InteractiveFly: GeneBrief

crooked legs : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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).

REGULATION

Transcriptional Regulation

The crol gene is induced by ecdysone during the onset of metamorphosis. CROL mRNA can be detected in mid-third instar larvae, consistent with the expression of beta-galactosidase in the CNS of crol4418 flies at this stage in development. The level of CROL mRNA then increases in late third instar larvae, in parallel with the high titer ecdysone pulse that triggers puparium formation. The levels of crol transcription decrease to low levels in mid-prepupae and then rise significantly in 12 hour prepupae, following the ecdysone pulse that triggers head eversion. This correspondence between the rises in ecdysone titer and the induction of crol transcription are consistent with crol being an ecdysone-inducible gene, subject to regulation by the Ecdysone receptor. To test this hypothesis more directly, salivary glands were dissected from mid-third instar larvae and cultured for 4 hours in the absence or presence of ecdysone. RNA was then isolated and crol transcription was analyzed by northern blot hybridization. This study revealed that CROL mRNA levels are induced approximately two-fold by ecdysone, similar to the level of induction seen in vivo in late third instar larvae. A similar induction of crol transcription is seen in cultures of mixed larval organs treated with ecdysone. These observations support the hypothesis that crol transcription is inducible by ecdysone, but the relatively low level of induction suggests that other factors may contribute to this regulation (D'Avino, 1998).

Targets of Activity

EcR and E74B are potential targets of Crol. In order to determine if crol functions in gene activation hierarchies during metamorphosis, the temporal patterns of transcription for a number of ecdysone primary- and secondary-response genes were examined in crol mutant animals. These include the EcR ecdysone receptor gene as well as the BR-C, E74A, E74B, E75A, E75B, DHR3 and betaFTZ-F1. E75A and E75B are two isoforms of the E75 early puff gene that encodes orphan members of the nuclear receptor superfamily. DHR3 and betaFTZ-F1 encode distinct orphan receptors, with DHR3 functioning as an inducer of ßFTZ-F1 expression in mid-prepupae. E75B inhibits this DHR3 activation function through direct heterodimerization. betaFTZ-F1, in turn, appears to function as a competence factor that facilitates the reinduction of the early genes by ecdysone in late prepupae. DHR3 is specifically expressed in early prepupae and is unaffected by crol mutations. In contrast, the other genes are all expressed at later stages and, interestingly, their transcription is selectively reduced in mid- and late crol 4418 mutant prepupae. EcR and E74B are both submaximally transcribed in crol 4418 mid-prepupae. Similarly, the peak of BR-C, E74A, E75A and E75B transcription in response to the prepupal ecdysone pulse is significantly reduced, while the earlier induction of these genes in response to the late larval ecdysone pulse is unaffected. Consistent with the stage-specificity of this mutant phenotype, a significant reduction in the transcription of the stage-specific early gene E93 is also seen. The timing of these transcriptional responses confirms that crol mutations have no effect on the duration of larval and prepupal development, but rather indicates that crol is required for the proper magnitude of ecdysone-induced gene expression in prepupae. The level of betaFTZ-F1 mRNA is also reduced in crol 4418 /Df mutants. However, crol 6470 homozygotes show only an approximate two-fold reduction in betaFTZ-F1 mRNA levels, yet the reduction in early gene transcription in these mutants is indistinguishable from that seen in crol 4418 mutants. This observation suggests that crol works independently of betaFTZ-F1 to regulate the prepupal genetic response to ecdysone (D'Avino, 1998).

DEVELOPMENTAL BIOLOGY

Larval and Pupal Stages

The crol 4418 allele was derived from a P-lacZ enhancer trap mutagenesis, and thus carries a lacZ reporter gene that should provide an indication of the temporal and spatial patterns of crol expression. lacZ is induced in leg imaginal discs, salivary glands, and the central nervous system (CNS) of crol 4418 /+ late third instar larvae and prepupae. This induction of lacZ expression is coincident with the high titer ecdysone pulse that triggers puparium formation suggesting that crol expression is regulated by ecdysone. Expression of lacZ can be detected initially in leg imaginal discs isolated from late third instar larvae (-4 hours) and is restricted to the precursors of the tarsal segments. This expression expands in early prepupal leg discs to the precursors of the femur and tibia. Other imaginal discs in crol 4418 animals do not express lacZ. Expression of lacZ in the salivary glands is induced at puparium formation, slightly later than lacZ induction in leg discs. An identical pattern of expression is present in both fat bodies and trachea. In contrast, beta-galactosidase can be detected in the ventral ganglion and presumptive optic lobes of the CNS in mid-third instar larvae. Expression in these cell types increases noticeably at puparium formation, in apparent synchrony with lacZ expression in the salivary gland. Interestingly, lacZ is also expressed specifically in the corpus allatum of the ring gland at all stages examined. The corpus allatum is the endocrine organ responsible for releasing juvenile hormone. A possible function for crol in this cell type is, however, difficult to predict since the role of juvenile hormone is not well understood during pre-adult Drosophila development (D'Avino, 1998).

