cap'n'collar

Transcriptional Regulation

cnc is maintained independently of the activity of homeotic genes. cnc is activated in labral primordia by the maternal genes bicoid and torso (through the torso pathway), as tailless expression retracts from the anterior pole. Activation in mandibular primordia requires zygotic gap gene products, including activation by Buttonhead, and repression by Orthodenticle and Snail (Mohler 1993).

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterized Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity (Janody, 2000).

The results indicate that early anterior expression of a labral determinant, cnc, and of two foregut determinants, wg and hh, is repressed when zygotic expression of hb is allowed to persist at the anterior pole of the Drosophila blastoderm embryo. Expression of cnc, wg and hh is under the positive regulation of bcd and torso but no zygotic gene has yet been implicated in this control. This suggests that the Hb protein is able to repress the three genes cnc, wg and hh, and that torso-induced anterior repression of hb is necessary for their positive control by torso. To determine whether the positive control of cnc, wg and hh by torso could be the result of a double negative control involving hb, expression of these genes was analysed in hb zygotic mutant embryos derived from torso females. If the lack of early anterior expression of cnc, wg and hh was solely due to the absence of repression of hb at the pole, expression of these genes should be recovered in hb minus embryos derived from torso females. Early anterior expression of cnc, wg and hh is not recovered in hb minus embryos derived from torso females whereas it is normal in hb minus embryos. This indicates that, although necessary, the anterior repression of hb is not sufficient to mediate Torso positive control on cnc, wg and hh early anterior expression (Janody, 2000).

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. The embryonic head phenotype of col¹ hemizygous mutant embryos indicates a loss of skeletal structures derived from the intercalary, and possibly mandibular, segments without transformation toward another segment identity. To investigate this segmentation phenotype in more detail, col expression was compared with that of the segment polarity genes hh and wg. At the blastoderm stage, the posterior limit of col expression is parasegmental (PS0/PS1), since it precisely abuts the mandibular stripe of hh-expressing cells. Whether its anterior limit is also parasegmental cannot be answered at this stage because the expression of segment polarity genes in pre-gnathal segments is not yet established at this stage. Examination of early stage 11 embryos shows that col expression overlaps the intercalary hh stripe and abuts the intercalary Wg spot, indicating a parasegmental anterior border for col expression. At this stage however, col expression has been lost from the posterior part of PS0, since it does not overlap mandibular Wg expression. The cnc gene, which codes for a b-ZIP transcription factor, has been postulated to act as a segment identity gene in the mandibular segment. Consistent with col being expressed in PS0, col and cnc expression only partly overlap, in the region corresponding to the anterior mandibular segment. Together, these data indicate a parasegmental register of col expression at the blastoderm stage, which is subsequently restricted to anterior PS0 (Crozatier, 1999b).

A determination was made of whether col mutations affect the expression of wg and En, which mark the anterior and posterior compartments of each segment, respectively. In col¹ hemizygous embryos, both the intercalary stripe of En and the spot of wg expression are missing. Since col expression does not overlap the intercalary Wg spot, the loss of this spot in col mutant embryos suggested that col does not regulate wg expression directly but possibly by an hh-dependent mechanism. It has indeed been found that in col mutant embryos, the intercalary stripe of hh is also absent, or much reduced. Together, these results show that col controls hh, en and wg expression in the intercalary segment and is required for establishing the PS(-1)/PS0 parasegmental border. The head skeleton structures ventral arm (VA) and lateral-gr”ten (LG), which are, respectively, either missing or reduced in col mutant embryos, are also affected in two other head mutants: crocodile (croc), which codes for a forkhead-domain protein, and cnc. These structures are also affected in embryos mutant for the homeotic genes Dfd and lab, which are expressed, respectively, in the mandibular and maxillary segments, and in the intercalary segment. col expression was examined in embryos mutant for croc, cnc, Dfd or lab. In none of these embryos was there a change in col transcription. Conversely, no changes could be detected for croc, Dfd or Lb expression in col¹ hemizygous embryos, indicating that expression of each of these three genes is independent of col. In contrast, col is required for cnc transcription in the posterior intercalary segment at stage 9-10. Because this region is anterior to the region of overlap between col and cnc expression at the blastoderm stage, it is concluded that this region corresponds to a secondary site of cnc expression initiated at stage 9, under control of col activity. In cnc mutant embryos, intercalary hh expression is normal, indicating that hh and cnc are regulated by col, independent of one another (Crozatier, 1999b).

