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Gene name - cryptocephal
Synonyms - Cytological map position-39C2-39C3 Function - transcription factor Keywords - molting, metamorphosis, response to neuropeptides and peptide hormones |
Symbol - crc
FlyBase ID: FBgn0000370 Genetic map position - Classification - CREB/ATF superfamily bZIP domain proteins Cellular location - nuclear |
The cryptocephal (crc) mutation causes pleiotropic defects in ecdysone-regulated events during Drosophila molting and metamorphosis. crc encodes a Drosophila homolog of vertebrate ATF4, a member of the CREB/ATF family of basic-leucine zipper (bZIP) transcription factors. Three putative protein isoforms were identified. CRC-A and CRC-B contain the bZIP domain, and CRC-D is a C-terminally truncated form. Seven new crc alleles were generated. Consistent with the molecular diversity of crc, these alleles show that crc is a complex genetic locus with two overlapping lethal complementation groups. Alleles representing both groups were rescued by a cDNA encoding CRC-B. One lethal group (crc1, crcR6, and crcRev8) consists of strong hypomorphic or null alleles that are associated with mutations of both CRC-A and CRC-B. These mutants display defects associated with larval molting and pupariation. In addition, they fail to evert the head and fail to elongate the imaginal discs during pupation, and they display variable defects in the subsequent differentiation of the adult abdomen. The other group (crcR1, crcR2, crcE85, crcE98, and crc929) is associated with disruptions of CRC-A and CRC-D; except for a failure to properly elongate the leg discs, these mutants initiate metamorphosis normally. Subsequently, they display a novel metamorphic phenotype, involving collapse of the head and abdomen toward the thorax. The crc gene is expressed throughout development and in many tissues. In third instar larvae, crc expression is high in targets of ecdysone signaling, such as the leg and wing imaginal discs, and in the ring gland, the source of ecdysone. Together, these findings implicate CREB/ATF proteins in essential functions during molting and metamorphosis. In addition, the similarities between the mutant phenotypes of crc and the ecdysone-responsive genes indicate that these genes are likely to be involved in common signaling pathways (Hewes, 2000).
The development of Drosophila and other insects is punctuated by several molts, during which the animal produces a new external cuticle and sheds the old one. The larval molts are initiated and coordinated by steroid hormones, the ecdysteroids (hereafter called ecdysone). At the onset of metamorphosis, a high titer pulse of ecdysone triggers pupariation, which is followed ~12 hr later by a brief ecdysone pulse that causes head eversion and the prepupal-pupal transition. Subsequently, a large, prolonged surge of ecdysone directs adult development. These remarkable developmental changes involve the programmed cell death of most larval tissues, extensive remodeling of other larval tissues, and the generation of adult tissues from islands of undifferentiated imaginal cells (Hewes, 2000).
Detailed studies of the responses to ecdysone in the larval salivary glands provided key insights into the gene regulatory pathways controlling molting and metamorphosis. Expression of a small set of about six early genes is triggered rapidly and directly by ecdysone. Together with ecdysone, these genes regulate numerous late genes. Four early genes have been molecularly characterized. E74 encodes a family of ETS proteins, E75 encodes a family of orphan nuclear receptors, the Broad-Complex (BR-C) encodes a family of zinc-finger proteins, and E63-1 encodes a novel Ca2+-binding protein. E74 and the BR-C control developmental responses to ecdysone in diverse larval and imaginal tissues at least in part through direct transcriptional regulation of the late genes. Thus, the early genes are near the top of a complex gene regulatory hierarchy (Hewes, 2000).
Mutations in several ecdysone-responsive genes reveal distinctive phenotypes that reflect important developmental functions. For example, an E74B mutant displays incomplete differentiation of the adult abdomen and failed gas bubble translocation at pupation. A mutation of ßFTZ-F1, which functions as a competence factor for pupal stage-specific responses to ecdysone, displays similar translocation defects. Mutations in E74, ßFTZ-F1, and two other ecdysone-response genes, crol and DHR3, each display several additional common features. These include defective eversion of the adult head and (with the exception of DHR3) incomplete leg disc elongation. The head eversion defect is called the 'cryptocephal' phenotype, named after cryptocephal (crc1), a mutation that displays all of the above-mentioned defects (Hadorn, 1943). These phenotypic parallels indicate that crc and the ecdysone-response genes are likely to be involved in common regulatory pathways (Hewes, 2000).
