic Scr function molecularly mapping to the region proximal (5') to ftz and those exhibiting polyphasic semilethality predominantly mapping distal (3') to ftz (Pattatucci, 1991a). Distal to ftz is Antennapedia, 30 kb from ftz, and 48 kb from Scr. Deformed is 20 kb downstream of Scr (Le Motte, 1989).

Genomic length - 23 kb

cDNA clone length - There are two transcripts, 3.6 and 5.2 kb consisting of alternative 3' ends (Andrew, 1995).

Bases in 5' UTR - 626

Exons - three

Bases in 3' UTR - 2261

PROTEIN STRUCTURE

Amino Acids - 417

Structural Domains

Scr has sequence homology to mouse HOXA5, B5 and C5 (see four paralogous Hox clusters of mammals). The greatest similarity is within the DNA-binding homeodomain. Two shorter conserved motifs are found: one near the N-terminus and a second in a region N-terminal to the homeodomain. The former consists of an MSSF sequence that differs by one amino acid from the MSSY consensus as with most of the D. melanogaster homeotic genes. The latter consists of a WPWM core sequence (LeMotte, 1989 and Andrew, 1995).

date revised: 10 February 98

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