Because knot encodes a transcription factor, it was of interest to determine whether kn is required for the proper regulation of segment-specific homeotic genes. Notably, kn mutant embryos (zygotic + maternal) show an altered expression of cnc in the early embryo. At early gastrulation, the mandibular stripe of cnc expression is approximately one cell narrower than normal. In stage 10, cnc expression is normally found throughout the anterior compartment of the mandibular segment. However, in kn mutant embryos at stage 10, mandibular cnc expression is restricted to the posterior portion of the anterior compartment of the mandibular segment (which later gives rise to the mandibular lobe) and absent from the anterior-most portion. In wild-type stage 12 embryos cnc is found in both the hypopharyngeal and mandibular lobes. In kn mutant embryos at stage 12, mandibular cnc expression is restricted to the mandibular lobes, while the hypopharyngeal lobes fail to form. Unfortunately, because the cells that normally form the hypopharyngeal lobe are no longer marked with cnc in the kn mutant embryos, it is not possible to determine their new, alternative fate. This early alteration of the homeotic gene cnc expression, prior to the manifestation of the hypopharyngeal lobe, indicates that kn functions in establishing the cell fate of hypopharyngeal lobe. In contrast, no alteration in the expression of the two HOX genes, lab and Dfd, expressed in flanking ectodermal regions was observed. In addition, no altered expression was found in the mandibular region of kn mutant embryos of kn itself prior to stage 11, when mandibular-specific expression of kn ceases and kn becomes expressed in a segmentally reiterated pattern (Seecoomar, 2000).

Targets of Activity

After stage 11, cnc and forkhead are required for the continued expression of dorsal pharyngeal domains of wingless and hedgehog (Mohler 1995).

The genetic function of the homeotic gene Deformed (< I>Dfd) is required in the cnc mutant background to produce ectopic mouth hooks, and it has been proposed that Dfd and cnc function in combination to specify mandibular identity. One of the downstream genes that is activated by Dfd in maxillary cells is Distal-less (Dll). Dll is required for the formation of the larval appendage primordia and the distal regions of adult appendages. In the maxillary segment, Dll is expressed in two patches of cells: a dorsal patch that gives rise to the maxillary sense organ and a ventral patch that consists of the primordia for the maxillary cirri. The dorsal maxillary domain of Dll expression is largely independent of Dfd function, while the ventral maxillary patch of Dll is activated by Dfd through a 3' enhancer (OíHara, 1993). In cnc^2E16 mutant embryos, Dll is ectopically expressed in ventral mandibular cells, suggesting that cncB, one of the transcripts coded for by cnc, represses Dll transcription in mandibular cells. In hs-cncB embryos 30 minutes after heat shock, when Dfd protein abundance is normal, Dll expression is repressed in the ventral maxillary segment but other domains of Dll expression in the head and thorax are relatively unaffected, indicating that CncB selectively represses the Dfd-dependent portion of the Dll expression pattern. In hs-cncC and hs-cncA embryos, the ventral maxillary expression of Dll is not selectively repressed. Reporter gene expression from the 3' enhancer follows the expression of Dll as the enhancer is ectopically activated in the ventral mandibular region in cnc mutants and repressed in hs-cncB embryos (McGinnis, 1998).

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Ruscha, 2000).