Fristrom (1965) examined chitin biosynthesis in the crc1 mutant and concluded that the head eversion defect is due to excess chitin deposition in (and increased stiffness of) the cuticle. Sparrow (1982) tested this hypothesis with a different crc1 strain and found normal chitin deposition. At pupation, crc1 mutants display contractions of the abdomen that are slower, more irregular, and weaker than in wild-type animals, indicating that behavioral abnormalities may cause at least some of the phenotypic defects observed in these mutants (Chadfield, 1985). However, behavioral abnormalities likely do not explain other aspects of the crc1 mutant phenotype, such as incomplete abdominal differentiation. In a discussion of the similar phenotypic defects displayed by E74B mutants, Fletcher (1995) hypothesized that premature muscle death accounts for the full range of defects observed in these cryptocephalic mutants. To distinguish among these and other competing models, it will be important to characterize crc gene function at the molecular and cellular level (Hewes, 2000).
This study shows that crc encodes multiple proteins in the activating transcription factor 4 (ATF4) subfamily of CREB/ATF basic-leucine zipper (bZIP) transcription factors. ATF4 proteins have been implicated in several important developmental and disease processes, including wound healing (Estes, 1995), long-term synaptic facilitation (Bartsch, 1995), stress responses (Fawcett, 1999), apoptosis (Kawai, 1998), and cancer (Mielnicki, 1996). The Drosophila ATF4 homologs play critical roles in molting and metamorphosis. Seven new crc alleles have been isolated, that reveal multiple functions of the gene in larval molting, pupariation, pupation, and adult differentiation. These tissues include several targets of ecdysone signaling as well as the endocrine source of ecdysone, the ring gland. These findings implicate CREB/ATF transcription factors for the first time in the hormonal regulation of molting and metamorphosis. Moreover, these results indicate that there are likely to be important interactions between signaling by crc and the ecdysone-response genes (Hewes, 2000).
crc is a complex locus encoding multiple mRNA and protein isoforms. The two most abundant forms are CRC-A and CRC-B; on the basis of their representation among ESTs, the transcripts encoding CRC-A outnumber those encoding CRC-B by approximately nine to one. Consistent with this observation, most of the in situ hybridization signal observed with a probe for both isoforms was attributable to the transcript encoding CRC-A. CRC-A and CRC-B differ at the N terminus, while a common C-terminal region contains identical bZIP protein dimerization and DNA-binding domains. Therefore, CRC-A and CRC-B likely share dimerization partners and show identical DNA-binding properties (Hewes, 2000).
In addition to the two major mRNA isoforms, there were three uncommon transcripts, crc-d-f, which may serve regulatory functions. crc-d encodes CRC-D, a C-terminally truncated form of CRC-A. Therefore, CRC-D lacks the bZIP domain and could function as a dominant negative regulator by competing with CRC-A (or other factors) for protein-binding sites. The expression of CRC-D may be essential for viability; the crcE85 mutation, which partially deletes a CRC-D-specific exon, displays significant lethality. The crc-e and crc-f transcripts have very small open reading frames that are preceded by suboptimal translational start sites, indicating that they may not be efficiently translated. Rather, these transcripts may participate in the regulation of the crc gene. For mammalian CREB, the expression of truncated forms has been proposed to interrupt a positive feedback loop involving autoactivation of the gene. Therefore, similar mechanisms may be involved in the regulation of crc expression (Hewes, 2000).