EFFECTS OF MUTATION

crooked legs (crol) mutants die during pupal development with defects in adult head eversion and leg morphogenesis. crol mutations have stage-specific effects on ecdysone-regulated gene expression. The EcR ecdysone receptor, and the BR-C, E74 and E75 early regulatory genes, are submaximally induced in crol mutants in response to the prepupal ecdysone pulse. These changes in gene activity are consistent with the crol lethal phenotypes and provide a basis for understanding the molecular mechanisms of crol action (D'Avino, 1998).

Three P-element-induced pupal lethal mutations were identified that define a single lethal complementation group: l(2)04418, l(2)06470 and l(2)k08217. These mutations all behave as recessive loss-of-function alleles. Based on the mutant phenotype, the corresponding locus has been named crooked legs. crol mutants show no delay in puparium formation and their imaginal discs are normal in size and morphology. Lethal phase analysis reveals that homozygous crol 4418 mutants survive through embryogenesis and hatch to form first instar larvae. Collections of crol 4418 first and third instar larvae progress normally through prepupal development, but die before adult eclosion. An identical lethal phase can be seen in animals that carry crol 4418 in combination with Df(2L)esc 10, a 380 kb deficiency that removes the crol locus (from 33A1-33B2). This observation suggests that crol 4418 represents a null allele for this locus, a conclusion that is consistent with the absence of crol transcription in this mutant (D'Avino, 1998).

crol mutants die during two stages of pupal development. The first lethal phase occurs at the beginning of pupal development, 14-16 hours after puparium formation, corresponding to stage P5. These mutants display severe defects in head eversion and leg elongation. The remaining 40-50% of the mutants die at the end of pupal development, stage P14, with a microcephalic phenotype and malformed legs. While the penetrance of the microcephalic phenotype is variable, the 'crooked legs' phenotype is highly penetrant and is similar in all animals examined. This can be easily seen in the third pair of legs, which are smaller than wild type and distorted. A kink near the middle of the femur is seen and a few missing bristles. The other two crol alleles display lethal phenotypes that are indistinguishable from those seen in crol 4418 mutants. The malformed leg phenotype is indicative of defects during leg disc elongation (D'Avino, 1998).

Accordingly, the morphology of 6 hr crol 4418 mutant leg discs was examined after puparium formation, by which time these discs should have completed their eversion and elongation. A range of defects are evident in legs dissected from crol 4418 prepupae. Of nine mutant animals examined, three contained legs with less severe defects. The 2nd-5th tarsal segments of these legs are slightly more expanded than wild type and the leg is not fully elongated. The remaining six crol 4418 mutants examined had legs that were severely distorted and reduced in length. It is possible that these legs arise from the class of crol mutants with more severe leg defects; these die early in pupal development. These observations indicate that at least part of the crooked legs phenotype is due to defects in leg disc elongation (D'Avino, 1998).

Drosophila imaginal discs are specified and patterned during embryonic and larval development, resulting in each cell acquiring a specific fate in the adult fly. Morphogenesis and differentiation of imaginal tissues, however, does not occur until metamorphosis, when pulses of the steroid hormone ecdysone direct these complex morphogenetic responses. The ecdysone regulatory pathway controls wing morphogenesis and integrin expression during Drosophila metamorphosis. Mutations in the EcR ecdysone receptor gene and crooked legs (crol), an ecdysone-inducible gene that encodes a family of zinc finger proteins, cause similar defects in wing morphogenesis and cell adhesion: this indicates a role for ecdysone in these morphogenetic responses. In some homo- and hetero-allelic crol combinations a few adult escapers can be recovered. All of these escapers also display wing defects. Fifty-seven percent of the adult escapers have held out wings with a partial (blister) or complete (balloon) separation of the dorsal and ventral wing surfaces, while the remaining 43% have either malformed or completely unfolded wings. The blisters in crol mutant wings are generally large and do not appear to have sharp boundaries. These phenotypes indicate a role for crol in cell adhesion and wing morphogenesis, raising the possibility that ecdysone signaling might play a role in regulating these processes. To test this hypothesis, the role of the ecdysone receptor in wing development was analyzed using the hypomorphic EcR^k06210 allele. The wings of EcR^k06210 homozygous mutants display cell adhesion and morphogenetic defects similar to those seen in crol mutants. At 18°C, 24% of the eclosed adults display venation defects, 32% have malformed wings, and 13% display wing blisters. The blisters are usually small and centrally located, although blistering of the entire wing (a balloon wing) can occasionally be seen. The most frequent venation defects include a small, extra vein that originates from the third longitudinal vein; an additional anterior crossvein, and a 'delta' thickening at the intersection between the posterior crossvein and the fourth longitudinal vein. crol and EcR mutations are shown to interact with mutations in genes encoding the integrin subunits. The frequency of blisters in if mutants is enhanced three- to four-fold by crol¹ and crol² and approximately sevenfold by crol³. In contrast, no interaction was observed between mys^nj42 and crol¹ or crol², whereas the frequency of blisters and balloons in mys^nj42 mutants is increased three- to four-fold by crol³, and the frequency of balloon wings increased approximately sevenfold (D'Avino, 2000).