The early phase reflects a requirement for gap gene function for normal expression of pb to occur during later stages. Specifically, btd, gt, and hb have been identified as being required for proper gnathal expression of pb. The function of the head gap gene btd has been shown to be required only during early stages of embryogenesis. The expression patterns of gt and hb are such that they are no longer expressed in the labial segment at the time when pb expression begins. This is taken as a strong indication that the gap genes influence pb indirectly. Consistent with this hypothesis, no gt or hb binding sites could be detected in the regulatory elements of the pb reporter. In the case of hb, the role that various trans-acting factors might play in mediating loss of pb expression in the labial segment was investigated. Expression of Scr, the positive regulator of pb in the labial segment, is not eliminated. Further, repression of pb is not attributable to expansion of tsh expression. One possibility is that another negative regulator is being expressed such that Scr can no longer activate pb. Given the negative regulatory interactions that occur between the gap genes, it is possible that one of the other gap genes might be misexpressed and downregulate pb. However, it may be misexpression of cnc or some other gene that has yet to be identified. Alternatively, the 'hit-and-run' hypothesis, proposed to explain the long-term repression of Ultrabithorax (Ubx) by hb, may describe how transient expression of the gap genes is required very early in development to permit later expression of pb. In this hypothesis, heritable changes in chromatin structure, mediated by the PcG genes, are invoked to explain how hb regulates Ubx long after hb expression has ceased. In the case of pb regulation, one or more of these gap genes may be required to alter chromatin structure in and around the pb locus, thereby allowing the various trans-acting factors access to the pb cis-acting regulatory elements (Rusch, 2000).

During the middle phase, the initial expression pattern of pb is set by a variety of trans-acting factors. Focus was placed on the identification of those factors that determine the ectodermal pattern of pb expression along the A/P axis of the embryo. The Hox genes Dfd and Scr act as positive regulators of pb and Dfd can bind to pb regulatory elements in vitro. It is thought likely that Scr also regulates pb directly based on the similarity with which mutations in Dfd and Scr affect expression of pb and the pb reporter. In addition to the Hox genes, the region-specific homeotics cnc and tsh have been identified as negative regulators of pb and serve to restrict pb expression to the gnathos. It is not clear whether these genes regulate pb directly, though in the case of tsh the sequence TGGAAAAGT has been identified in the 500-bp regulatory fragment used in the pb reporter; this sequence is very similar to the identified tsh binding site. While this regulatory paradigm does not completely describe the regulation of the endogenous gene, based on the presence of pb residual expression, it is sufficient to explain the behavior of the 500-bp pb reporter. This mechanism of regulation places pb downstream of the Hox genes and is the first instance in Drosophila where one Hox gene is positively and directly regulated by another, a distinction previously accorded only to vertebrate Hox genes. Studies by others have suggested that wg may be mediating the nonautonomous residual expression of pb that is uncovered by mutations in Dfd and/or Scr. With the exception that wg has the strongest effect on pb expression of the segment polarity genes tested, the results shed little light on the mechanism that underlies this phenomenon. However, non-cell autonomous signaling has been implicated to explain regulation of ectodermal pb function by mesodermal expression of Scr; perhaps the residual expression in the embryo is an example of this pathway. Further experiments, including identification of an enhancer that mediates this residual expression, are needed (Rusch, 2000).

Protein Interactions

cnc and Deformed act in overlapping sets of structures. Nevertheless, cnc expression is independent of Deformed (Mohler 1995).

The cnc locus encodes three transcript and protein isoforms. The cncB transcript is expressed in an embryonic pattern that includes the labral, intercalary and mandibular segments, while cncA and cncC are expressed ubiquitously. CncB suppresses the segmental identity function of the Hox gene Deformed (Dfd) in the mandibular segment of Drosophila embryos. Evidence has been provided that the CncB-mediated suppression of Dfd requires the Drosophila homolog of the mammalian small Maf proteins, Maf-S, and that the suppression occurs even in the presence of high amounts of Dfd protein. Interestingly, the CncB/Maf-S suppressive effect can be partially reversed by overexpression of Homothorax (Hth), suggesting that Hth and Extradenticle proteins antagonize the effects of CncB/Maf-S on Dfd function in the mandibular segment. In embryos, multimers of simple CncB/Maf-S heterodimer sites are transcriptionally activated in response to CncB, and in tissue culture cells the amino-terminal domain of CncB acts as a strong transcriptional activation domain. There are no good matches to CncB/Maf binding consensus sites in the known elements that are activated in response to Dfd and repressed in a CncB-dependent fashion. This suggests that some of the suppressive effect of CncB/Maf-S proteins on Dfd protein function might be exerted indirectly, while some may be exerted by direct binding to as yet uncharacterized Dfd response elements. Ectopic CncB is sufficient to transform ventral epidermis in the trunk into repetitive arrays of ventral pharynx. The functions of CncB are compared to those of its vertebrate and invertebrate homologs, p45 NF-E2, Nrf and Skn-1 proteins, and it is suggested that the pharynx selector function of CncB is highly conserved on some branches of the evolutionary tree (Veraksa, 2000).

