BIOLOGICAL OVERVIEW

Even a cursory look at the Drosophila head [Images] reveals an incredible elaboration of numerous structures: discrete, diverse and unique. What produces these complex yet well organized and differentiated results? Early in head differentiation the anterior posterior axis is marked by a subdivision into seven segments. The element primarily responsible for this subdivision is the action of gap genes, expressed in well defined anterior-posterior positions. Among the gap genes, buttonhead regulates cnc activation, and subsequently cnc regulates genes responsible for labral and mandibular development, specifically in the dorsal portion of the labral segment and the posterior lateral and ventral portion of the mandibular segment. cnc also functions in conjunction with Deformed, a homeotic gene expressed in the head.

In the trunk, segment polarity genes are activated by pair-rule genes, but this is not the case in the anterior of the embryo. Here gap genes activate segment polarity genes. The segment polarity genes hedgehog and wingless are two important targets of cnc and forkhead, expressed in the anterior and posterior gut anlagen. cnc is expressed in the labral region of the foregut, fated to give rise to the dorsal pharynx and fkh is expressed in the adjacent esophagus. fkh is responsible for the maintenance but not the initiation of wg synthesis in the invaginating esophageal primordium. cnc is responsible for the maintenance of wg in the dorsal pharyngeal domain of wingless expression. Expression of hedgehog is similarly affected in cnc and fkh mutants. It is not known whether the actions of cnc and fkh on hh and wg are direct or indirect (Mohler, 1995).

Deletion mutants of cnc coding sequences indicate that cnc functions are required for the normal development of both labral and mandibular structures (Mohler, 1995). In place of the missing mandibular structures, some maxillary structures - mouth hooks and cirri - are ectopically produced (Harding, 1995; Mohler, 1995). The genetic function of the homeotic gene Deformed (Dfd) is required in the cnc mutant background to produce ectopic mouth hooks, and Mohler (1995) have proposed that Dfd and cnc function in combination to specify mandibular identity. A protein isoform (CncB) from the Drosophila cap ëní collar locus has been characterized that selectively represses cis-regulatory elements that are activated by the Hox protein Deformed. Analysis of the cnc gene reveals the presence of three isoforms: cncA, cncB, and cncC. The expression patterns of the three transcript isoforms were analyzed using exon-specific probes both on wild-type and EMS-induced cnc mutants. In wild-type embryos, a cncB probe detects cytoplasmic transcripts limited to the mandibular and labral segments from cellular blastoderm to the end of embryogenesis. The cncB transcripts are expressed throughout both anterior and posterior regions of the mandibular lobes. In contrast, a cncA-specific probe detects a ubiquitous distribution of presumably maternal RNA at syncytial and early cellular blastoderm stages. After cellular blastoderm, cncA transcripts are not detectable until stage 14, when the level of ubiquitous cytoplasmic transcript increases and remains high for the remainder of embryogenesis. cncC-specific probes also detect a ubiquitous distribution of mRNA in syncytial stage embryos and a low level ubiquitous expression pattern in embryos after stage 14 (McGinnis, 1998).

Based on the above results, the labral and mandibular stripes of transcription that were detected by Mohler, (1991) using a probe including the cnc common exons (A2 and A3), correspond primarily to cncB transcripts. Since cncB is the transcript isoform that is expressed throughout the entire mandibular segment during mid-embyronic stages, cncB is likely to encode the principal function that modulates Dfd function in the mandibular segment (Harding, 1995). To further test this hypothesis, an assay was carried out to see whether cncB transcript or protein abundance is altered in embryos homozygous for the cnc^2E16 and cnc^C7, mutations known to reveal interaction with Dfd. The pattern of zygotic RNA expression detected with a cncB probe is unaltered in the EMS-induced cnc mutant embryos. The signal due to cncA and cncC transcripts is also unchanged in these mutants. However, the use of polyclonal antiserum raised against the common domain of the cnc isoforms (anti-Cnc) indicates that CncB protein expression is strikingly reduced in both cnc^2E16 and cnc^C7 mutant embryos. In wild-type embryos, the anti-Cnc antiserum exhibits a low-level ubiquitous staining in syncytial embryos, presumably due to maternally deposited CncA and CncC isoforms. From cellular blastoderm (stage 5) until stage 14, the staining detected by the anti-Cnc antiserum is localized in the nuclei of mandibular and labral cells. Although the anti-Cnc antiserum used in these experiments cross-reacts with all three Cnc proteins, only cncB RNA expression is localized in mandibular and hypopharyngeal regions from stages 6 through 14. cnc^2E16 mutants (and cnc^C7 mutants) accumulate much lower levels of Cnc antigen in both mandibular and labral cells of stage 11 embryos. These results provide further evidence that the cnc^2E16 and cnc^C7 mutations result in a loss of cncB function, and is consistent with the idea that CncB protein is required to prevent the maxillary-promoting function of Dfd from being active in mandibular cells (McGinnis, 1998).

