Gene name - odd-paired
Cytological map position - 82E1-E3
Function - transcription factor
Keywords - pair-rule
Symbol - opa
Genetic map position - 3-[47.1]
Classification - zinc finger
Cellular location - nuclear
|Recent literature|| Clark, E. and Akam, M. (2016). Odd-paired controls frequency doubling in Drosophila segmentation by altering the pair-rule gene regulatory network. Elife [Epub ahead of print]. PubMed ID: 27525481
The Drosophila embryo transiently exhibits a double segment periodicity, defined by the expression of seven "pair-rule" genes, each in a pattern of seven stripes. At gastrulation, interactions between the pair-rule genes lead to frequency doubling and the patterning of fourteen parasegment boundaries. In contrast to earlier stages of Drosophila anteroposterior patterning, this transition is not well understood. By carefully analysing the spatiotemporal dynamics of pair-rule gene expression, this study demonstrates that frequency-doubling is precipitated by multiple coordinated changes to the network of regulatory interactions between the pair-rule genes. The broadly expressed but temporally patterned transcription factor, Odd-paired (Opa/Zic), was identified to be the cause of these changes. The patterning of the even-numbered parasegment boundaries relies on Opa-dependent regulatory interactions. These findings indicate that the pair-rule gene regulatory network has a temporally-modulated topology, permitting the pair-rule genes to play stage-specific patterning roles.
|Mendoza-Garcia, P., Hugosson, F., Fallah, M., Higgins, M. L., Iwasaki, Y., Pfeifer, K., Wolfstetter, G., Varshney, G., Popichenko, D., Gergen, J. P., Hens, K., Deplancke, B. and Palmer, R. H. (2017). The Zic family homologue Odd-paired regulates Alk expression in Drosophila. PLoS Genet 13(4): e1006617. PubMed ID: 28369060
The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) this study identified Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, modulation of Alk expression by Opa was shown in the embryonic AS, epidermis and VM. In addition, enhancer elements were identified that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, these data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression.
odd-paired appears to be an oddity among the pair-rule transcription factors. During early development of the embryo, odd-paired is not found in a pair-rule, strongly zebra-striped expression pattern, as are other pair-rule genes. Instead, it is transcribed ubiquitously in the central 60% of the embryo. Only later does it assume a banded appearance, in this case, 14 weakly staining zebra stripes. Since all other pair-rule proteins appear in only 7 stripes, OPA is indeed an anomaly. Its classification as a pair-rule gene arises from its mutant phenotype and not its expression pattern (Benedyk, 1994).
Despite its peculiarities, opa functions like other pair-rule genes, and is involved in the induction of wingless into the posterior compartments of parasegments. Because opa's effects are localized, while its expression is not, the positive regulation of wingless must be permissive and not instructive. opa doesn't signal location but simply gives permission. opa appears to act in conjuction with other transcription factors that provide positional signals.
One odd-paired function should be highlighted because it takes place in the mesoderm, rather than the ectoderm. opa is required for the formation of midgut constrictions. It is expressed ubiquitously in segmented regions of both ectoderm and mesoderm. This expression ceases and later reinitiates in restricted portions of the mesoderm. This expression is positively regulated by homeotic genes Antennapedia and abdominal-A, and negatively regulated by Ultrabithorax and decapentaplegic in opa negative areas (Cimbora, 1995).
A crucial step in generating the segmented body plan in Drosophila is establishing stripes of expression of several key segment-polarity genes, one stripe for each parasegment, in the blastoderm stage embryo. It is well established that these patterns are generated in response to regulation by the transcription factors encoded by the pair-rule segmentation genes. However, the full set of positional cues that drive expression in either the odd- or even-numbered parasegments has not been defined for any of the segment-polarity genes. Among the complications for dissecting the pair-rule to segment-polarity transition are the regulatory interactions between the different pair-rule genes. An ectopic expression system that allows for quantitative manipulation of expression levels was used to probe the role of the primary pair-rule transcription factor Runt in segment-polarity gene regulation. These experiments identify sloppy paired 1 (slp1), most appropriately classified as segment polarity genes, as a gene that is activated and repressed by Runt in a simple combinatorial parasegment-dependent manner. The combination of Runt and Odd-paired (Opa) is both necessary and sufficient for slp1 activation in all somatic blastoderm nuclei that do not express the Fushi tarazu (Ftz) transcription factor (see The pair-rule to segment-polarity transition). By contrast, the specific combination of Runt + Ftz is sufficient for slp1 repression in all blastoderm nuclei. Furthermore Ftz is found to modulate the Runt-dependent regulation of the segment-polarity genes wingless (wg) and engrailed (en). However, in the case of en the combination of Runt + Ftz gives activation. The contrasting responses of different downstream targets to Runt in the presence or absence of Ftz is thus central to the combinatorial logic of the pair-rule to segment-polarity transition. The unique and simple rules for slp1 regulation make this an attractive target for dissecting the molecular mechanisms of Runt-dependent regulation (Swantek, 2004; full text of article).