Mammalian homologs of Cnc (p45 NF-E2 and Nrf proteins) have been shown to bind DNA as obligate heterodimers with small Maf proteins (MafK/p18, MafF and MafG). An apparent Drosophila ortholog of the mammalian small Mafs was identified in a yeast two-hybrid screen for genes encoding peptides capable of interacting with the common b-Zip domain of Cnc proteins. BLAST searches with a corresponding cDNA sequence detect only one small maf gene, corresponding to CG9954, in the near complete Drosophila genome. The gene has been called Drosophila maf-S (S for Small), and the protein Maf-S. In the basic region that contacts DNA, Maf-S shares a domain of perfect identity with mammalian small Maf proteins, and partial identity in the leucine zipper and other protein regions. In situ hybridizations show that maf-S RNA is maternally deposited in the Drosophila egg, and the transcripts are present in all or nearly all embryonic cells throughout development, making the Maf-S protein available for potential interactions with all Cnc isoforms. In addition, there is an enrichment in maf-S transcripts in certain areas of the embryo, against the ubiquitous expression background. At stage 8, slightly higher levels are detected around cephalic furrow and in the mesoderm. At stage 11 and beyond, there is an enrichment in maf-S transcripts in the anterior and posterior midgut, in the nervous system, as well as in the involuting intercalary segment that will give rise to ventral pharynx (Veraksa, 2000).

Double stranded RNA interference was used in an attempt to test the phenotypic effects of loss of maf-S function. After injection of maf-S dsRNA into the head region of precellular blastoderm embryos, only 1.5% of embryos hatched as first instar larvae. The cuticles of injected embryos show head defects that are remarkably similar to those found in cncB mutants: duplications of mouth hooks, shortened lateralgraten, missing or deformed median tooth and truncated ventral pharynx. Immunostaining of maf-S dsRNA-injected embryos with Cnc-specific antibody reveals that the levels of CncB protein are not significantly different from wild type. Results of the dsRNA interference experiment suggest that Maf-S is an obligate functional partner of CncB. Without the small Maf subunit, CncB is unable to promote the development of intercalary, mandibular and labral head structures and to repress the function of Dfd. The maf-S gene, which maps to chromosomal segment 57A, is uncovered by Df(2R)exu². Homozygotes for this deletion, which lack the zygotic dose of maf-S and other nearby genes, exhibit mild head defects that include disruptions of the pattern of ventral pharyngeal transverse ribs (T-ribs). The weakness of this phenotype relative to the RNAi result may be due to the maternal contribution from maf-S, which is unaffected in the zygotic deletion mutants (Veraksa, 2000).

Despite a high degree of primary sequence similarity between the DNA binding domains of CncB and NF-E2, the optimal recognition site for the Cnc isoforms in association with Drosophila Maf might be different. CncA and CncB optimal binding sites were sought by selecting specific sequences from a pool of degenerate oligonucleotides by Cnc/Maf-S heterodimers. Flag-tagged full-length CncA or CncB proteins were cotranslated in vitro with Maf-S and bound to an oligonucleotide containing a core of 14 ambiguous positions. All selected sites start from the same position (T), suggesting that this and nearby nucleotides influence the selection process. The sequence selected in this experiment by Cnc/Maf-S heterodimers (TGCTGAGTCAT) is identical to the preferred site selected by TCF11/MafG, and is very similar to the consensus binding site derived for p45 NF-E2/p18 MafK from gel shift competition experiments. The TGCTGAG part of the site corresponds to the Maf-bound sequences in the sites for mammalian CNC family proteins, and also to the T-MARE (TRE[phorbol-12-O-tetradecanoate-13-acetate (TPA)-responsive element]-type Maf recognition element) consensus half-site for large Maf homodimers. The GTCAT half-site is contacted by Cnc homologs, and also is a perfect match to the binding site for the C. elegans Skn-1 protein that binds DNA as a monomer. Monomers of CncA or CncB do not appreciably bind to the Cnc/Maf consensus. Maf-S does not bind as a homodimer either to this sequence or to the T-MARE consensus. This property of the Drosophila small Maf protein is different from mammalian small Mafs, which form stable homodimer complexes on T-MARE-like sites. When CncA or CncB are cotranslated with Maf-S, abundant and stable complexes form on Cnc/Maf binding sites, suggesting that DNA binding is achieved only by Cnc/Maf heterodimers and that such binding is highly cooperative (Veraksa, 2000).