In another test of the functions of the Cnc protein isoforms, each of the cncA, cncB and cncC open reading frames were placed under the control of the heat-shock promoter in P-element vectors and transgenic fly strains were generated carrying these constructs. Using the Cnc common-region antiserum to stain heat-shocked embryos, it appears that all three isoforms are produced at similar levels, localized in nuclei and possess similar stabilities after ectopic expression. However, their morphogenetic and regulatory effects are quite dissimilar. Heat-shock-induced ectopic expression of CncA during embryogenesis has no effect on embryonic morphology. Nearly all of the hs-cncA embryos hatch and proceed through larval development, and many eclose as viable adults. In contrast, ectopic expression of CncB at mid-stages (4-10 hours) of embryonic development is lethal. When ectopic expression is induced at 6 to 8 hours after egg lay, a defective embryonic head phenotype, which resembles the mutant phenotype of strong Dfd hypomorphs is produced. These hs-cncB embryos develop with rudimentary mouth hooks, H-piece and cirri. In addition, the anterior portion of the lateralgr”ten are truncated. All of these structures are components of the head skeleton that are absent or abnormal in Dfd mutant embryos. The head defects seen in the hs-cncB embryos also include an absent or abnormal dorsal bridge, a structure that is usually unaffected in Dfd mutant embryos. Many other head structures that develop in a Dfd-independent manner, such as the antennal sense organ, vertical plates and T-ribs develop normally in the hs-cncB embryos. The hs-cncB head defects are produced at high penetrance (>95%) by heat shocks in mid-embryogenesis (4-10 hours). In 10%-70% of these embryos, depending on the stage of heat shock, abdominal denticles near the ventral midline are replaced with naked cuticle. Ectopic induction of hs-cncC at 6-8 hours of development also results in highly penetrant defects in head development that include the loss of maxillary mouth hooks and cirri as well as head involution defects that are more profound than those induced by hs-cncB. In addition to the morphological defects described for CncB, ectopic CncC induces the formation of an abnormal head sclerite that develops as an extension of the normal lateralgr”ten. The position and appearance of this extra fragment of head skeleton suggests that it might correspond to ectopic production of lateralgr”ten or longitudinal arms of the H-piece (McGinnis, 1998).

Since CncB encodes a function that is required and sufficient to antagonize the maxillary-promoting effects of the Hox gene Dfd, it is reasonable to ask if CncB protein acts upstream to repress Dfd transcription, or in parallel to inhibit Dfd protein function? It is possible for CncB to do both, since Dfd protein function is required to establish an autoactivation circuit that provides persistent Dfd transcription in maxillary and mandibular cells. In wild-type embryos at stage 9, both Dfd and CncB proteins are expressed throughout the entire mandibular segment. By stage 11, Dfd protein is present at lower levels in the anterior, when compared to posterior mandibular nuclei, while CncB protein persists at relatively high levels throughout the segment. Finally, at stage 13, Dfd protein expression is no longer detected in anterior mandibular nuclei, although it is still abundant in posterior nuclei. cnc is required for this progressive repression of Dfd expression in the anterior mandibular segment, since cnc null mutants as well as the EMS-induced mutants show inappropriate persistence of Dfd transcripts and protein after stage 11 in anterior mandibular cells. All of these data suggest that CncB is not capable of repressing Dfd expression before stage 11. But after this stage, CncB represses the maintenance phase of Dfd transcription in mandibular cells, perhaps by repressing the autoactivation circuit that is normally established during stages 9 and 10 (Zeng et al., 1994). CncB is found to be sufficient to repress Dfd transcription outside the mandibular segment. When CncB is ectopically expressed in embryos, Dfd transcript levels in the maxillary segment are reduced, especially in the anterior region of the segment. Only the CncB isoform is capable of repressing Dfd transcription. Neither the ectopic expression of CncA nor CncC have an effect on the abundance or pattern of Dfd transcripts in the maxillary epidermis. Since the phenotypic effect of hs-cncC in epidermal cells strongly resembles that of hs-cncB, this indicates that the effect of Cnc gene products on maxillary epidermal development may not require repression of Dfd transcription per se. However, various experiments show that the maxillary-promoting function of Dfd protein is reduced in the presence of CncB; this could either be due to CncB-mediated repression of the Dfd autoactivation circuit in ectopic positions or to CncB repression of downstream target elements of Dfd protein, or to both of these effects. It is concluded that CncB provides a mechanism to modulate the specificity of Hox morphogenetic outcomes, which results in an increase in the segmental diversity in the Drosophila head. (McGinnis, 1998).