A somewhat surprising result from these experiments is that slp1 expression in odd-numbered parasegments is lost in opa mutant embryos (see Runt and Opa cooperate to activate slp1 transcription). The importance of Opa is surprising since expression of other segment-polarity genes is reduced, but not eliminated in opa mutants (Benedyk, 1994; Cimbora, 1995). Moreover, Opa is expressed at uniform levels throughout the pre-segmental region of the embryo, and thus does not provide positional information that defines the placement of slp1 stripes relative to other pair-rule transcription factors. The odd-numbered slp1 stripes require Runt, and are interpreted to expand in response to ectopic Runt. The requirement for Opa in this Runt-dependent activation was tested by examining slp1 expression in embryos that have high levels of NGT-driven (NGT stands for nanos-GAL4-tubulin) Runt and that are also mutant for opa. Expression of slp1 within the pre-segmental region is lost in these embryos. This result corroborates the interpretation that the expanded slp1 stripes produced by NGT-driven Runt correspond to the odd-numbered stripes and further confirms the importance of Opa for Runt-dependent activation (Swantek, 2004).
A useful feature of the GAL4 expression system is that expression levels can be varied by changing the strengths of either the GAL4 driver, or the responding UAS transgene. Advantage of this feature was taken to further investigate the relative roles of Runt and Opa in slp1 regulation by generating a co-expression matrix with a panel of different UAS-runt and UAS-opa lines. Increasing the level of Opa in embryos with the same low level of NGT-driven Runt alters slp1 in a manner similar to that obtained by increasing Runt alone. Thus, Opa potentiates Runt-dependent regulation in a concentration-dependent manner. Concentration-dependent effects of Opa are also observed at both intermediate and high levels of NGT-driven Runt. In order to interpret these changes, it is useful to first consider the relatively simple, yet striking response of slp1 to high levels of both Runt and Opa. In these embryos, slp1 is expressed throughout the anterior head region and is nearly uniformly repressed throughout the pre-segmental region of the embryo. The anterior activation is particularly informative since none of the other pair-rule or segment-polarity gene shows this response to Runt and Opa. Thus, anterior activation of slp1 by Runt and Opa occurs in the absence of regulatory inputs from other segmentation genes. It is notable that anterior activation can be triggered either by increasing the level of Runt in embryos with constant intermediate levels of Opa, or by increasing the levels of Opa in embryos with constant intermediate levels of Runt. The observation that Runt and Opa are both obligatory for anterior activation, coupled with this mutual dose-dependent cooperation strongly suggests that these two factors function together in a concentration-dependent complex to activate slp1transcription (Swantek, 2004).
The other notable response to high levels of Runt and Opa is the nearly complete repression of slp1 throughout the presegmental region of the embryo. slp1 and ftz are expressed in complementary patterns in embryos with high uniform levels of Runt. Examination of the response of ftz to the co-expression of Runt and Opa indicates a perfect correlation between the elimination of slp1 and the expansion of ftz. These observations indicate that Opa potentiates the ability of Runt to activate ftz. Moreover, these results strongly suggest that Ftz plays a key role in slp1 repression (Swantek, 2004).
The eye/antennal discs of Drosophila form most of the adult head capsule. The role of the BMP family member decapentaplegic (dpp) in the process of head formation is being analyzed, since a class of cis-regulatory dpp mutations (dpps-hc) have been identified that specifically disrupts expression in the lateral peripodial epithelium of eye/antennal discs and is required for ventral head formation. This study describes the recovery of mutations in odd-paired (opa), a zinc finger transcription factor related to the vertebrate Zic family, as dominant enhancers of this dpp head mutation. A single loss-of-function opa allele in combination with a single copy of a dpps-hc produces defects in the ventral adult head. Furthermore, postembryonic loss of opa expression alone causes head defects identical to loss of dpps-hc/dpps-hc, and dpphc/+;opa/+ mutant combinations. opa is required for dpp expression in the lateral peripodial epithelium, but not other areas of the eye/antennal disc. Thus a pathway that includes opa and dpp expression in the peripodial epithelium is crucial to the formation of the ventral adult head. Zic proteins and members of the BMP pathway are crucial for vertebrate head development, since mutations in them are associated with midline defects of the head. The interaction of these genes in the morphogenesis of the fruitfly head suggests that the regulation of head formation may be conserved across metazoans (Lee, 2007).
