BIOLOGICAL OVERVIEW

Drosophila imaginal discs undergo extensive pattern formation during larval development, resulting in each cell acquiring a specific adult fate. The final manifestation of this pattern into adult structures is dependent on pulses of the steroid hormone ecdysone during metamorphosis, which trigger disc eversion, elongation and differentiation. The gene crooked legs codes for a zinc finger protein. crol mutants exhibit both a morphological defect in leg morphogenesis and altered gene expression during morphogenesis. A range of defects are evident in legs dissected from crol 4418 prepupae. The 2nd-5th tarsal segments of legs from mutant flies are slightly more expanded than wild type and the leg is not fully elongated. In other mutant flies, legs are severely distorted and reduced in length. These observations indicate that at least part of the crooked legs phenotype is due to defects in leg disc elongation. Although at least part of the leg phenotype associated with crol mutations can be attributed to defects in leg elongation during early prepupal development, crol appears to affect leg development during later stages as well. One manifestation of this is a kink near the middle of the mutant femur. The femur and tibia are initially fused at 18 hours after puparium formation and are divided into distinct segments between 21 and 36 hours after puparium formation. It is possible that the kink in crol mutant femurs is due to a defect in this morphogenetic process. In addition, some bristles are occasionally missing from crol mutant legs, indicating defects in the final stages of leg differentiation. crol mutants also exhibit a defect in head eversion. Altered expression of EcR and E74B (Ecdysone-induced protein 74EF) during the pupal period suggests that these genes are potential targets of Crol (D'Avino, 1998). This essay will analyze the role of Crol in leg elongation and consider the affect of Crol on gene expression.

The changes in ecdysone-regulated gene expression seen in crol mutant prepupae provide a framework for understanding the molecular basis of the crol mutant phenotypes. Four genes were examined that are expressed in imaginal discs and that appear to play a role in imaginal disc development: IMP-E1, Sb, Brg-P9 and EDG-84A. Of these genes, only Brg-P9 was affected in crol mutants: Brg-P9 is expressed at higher levels, and for a longer duration, in crol mutant prepupae, suggesting that crol may normally repress Brg-P9. Interestingly, Brg-P9 encodes a protein with sequence similarity to the kunitz class of serine protease inhibitors (Emery, 1995). Several studies predict an important function for proteases in imaginal disc morphogenesis during prepupal development. Mutations in the Stubble gene (Sb), which encodes an apparent transmembrane serine protease, interact with the Broad Complex to regulate appendage elongation (Beaton, 1988 and Appel, 1993). Some heteroallelic combinations of Sb alleles lead to defects in leg elongation that are similar to those seen in crol mutants; proper Sb leg elongation can be restored by simply culturing the mutant leg discs in the presence of trypsin (Appel, 1993). It is possible that increased levels of Brg-P9 expression, and perhaps other serine protease inhibitors, could block the activity of Sb and other serine proteases in the leg discs, and thus prevent proper leg elongation during prepupal development. It is also likely that crol regulates other, as yet unidentified, target genes that function during leg elongation. The identification of other secondary-response genes that are regulated by crol and expressed in leg imaginal discs should provide a better understanding of crol function in this tissue (D'Avino, 1998).

crol mutations lead to stage-specific effects on ecdysone-induced regulatory gene expression during the onset of metamorphosis. The levels of EcR and E74B transcription are reduced in 6-10 hour crol mutant prepupae, and the BR-C, E74A, E75A, E75B and E93 early genes are submaximally induced in response to the ecdysone pulse in 10 hour prepupae. These effects on gene expression provide a molecular basis for understanding the defects in adult head eversion seen in crol mutants. E74B, formally known as Ecdysone-induced protein 74EF, has been shown to be required for head eversion, although the mechanism(s) by which E74B regulates this response remains unknown. E74B codes for an Ets domain transcription factor. The reduced expression of E74B in crol mutant prepupae thus provides one means of interpreting the effect of crol mutations on adult head development. Alternatively, reduced levels of EcR expression in crol mutants could attenuate early gene induction by ecdysone and thereby indirectly affect head eversion. It is also possible that crol directly regulates early gene expression in prepupae. In this regard, it is interesting to note that preliminary studies have shown that crol is expressed normally in BR-C and E74 mutants, confirming that crol functions either upstream from, or in parallel with, these regulatory genes (D'Avino, 1998).

GENE STRUCTURE

Two mRNA isoforms have been identified and called crolß and crolg. The 5' sequences of the crolß and crolg cDNAs overlap an independent clone that contains the 5' non-coding exons 1 and 2. The crolß and crolg isoforms are generated by differential splicing such that crolß lacks the sixth exon of crolg, and a splice donor site within the third exon of crolß is used to generate the crolg mRNA. A third mRNA isoform was also identified by RT-PCR, designated crola, which utilizes the distal exon 3 splice site of crolß and carries the sixth exon of crolg. The crol gene thus encodes at least three different mRNA isoforms: crola is 6263 nucleotides in length; crolß is 6050 nucleotides in length, and crolg is 5645 nucleotides in length. It is unclear, however, how these isoforms relate to the 6.0 and 5.3 kb RNAs detected by northern blot hybridization. Only the 6.0 kb RNA corresponds in length to a crol mRNA isoform -- crolß. Probes derived from exons 1 and 2, the 3' end of exon 7, or 3' sequences of exon 3, which are specific to crola and crolß, all detect both 6.0 and 5.3 kb RNA size classes on northern blots. This observation suggests that the crol mRNA isoforms may utilize more than one promoter and/or 3' polyadenylation signal (D'Avino, 1998).

Bases in 5' UTR - 1418

Exons - 7

PROTEIN STRUCTURE

Amino Acids - 756, 891 and 962 for a, ß and g isoforms

Structural Domains

crol encodes at least three protein isoforms that contain 12-18 C2H2 zinc fingers and have identical amino- and carboxy-terminal regions flanking a variable number of internal C2H2 zinc fingers. Translation of the three Crol protein isoforms initiates with the same AUG triplet, which lies in a near ideal context for initiation. The longest isoform, Crola, is 962 amino acids in length and contains 18 zinc fingers while the other two isoforms, Crolß and Crolg, are 891 and 756 amino acids in length and contain 16 and 12 zinc fingers, respectively. Crolß is missing one and a half of the C-terminal zinc fingers found in Crola, while Crolg is missing the first six zinc fingers of Crola (D'Avino, 1998).

date revised: 2 July 98

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