Previous genetic and molecular data have established that CncB has several functions in the developing Drosophila embryo: it is required for the formation of labral and intercalary structures such as median tooth and pharynx, and it is required to inhibit Dfd function in the mandibular segment. A summary model has been proposed in which these functions are mediated in part by the transcriptional activator properties of CncB, and are achieved through specific DNA binding with Maf-S to an 11-bp CncB/Maf-S heterodimer binding site. In the intercalary and anterior mandibular segments of the embryo, heterodimerization of CncB with Maf can activate some downstream target genes, and the heterodimers are required to develop ventral pharyngeal structures (T-ribs and ventral arms). Furthermore, CncB, in the context of the ventral epidermis of other head and trunk segments, is sufficient to promote pharynx identity, and thus cncB functions as a pharynx selector gene (Veraksa, 2000).

The function of CncB in suppressing the maxillary-promoting function of Dfd is carried out in the mandibular cells. Early in development CncB and Dfd are coexpressed throughout the mandibular segment, but by stage 13 Dfd expression retracts and becomes localized to a row of cells in the posterior of the segment. In these cells, CncB and Dfd are both required to specify lateralgraten and the base of the mouth hook. Although Dfd function is required for the normal structures that derive from the posterior mandibular segment, the maxillary-promoting function of Dfd is inhibited in these cells. In the anterior mandibular segment, CncB represses both the function and expression of Dfd. Since Dfd protein activates its own transcription, inhibition of Dfd function inevitably results in silencing the endogenous transcription of the Dfd gene. Persistent expression of Dfd in the posterior mandibular cells is likely to be mediated by regulatory regions that are different from the autoactivation enhancers and are insensitive to CncB-mediated repression. Based on the present data, it is suggested that CncB-mediated repression of Dfd function is at least in part indirect, for several reasons. (1) There are no close matches to the consensus CncB/Maf-S binding site in known Dfd response elements, despite the fact that these elements can be repressed by ectopic expression of CncB, e.g. modules C, E, and F from the 2.7 kb Dfd epidermal autoregulatory enhancer. (2) The CncB-specific amino terminal region, crucial for the suppression of Dfd function, contains a strong transcriptional activation domain. (3) A CncA fusion protein with a simple heterologous transcriptional activation domain from VP16 can also partially repress Dfd-dependent maxillary structures upon overexpression. (4) Chimeric Dfd response elements containing Cnc/Maf binding sites adjacent to Dfd binding sites are not repressed by overexpression of CncB, as might be expected if the action of CncB on Dfd were direct. Instead, the endogenous Dfd-dependent maxillary expression of 4CE is enhanced in HS-CncB embryos when compared to non-heat-shocked controls. Suppression of Dfd function by CncB may therefore be achieved by transcriptional activation of an intermediate gene encoding a repressor or corepressor that would interfere with the function of Dfd. Another possibility is that CncB might compete for a common cofactor or coactivator (such as Exd, Hth or CBP), or it might activate a specific Dfd modifying enzyme such as a kinase or phosphatase. It is still possible that the suppressive effect that CncB protein exerts on Dfd function may involve CncB mediated transcriptional repression on as yet uncharacterized response elements. A binding interaction between Dfd and CncB proteins has been observed in GST pull-down assays, but the biological significance of this result is as yet unclear (Veraksa, 2000).

Repression of Dfd function by CncB is analogous to the phenomenon of phenotypic suppression (also known as posterior prevalence), which denotes the ability of more posterior Hox proteins to suppress the function of more anterior Hox proteins in the same cells. 'Anterior prevalence' of CncB over Dfd provides additional morphological diversity in the mandibular and maxillary segments. The indirect way by which CncB acts on Dfd maxillary function has a precedent in Hox phenotypic suppression. The inhibitory effects of posterior Hox proteins on Antennapedia function can apparently be exerted via an indirect pathway involving phosphorylation of Antp protein by casein kinase II. Dfd protein is also phosphorylated in vitro by CKII, but whether this phosphorylation is relevant to the suppression by CncB is unknown (Veraksa, 2000).