Genetic analysis demonstrated that crc is a complex locus consisting of at least two overlapping lethal complementation groups. These complementation groups correlate with the molecular structure of the crc gene, indicating that the different CRC protein isoforms have overlapping, but distinct functions. The following hypothesis is proposed to explain the correlation between the molecular and genetic results. The 3' complementation group phenotypes reflect the functions of both CRC-A and CRC-B. Consistent with this prediction, the one observed sequence alteration in crc1 (Q171R) was found in a region common to CRC-A and CRC-B. The 5' group phenotypes reflect CRC-A- and/or CRC-D-specific functions that do not require CRC-B (Hewes, 2000).
Nevertheless, some overlap is anticipated in the functions of the different CRC proteins. The lethal phenotypes of both crc complementation groups were rescued by ectopic expression of a single RNA isoform encoding CRC-B. Furthermore, the C-terminal 288 amino acids of CRC-A and CRC-B are identical, and both lethal complementation groups displayed similar defects in leg disc elongation. R6, which deleted this common region, failed to complement both 5' and 3' group alleles (Hewes, 2000).
crc mutant alleles displayed several defects associated with molting and metamorphosis. The mutant phenotypes associated with the two lethal complementation groups are distinct, although there was some overlap. Therefore, these mutations define multiple roles for crc during development (Hewes, 2000).
In insects, the molts between successive larval stages are initiated and coordinated by pulses of ecdysone. This process appears to require crc. As previously described for the crc1 allele (Chadfield, 1985), both crc1 and R6 displayed larval lethality associated with failure to shed the old larval mouthparts. These alleles comprise the 3' complementation group and involve disruptions common to the crc-a, crc-b, and crc-c transcripts. Therefore, CRC-A and/or CRC-B perform an important role(s) in the regulation of larval molting. Similar larval phenotypes have been described for mutations in the dare gene, which encodes an adrenodoxin reductase likely to be involved in ecdysone biosynthesis. Likewise, mutants in EcR-B, which is a component of heterodimeric ecdysone receptors, and PHM, an enzyme involved in neuropeptide biosynthesis, both displayed this larval molting phenotype. These similarities indicate that CRC-A and CRC-B may perform necessary functions in the peptidergic neurons that stimulate ecdysone biosynthesis, in the ecdysone-producing prothoracic gland cells, and/or in the tissues that respond to the ecdysone signal (Hewes, 2000).
During the third larval instar, pulses of ecdysone trigger the onset of metamorphosis. A late high titer pulse of ecdysone triggers puparium formation. Approximately 12 hr later, a subsequent brief pulse of ecdysone directs pupation. crc mutants displayed defects in pupariation and pupation, indicating that both of these developmental transitions require crc. The pupariation defects seen in R6 hemizygotes -- retention of the larval shape, failure to form the abdominal gas bubble, and incomplete eversion of the anterior spiracles -- are reminiscent of similar defects described for late-arrested EcR-B mutants and for mutations in E74B and DHR3 (Hewes, 2000).
At pupation, crc1 and R6 both displayed the cryptocephal phenotype as well as defects in imaginal disc elongation. Similar pupation defects are associated with mutations in several ecdysone-response genes, including E74B, crol, ßFTZ-F1, DHR3, and the BR-C. Unlike crc1 and R6, the leg and wing discs in the 5' group mutants remained bulbous and undifferentiated, and often discolored. A phenotype similar to that of 5' group mutants has been reported for ßFTZ-F1. Therefore, lesions in crc and the ecdysone-response genes generate common defects in the larval, prepupal, and pupal responses to ecdysone signaling. These similarities indicate that crc has a central role in the regulation of ecdysone biosynthesis/secretion or in determining the responses of target tissues to the steroid signals. As an important next step in the analysis of crc function, whether crc is also an ecdysone-response gene will be examined (Hewes, 2000).
After comparing aspects of the E74B pupal phenotype and the phenotypes of mutations affecting larval muscle development, it has been proposed that premature death of the larval muscles might account for the defects observed at pupariation and pupation in those mutants. Due to similarities in phenotype between E74B and crc, this model could also account for the pupariation and pupation defects observed in crc mutants, but it probably does not explain the crc larval molting and adult fecundity defects. Moreover, as is true for E74B, most crc1 and R6 mutants display normal larval locomotion, indicating that the larval muscles develop and function normally prior to metamorphosis (Hewes, 2000).
