Expression of the Drosophila secreted cuticle protein 73 (dsc73) requires Shavenbaby

Abstract

Low stringency genomic library screens with genomic fragments from the sex determination gene doublesex identified the Drosophila secreted cuticle protein 73 (dsc73) gene, which encodes an 852-residue protein with an N-terminal signal sequence. In embryos, dsc73 RNA and protein are expressed to high levels in the epidermal cells that secrete the larval cuticle as well as in other cuticle-secreting tissues such as the trachea and salivary duct. Embryonic expression of dsc73 requires Shavenbaby, a transcription factor regulating cuticle formation. Double-labeling experiments with alphaCrb and alphaSAS reveal that, as with chitin and other known cuticle proteins, Dsc73 is secreted apically. Zygotic loss of dsc73 results in larval lethality but loss does not result in overt patterning defects or overt morphological defects in the embryonic tissues in which it is expressed. Thus, dsc73 encodes a novel secreted protein, and it is conserved within the Drosophila group. dsc73 may serve as a useful embryonic marker for cuticular patterning.

Fig. 1

A: dsc73 (green bar, top figure) maps distal to the argos gene (red bar, top figure) and encodes a single transcript with five exons (boxed areas, bottom figure). The open reading frame (green areas inside boxed region, bottom figure) encodes 852 residues. An inactivating P-element insertion in dsc73, l(3)j10E8, maps within the 5′untranslated region and an inactivating Piggy-bac element insertion in dsc73, l(3)e01027, maps ~4.8 kb upstream of the splice acceptor site of exon 2. B: The open reading frame of dsc73 has an N-terminal signal sequence (blue), several N-linked gycosylation consensus sites (red), and four repeats of CXCPX(1-2) aromatic (brown). The sequence with yellow background was used to generate antiserum to Dsc73. The sequence with green background is the most highly conserved and highly structured domain of the protein. C: A 147-residue domain in Dsc73 is conserved in all insects and in the arthropod Daphnia pulex. Of interest, this domain spans four exons in Drosophila melanogaster. The exon boundaries are indicated by arrows.

Fig. 2

A: dsc73 RNA and protein are expressed to high levels in embryos. a–j show dorsal–ventral views of stage 12 (st 12) through 17 (st 17) embryos. k–t show lateral views of stage 12 through 17 embryos. a–e and k–o were hybridized with an RNA probe corresponding to a dsc73 cDNA. f–j and p–t were immunostained with a rat antiserum directed against a fragment of the Dsc73 protein. Expression is first observed in the posterior spiracle (ps) and limited domains in the head (hc; a,f,k,p). Expression is subsequently observed in epidermal cells (epid) and hindgut (hg; b,g,l,q), with high levels persisting in the head and epidermis through embryogenesis. During stages 15 and 16, expression is also observed in internal tissues, including the pharynx (ph; i,r), dorsal trunk (dt) cells of the trachea (i and s), salivary duct (sd; d and h), esophagus (es; c,d), an anterior subset of midgut cells (mg; c), and a subset of cells in all segments of the central nervous system (j). During stage 17, expression is also observed in the ventral and lateral black dots (vbd and lbd; o,t). Epidermal expression (epid) of dsc73 is highest in cells that form the denticles and hairs (b– e,i,l,j,n,o,s,t). B: Northern analysis of sex-specific polyA+-selected RNA revealed abundant dsc73 expression during the pupal stages (P), with very little expression in third instar larvae (L) and adults (A). C: Colocalization of Dsc73 with known apical proteins (Crb and/or SAS) in epidermal cells (a–a″), dorsal trunk tracheal cells (b– c″), and salivary duct (d– d″) reveals that Dsc73 is secreted apically. Note that levels of Dsc73 are higher in the part of the ventral epidermis that forms the denticles than in the part that forms naked cuticle (* and , respectively, in a′ and a″). Although Dsc73 is expressed in the dorsal trunk cells of the trachea (DT), it is not detected in the dorsal branch (DB) or transverse connective (TC) (b′, b″, c′ and c″).

Fig. 3

Expression of dsc73 requires ovo/shavenbaby. Wild-type larvae have high levels of dsc73 starting around embryonic stage 13 (left panels), whereas ovo/svb2 homozygotes express very low levels of the transcript (right panels), which is limited to the posterior spiracle precursors during stage 13 (upper right panel) and head structures at later stages (lower two right panels).

Fig. 4

Expression of βgal from the P-element insertion in the l(3)j10E8 line is nuclear and accumulates in the same tissues as the dsc73 transcript. Expression in internal tissues including the esophagus and midgut cells is evident in the top panels, whereas expression in the epidermal cells is evident in the lower four panels.

Fig. 5

A: A wild-type first instar larval cuticle is shown as well as a magnified view of the denticles from the fourth abdominal segment (A4). B–E: Larval cuticles from dsc73 mutant larvae have a completely normal pattern of denticles and hairs, and the A4 denticles appear morphologically normal. F: Larval cuticles from the viable dsc73 excision allele are also normal.