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of cnc at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site

cnc functions as a required homeotic segment-specific selector gene, continually expressed in labral and mandibular parasegments [Image] of the head. Earliest expression is in the late blastoderm, found in the anterior portion of the mandibular segment (parasegment 0), just anterior to the cephalic furrow, and in the clypeolabral lobe (parasegment -4). labial and spalt, two other homeotic genes affecting the head, are independent of cnc. cnc represses maxillary development in the mandibular segment. cnc defines the dorsal pharyngeal region and the posterior lateral and ventral portion of the pharynx (Mohler, 1995).

cap'n'collar expression in the anterior compartment of the mandibular segment has two roles, depending on whether or not Deformed is expressed. Where Deformed is not expressed, cnc promotes hypopharyngeal development of the mandibular segment. Where Deformed is expressed, cnc promotes mandibular development in combination with Deformed (Mohler, 1995).

Effects of Mutation or Deletion

Embryonic lethal mutants lack mandibular and labral head structures (Mohler, 1995). cnc mutant larvae possess head skeletons missing the labrum and dorsal pouch (labral structures), the transverse-ribs and ventral arms (hypopharyngeal structures) and the lateralgräte (mandibular structure). cnc mutants are also missing the posterior pharyngeal wall (labral structure), as well as having some duplicate maxillary structures. Such duplication is due to the fact that CNC represses maxillary development in the mandibular segment (Mohler, 1995).

Proteins produced by the homeotic genes of the Hox family assign different identities to cells on the anterior/posterior axis. Relatively little is known about the signaling pathways that modulate their activities or the factors with which they interact to assign specific segmental identities. To identify genes that might encode such functions, a screen was carried out for second site mutations that reduce the viability of animals carrying hypomorphic mutant alleles of the Drosophila homeotic locus, Deformed. Genes mapping to six complementation groups on the third chromosome were isolated as modifiers of Deformed function. Products of two of these genes, sallimus and moira , have been previously proposed as homeotic activators since they suppress the dominant adult phenotype of Polycomb mutants. Mutations in hedgehog, which encodes secreted signaling proteins, were also isolated as Deformed loss-of-function enhancers. hedgehog mutant alleles also suppress the Polycomb phenotype. Mutations were also isolated in a few genes that interact with Deformed but not with Polycomb, indicating that the screen identified genes that are not general homeotic activators. Two of these genes, cap 'n' collar and defaced, have defects in embryonic head development that are similar to defects seen in loss of function Deformed mutants (Harding, 1995).

In Drosophila, dorsoventral polarity is established by the asymmetric positioning of the oocyte nucleus. In egg chambers mutant for cap 'n' collar, the oocyte nucleus migrates correctly from a posterior to an anterior-dorsal position, where it remains during stage 9 of oogenesis. However, at the end of stage 9, the nucleus leaves its anterior position and migrates towards the posterior pole. The mislocalization of the nucleus is accompanied by changes in the microtubule network and a failure to maintain Bicoid and Oskar mRNAs at the anterior and posterior poles, respectively. Gurken mRNA associates with the oocyte nucleus in cap 'n' collar mutants and initially the local secretion of Gurken protein activates the Drosophila EGF receptor in the overlying dorsal follicle cells. However, despite the presence of spatially correct Grk signaling during stage 9, eggs laid by cap 'n' collar females lack dorsoventral polarity. cap 'n' collar mutants, therefore, allow for the study of the influence of Grk signal duration on DV patterning in the follicular epithelium (Guichet, 2001).