Transgenic UAS-crc lines rescued the lethal phenotype of both the 5' and 3' lethal complementation groups (929 and crc1), confirming the identification of crc. The rescue was partial, and some aspects of the mutant phenotype, such as the reduction in female fecundity and the defects in adult wing expansion and tanning, showed no improvement. Several factors may have contributed to the incomplete rescue. These include requirements for expression of the CRC-A and CRC-D isoforms, or for more precise temporal and/or spatial regulation of CRC expression (Hewes, 2000).
One aspect of the rescue experiments did not fit simple predictions but may be explained by technical details of the transgene expression. The rank order of potency for the rescue of crc1 by the different UAS-crc lines was reversed for the rescue of 929. The variation in the degree of crc1 rescue was likely due to position effects that led to constitutive, low level expression of the transgene. By contrast, c929 is an enhancer trap P element that drives heterogeneous GAL4 reporter gene expression in several tissues. Thus, to explain the second observation, c929 may rescue the wild-type pattern of crc expression to a significant degree, while minimizing the negative effects of crc misexpression in other tissues (Hewes, 2000).
By analogy to other CREB/ATF proteins, the roles of crc during molting and metamorphosis are likely to involve heterodimerization with other bZIP proteins and competition with them for DNA-binding sites. Similarly, it is hypothesized that ecdysone-responsive signaling pathways include crc. For example, by convergence on the transcriptional coactivator, CREB-binding protein (CBP), CREB/ATF proteins can antagonize the activity of members of the nuclear receptor superfamily. This family includes several ecdysone-response genes. Therefore, further analysis of crc may elucidate several points of interaction between crc and these hormonal signaling pathways (Hewes, 2000).
The cytological location of crc1 is 39C2-4. The cloning of crc1 was initiated using a P-element insertion, c929, which was mapped to 39C4 by in situ hybridization. Subsequent to plasmid rescue, several overlapping genomic clones were obtained covering a region of ~50 kb. The DS01560 Berkeley Drosophila Genome Project (BDGP) clone covers this region. Seven cDNAs were isolated from diverse libraries. These clones represent six distinct mRNAs. Southern analysis showed that the crc locus is represented only once in the genome. The BDGP has generated >40 crc expressed sequence tags (ESTs) derived from several stages and tissues, indicating crc expression throughout development. Among clones from adult head and embryo cDNA libraries, the isoforms crc-a and crc-b are the most abundant, representing ~85% and ~10% of the total, respectively. Each of the other crc mRNA isoforms is represented by a single cDNA (Hewes, 2000).
The predicted crc open reading frames are encoded by crc-a, whereas crc-b and crc-c encode an identical isoform, CRC-B. CRC-A and CRC-B differ only at the N terminus. The crc-d transcript encodes CRC-D, a truncated isoform of CRC-A. The C terminus of the 288-amino acid region common to CRC-A and CRC-B contains basic DNA-binding and leucine zipper protein dimerization motifs. The basic DNA-binding region is immediately preceded by a PEST-like sequence (PEST score 8.21). Thus, CRC may display PEST-mediated instability (Hewes, 2000).
The bZIP domain of CRC displays the strongest homology with other members of the CREB/ATF superfamily of transcription factors. CRC belongs to the ATF4 subfamily, on the basis of phylogenetic analysis and the conservation of several characteristic residues in the bZIP domain. CRC is most closely related to mouse and human ATF-4 (>40% sequence identity within the bZIP domain); CRC is much more distantly related to Drosophila CREB-A and CREB-B (Hewes, 2000).
See CrebB-17A for information on CREB/ATF superfamily bZIP domain protein evolutionary homologs
See PubMed articles for information about vertebrate ATF4.
date revised: 2 September 2007
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.