Three EMS-induced mutant alleles of cnc (cnc^2E16, cnc^C7 and cnc^C14) in a screen for mutations that interact with the Hox gene Dfd (Harding, 1995). Embryos homozygous for these EMS-induced alleles have ectopic duplications of maxillary mouth hooks and cirri, but retain normal labral structures and some normal mandibular structures, e.g. the lateralgr”ten and median tooth. This contrasts with the phenotype of deletion mutants of cnc, which lack all mandibular and labral derivatives. The difference between the phenotypes of the EMS-induced alleles when compared to the deletion alleles prompted a consideration of the possibility that multiple functions are encoded in the cnc locus. Previous studies detected one transcript isoform at cnc, but the current molecular analyses of the locus indicates that three transcript and protein isoforms are produced from the cnc gene. A probe homologous to the region that encodes the b-ZIP region of cnc detects three different sizes of polyadenylated RNAs on embryonic northern blots. These will be referred to as the cncA, cncB and cncC transcripts. The 3.3 kb cncA transcript is present in 0-2 hour embryos, presumably from maternal stores and is also abundantly expressed in 12-24 hour embryos. The 5.4 kb cncB transcript is absent from 0-2 hour embryos, but present at all other embryonic stages. The 6.6 kb cncC transcript is present in 0-2 hour embryos, is barely detected in 2-12 hour embryos and is detected at relatively higher levels in 12-24 hour embryos. The sequence of all of the coding exons and exon/intron boundaries for all isoforms on the cnc^2E16 and cnc^C7 mutant chromosomes was determined in an attempt to find the molecular lesion responsible for the decreased amount of CncB protein in the mutant embryos. However, no nucleotide substitutions were detected when the coding and splice site sequences were compared with parental chromosome sequence. Though the location of the mutations that alter CncB protein expression are not yet known, they could plausibly reside in translational regulatory sequences for cncB (McGinnis, 1998).

To identify cDNAs corresponding to the cncA, cncB and cncC transcripts, 212 cDNA clones from libraries covering all stages of Drosophila embryonic development were isolated and characterized. The first class of cDNAs corresponds to the cncA transcript. This is the same class characterized by Mohler, 1991, and is distinguished by the incorporation of exon A1. Exons A2 and A3, which encode the CNC and b-ZIP domains, are present in cncA and the other two isoforms of cnc. A probe containing exon A1 sequences specifically hybridizes the 3.3 kb cncA transcript on northern blots. The cncA open reading frame begins with an ATG codon near the 5' end of exon A2 and is predicted to encode a 533 amino acid protein (Mohler, 1991). A second class of cDNAs from the locus corresponds to cncB transcripts. Such cDNAs lack sequences from exon A1, but contain five additional exons (B1-B5) spliced onto the 5' end of exon A2. Each of these additional exons is found upstream of exon A1. A probe containing the B1-B4 exons detects the 5.4 kb cncB transcript and the 6.6 kb cncC transcript on Northern blots. The total extent of the cncB transcription unit is approximately 17 kb. Since exon A2 sequences contain no stop codons upstream of the initiating ATG for the CncA codons, the open reading frame in cncB transcripts includes the entirety of the CncA protein, as well as an additional 272 codons from exons B3, B4, B5 and A2. The predicted 805 amino acid CncB protein thus is distinguished from CncA by a 272 amino acid region that includes His-Pro repeats, Ala-repeats, a Pro-repeat and Val-Gly repeats, but the region exhibits no extended sequence similarity to other proteins in database searches, other than the CNC/b-ZIP domain that it shares with CncA. The third class of cDNAs from the locus corresponds to cncC transcripts. These cDNAs have identical sequences as the cncB cDNAs, except that exon B1 is absent, and five additional exons (C1-C5) are spliced onto the 5' end of exon B2. Each of these five additional exons are found upstream of the B1 exon. A probe containing the C1-C4 exons detects the 6.6 kb cncC transcript on Northern blots. Since exon B2 and the 5' end of exon B3 contain no stop codons upstream of the initiating ATG for the CncB codons, the ATG-initiated open reading frame in cncC transcripts includes the entirety of the CncB protein, as well as an additional 491 codons that derive from the C3, C4, C5, B2 and B3 exons. The extent of the entire cncC transcription unit is approximately 39 kb. The 491 amino acid CncC-specific domain at the N terminus of the predicted 1296 residue CncC protein includes regions that are rich in Ser and Thr residues, other regions with abundant concentrations of Glu and Asp residues, but exhibits no extended sequence similarity to other proteins in database searches. Interestingly, the fuzzy onions gene, which encodes a testis protein required for mitochondrial fusion in Drosophila spermatids (Hales, 1997), is encoded in the sequence interval between the C5 and B1 exons (McGinnis, 1998).

Bases in 5' UTR - 94

Bases in 3' UTR - 1028

PROTEIN STRUCTURE

Amino Acids - 533

Structural Domains

The leucine zipper of CNC is longer than bZIP proteins and contains six heptad repeats. The function of the leucine zipper is considered as a protein interaction domain. The leucine zipper of CNC is divergent from the typical sequence (Mohler, 1991).

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