cnc is a complex locus coding for three protein isoforms (CncA, CncB, CncC) which share a basic-leucine zipper domain at the carboxy terminus. While CncA and CncC are expressed ubiquitously, CncB is expressed specifically in the head region of early embryos where it is required for the repression of deformed function and the formation of intercalary and labral structures. Double-stranded RNA interference experiments have shown that CncA and CncC are dispensible for embryonic development. The two P-insertions used in this study affect all three isoforms. CncB is not expressed during oogenesis, thus the mutant phenotypes observed are due to a lack of either CncA, CncC, or both isoforms. Judging from their structure, both proteins probably function as transcription factors, as has been demonstrated for CncB and the Cnc homologs of vertebrates and other invertebrates. At present, no genes are known to be regulated by Cnc proteins during oogenesis. However, the cnc phenotype reveals two new aspects as to how DV polarity is established during oogenesis. (1) The initial asymmetric movement of the oocyte nucleus has to be followed by a separate process of stable anchoring of the nucleus at the anterior cortex. (2) An early pulse of asymmetric Egf signaling is insufficient to induce stable DV follicle cell patterning, indeed Egf receptor activation by Gurken has to persist until stage 10A to establish the DV axis of the Drosophila egg (Guichet, 2001).

In cnc mutant egg chambers, nuclear movement occurs normally. The nucleus remains cortically localized even after its posterior displacement. Since interference with components of the dynactin complex leads to the dissociation of the nucleus from the cortex, it is believed that the dynactin complex is not affected by the loss of cnc function. However, the polarization of the microtubule network is aberrant in stage 10A cnc oocytes. Higher numbers of microtubules accumulate in the posterior region of the oocyte at the expense of the anterior cortical ring, which dominates the microtubule network of wild-type stage-9 to -10A egg chambers. This second microtubule reorganization could either be the cause of or result from the late displacement of the nucleus. In the first case, cnc would be required for a process that stabilizes and maintains the microtubule polarity after stage 8. Prolonged signaling from posterior follicle cells might be necessary to suppress the reestablishment of microtubule organizing centers (MTOCs) at the posterior pole. The reception of such a signal or its transmission to the cytoplasm might be impaired in the absence of cnc function. In this model, the reassembly of MTOCs in posterior regions would lead to the redistribution of free tubulin and consequently weaken anterior MTOCs. The nucleus would subsequently migrate towards these ectopic posterior MTOCs. BCD mRNA also would become mislocalized since it is known to move, like the nucleus, towards the minus ends of microtubules, i.e., towards the MTOCs (Guichet, 2001).

In the other scenario, cnc would be required specifically for oocyte nucleus anchoring at the anterior cortex. Anterior anchoring might be necessary since there is a massive influx of cytoplasm from the nurse cells to the anterior pole of the oocyte during egg chamber growth. If the nucleus is not properly anchored, these transport processes might dislodge the nucleus from the anterior pole. Why would this mispositioning of the nucleus lead to the reorganization of the microtubule network? Such microtubule reorganizations have not been described in other mutant backgrounds where the nucleus does not reach the anterior cortex, such as grk, cni, mago, and DLis-1. It has been shown that the nucleus gets encaged by microtubules when it arrives at the anterior pole in wild-type oocytes, indicating that the anteriorly localized nucleus acquires a microtubule-nucleating activity. This activity might remain associated with the mispositioned nucleus in cnc egg chambers and might subsequently cause the increased microtubule density in the posterior half of the cnc oocytes (Guichet, 2001).

In both scenarios, the mislocalization of OSK mRNA remains somehow enigmatic. OSK should not localize to the same region to which BCD is transported. However, normal OSK transport from the anterior to the posterior might just be blocked by the mispositioned nucleus and its associated microtubules. Thus OSK might be trapped in the vicinity of the ectopic nucleus on its way to the posterior pole (Guichet, 2001).

Although at present it is impossible to distinguish between these two explanations for the cnc phenotype, the observed correlation between ectopic microtubules and nuclear position supports the second scenario. It is therefore proposed that two processes of oocyte nucleus anchoring can be distinguished: the general anchoring to the oocyte cortex and the spatially restricted anchoring to the anterior surface of the oocyte. The first process involves the dynein/dynactin complex, which controls cortical anchoring and nuclear movement. This process is likely to be unaffected in cnc. The second process is required after migration to tether the nucleus to the anterior surface of the oocyte. It keeps the nucleus in place during the growth of the oocyte after stage 9 of oogenesis. This process might be affected in cnc mutant egg chambers. As a transcription factor, Cnc might control the expression of proteins, which function as components of the anterior anchor (Guichet, 2001).

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date revised: 10 December 2005

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