This work demonstrates that opa is an upstream activator of dpp in the peripodial epithelium, and acts in a cell-autonomous fashion. It is not known whether this role is direct, with Opa acting as a transcription factor for dpp, or through other proteins. This ability to activate dpp appears limited to the peripodial epithelium of the eye/antennal disc, since misexpression of Opa in the disc proper does not induce expression. Furthermore, Opa acts only on a dpp reporter that has expression restricted to the peripodial epithelium of the eye/antennal disc. With the exception of antennal defects, loss-of-function clones of opa produce identical head defects to homozygous dpps-hc mutants, and ectopic expression of either Dpp or Opa in the peripodial epithelium produces a similar spectrum of misplaced sensory structures. These data suggest that dpp is the major target of opa in the peripodial epithelium (Lee, 2007).
Both opa and dpp are involved in embryonic midgut development, where dpp is a negative regulator of opa in the visceral mesoderm. In addition, BMP2 and BMP4 are negative regulators of Zic proteins in zebrafish, but the exact mechanism of this regulation is unclear. Thus, Zic family proteins are often seen in regulatory networks with BMP proteins, but there does not seem to be a canonical regulatory relationship. These data indicates that during eye/antennal disc development opa exerts a positive effect on peripodial dpp (Lee, 2007).
Both opa and dpp exert their role on ventral head development through expression limited to the peripodial epithelium of the eye/antennal disc. The structures affected in ventral head capsule mutations, such as palps and vibrissae, are reported to arise from the disc proper in the fate map of the eye/antennal disc; thus the effect of Opa-Dpp signal transduction could be to cross epithelial layers, from the peripodial epithelium to the disc proper. Loss of lateral peripodial Dpp expression results in apoptosis in the underlying disc proper, which further suggests a role for peripodial signaling to support disc proper cell viability and morphogenesis. However, when the descendants of peripodial cells are followed by the perdurance of ß-galactosidase expression through metamorphosis, significant contributions of lateral peripodial cells are found in areas of the ventral head where defects are observed in dpps-hc or opa mutations, suggesting that the ventral adult head is formed from descendants of both disc proper and peripodial cells. Adult head expression has also been seen with the MS1096-Gal4 driver, of which expression in the eye disc is limited to the lateral and medial peripodial epithelium and margin cells. These data provide further support to the idea that the peripodial epithelium provides more than passive or purely mechanical functions during disc development. The role of the peripodial epithelium in imaginal disc development has begun to receive more attention, and there is evidence that peripodial-specific signaling affects the patterning of the eye, growth control and the fusion of discs at metamorphosis. It now seems likely that in addition to providing such support to cells of the disc proper, peripodial cells contribute directly to the cuticle of the adult head (Lee, 2007).
In mice and humans, Zic genes are associated with holoprosencephaly, a congenital head defect the extreme manifestation of which is cyclopia. In holoprosencephaly there is variable loss or disruption in the development of the ventral forebrain, and midline facial structures. Holoprosencephaly is a common defect in humans, and genes in the TGF-ß and hedgehog pathways are also associated with both the human and mouse condition. Relevant to this work, a significant number of holoprosencephaly cases result from autosomal dominant inheritance, and often, obligate carriers of these autosomal dominant pedigrees are clinically normal. This incomplete penetrance suggests extreme dose sensitivity and the presence of multiple modifying loci. The ability of a genetic screen to recover multiple dominant enhancers of the dpp ventral head defect, including opa, suggests that this may be a model for the kind of digenic inheritance seen with holoprosencephaly. The hedgehog pathway is known to be crucial to adult head development in Drosophila, and this work adds TGF-ß and opa to this process in the fruitfly. It will be of interest to see how many other connections exist between vertebrate and fly head malformations (Lee, 2007).
cDNA clone length - 2959
Bases in 5' UTR -293
Exons - four
Bases in 3' UTR - 820
opa encodes a zinc finger protein with five fingers homologous to those of the Drosophila segment polarity gene cubitus interruptus, the human glioblastoma gene GLI and the C. elegans sex determination gene tra-1 (Benedyk, 1994).
date revised: 20 January 2007
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