alpha-Integrin transcription is regulated by ecdysone in cultured larval organs and some changes in the temporal patterns of integrin expression correlate with the ecdysone titer profile during metamorphosis. Transcription of alpha- and beta-integrin subunits is also altered in crol and EcR mutants, indicating that integrin expression is dependent upon crol and EcR function. The expression patterns of alphaPS1 and alphaPS2 in culture appear almost identical. After ~1 h of culture in the presence of ecdysone, alphaPS1 and alphaPS2 transcript levels decrease rapidly, becoming very low by 8 h after hormone addition. The alphaPS3 (scab) gene consists of two transcription units [Long-alphaPS3 (L-alphaPS3) and Short-alphaPS3 (S-alphaPS3)] that initiate from different start sites. Interestingly, L-alphaPS3 mRNA accumulates rapidly in response to ecdysone, peaking by 6-8 h after hormone addition, while S-alphaPS3 transcription appears unaffected by the hormone. Similar to S-alphaPS3, betaPS mRNA levels remain uniform throughout the time course. Thus, only alpha-integrin subunits are regulated by ecdysone in cultured larval organs, and they are either induced or repressed in response to the hormone (D'Avino, 2000).

These findings suggest that altered integrin gene expression in crol and EcR mutants lead to the defects observed in wing morphogenesis and cell adhesion. However, integrins also function in a wide range of other biological pathways during development, including tissue morphogenesis, cytoskeletal reorganization, memory, and gene expression. These widespread functions raise the possibility that ecdysone-regulated integrin expression may control multiple events during metamorphosis. For example, the if ^V2 semilethal allele displays a misshapen leg phenotype that resembles the defective legs seen in crol mutants, indicating that alphaPS2 functions may be recruited by the ecdysone pathway to regulate leg morphogenesis. Furthermore, since alphaPS3 has been proposed to mediate synaptic rearrangements, its ecdysone-induced expression in late third instar larvae may contribute to the extensive neuronal remodeling that occurs in the central nervous system during metamorphosis. Further studies of the tissue-specific functions of integrins during metamorphosis will provide a better understanding of how these critical cell surface receptors exert their multiple effects during development (D'Avino, 2000).

Studies of Drosophila metamorphosis have been hampered by the inability to visualize many of the remarkable changes that occur within the puparium. To circumvent this problem, GFP was expressed in specific tissues of living prepupae and pupae and images of these animals were compiled into time-lapse movies. These studies reveal the dynamics and coordination of morphogenetic movements. Responses that have not been described previously include an unexpected variation in some wild-type animals, where one of the first pairs of legs elongates in the wrong position relative to the second pair of legs and then relocates to its appropriate location. At later stages, the antennal imaginal discs migrate from a lateral position in the head to their final location at the anterior end, as leg and mouth structures are refined and the wings begin to fold. The larval salivary glands translocate toward the dorsal aspect of the animal and undergo massive cell death following head eversion, in synchrony with death of the abdominal muscles. These death responses fail to occur in rbp⁵ mutants of the Broad-Complex, and imaginal disc elongation and eversion is abolished in br⁵ mutants of the BR-C. Leg malformations associated with the crol³ mutation can be seen to arise from defects in imaginal disc morphogenesis during prepupal stages. This approach provides a new tool for characterizing the dynamic morphological changes that occur during metamorphosis in both wild-type and mutant animals (Ward, 2003).

REFERENCES

Search PubMed for articles about Drosophila crooked legs

Appel, L. F., Prout, M., Abu-Shumays, R., Hammonds, A., Garbe, J. C., Fristrom, D. and Fristrom, J. (1993). The Drosophila Stubble-stubbloid gene encodes an apparent transmembrane serine protease required for epithelial morphogenesis. Proc. Natl. Acad. Sci. 90: 4937-4941.

Beaton, A. H., Kiss, I., Fristrom, D. and Fristrom, J. W. (1988). Interaction of the Stubble-stubbloid locus and the Broad-Complex of Drosophila melanogaster. Genetics 120: 453-464

D'Avino, P. P. and Thummel, C. S. (1998). crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis. Development 125: 1733-1745.

D'Avino, P. P. and Thummel, C. S. (2000). The ecdysone regulatory pathway controls wing morphogenesis and integrin expression during Drosophila metamorphosis. Dev. Biol. 220(2): 211-224

Emery, J. G. (1995). Identification and characterization of genes regulated by the Broad-Complex, a transcription factor necessary for Drosophila metamorphosis. Ph.D. thesis, University of Pennsylvania, Philadelphia, PA.

Ward, R. E., et al. (2003). GFP in living animals reveals dynamic developmental responses to ecdysone during Drosophila metamorphosis. Dev. Biol. 256: 389-402. 12679111