Cnidarian Zic Genes
3.4 Role of opa During Development
Genetic screens have identified opa as having various potential roles in Drosophila development. opa was recovered in a screen for genes controlling germ cell migra- tion and gonad formation (Moore et al. 1998). However, it is not clear that this is a direct role or secondary to opa’s function as a pair-rule gene. opa was also recovered
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in a screen for Notch pathway modifiers through a genetic interaction with Presenilin (Psn) mutations (Mahoney et al. 2006), but the mechanism by which opa interacts with Psn is unknown. Only a few biological roles of opa have been subject to detailed analysis by genetic or molecular methods. These are summarized below.
3.4.1 Embryonic Segmentation
As predicted by its recovery in a screen for essential genes specifying anterior- posterior structure of the embryo, opa plays a role in the process of segmentation.
Unlike in vertebrates, where segmentation is a sequential operation, with somites being added one by one, the segmental repeats of Drosophila embryos are formed simultaneously. This metameric pattern is set up early in development by the action of a hierarchy of zygotic gene activities that divides the anterior-posterior axis of the embryo into progressively smaller domains. Gap genes interpret patterns set down maternally and divide the embryo into large domains. These domains are read by the second group of genes, the pair-rule genes, which specify the periodic repeats of the thoracic and abdominal portions of the body plan. This pattern is further refined by segment polarity genes, which define the boundaries and polarity of given seg- mental elements (Fig. 3.3).
opa is a member of the intermediate group of the hierarchy, the pair-rule genes.
Most of the pair-rule genes are transcription factors. Combinatorial expression pat-
Fig. 3.3 opa in conjunction with other pair-rule genes regulates segment polarity genes. Opa and run induce the expression of slp1 in odd segments, while eve and ftz negatively regulate slp1. opa is also required for wg and en expression; however whether this is a direct interaction is unclear (Modified from Swantek and Gergen 2004). Bracket indicates segmental register, solid lines iden- tify parasegments
3 Odd-Paired: The Drosophila Zic Gene
terns of these transcriptional activators and repressors establish the repeating seg- mental pattern of the next level in the hierarchy, the segment polarity genes (Swantek and Gergen 2004; Schroeder et al. 2011; Jaynes and Fujioka 2004). Some of this expression follows a repeating organization that is offset from the metameric body segments of the embryo; these units are referred to as parasegments (PS). opa is genetically required for the expression of wingless (wg), the major Drosophila Wnt homolog, and the homeodomain transcription factor, engrailed (en); both these genes act as critical segment polarity genes (Benedyk et al. 1994). It is not clear whether this is a direct effect of opa or part of the complex gene regulatory network that mediates the segmentation gene network. However, in concert with the pair-rule gene run, opa activates yet another pair-rule gene, the forkhead domain transcrip- tion factor sloppy paired 1 (slp1) (Fig. 3.3), indicating its direct transcriptional role in the segmentation process (Swantek and Gergen 2004; Sen et al. 2010).
Interestingly, all the identified pair-rule genes, with the exception of opa, are initially expressed in seven evenly spaced but phase-shifted stripes. The overlap between the stripes sets up the unique transcriptional code that specifies the 14 seg- ments of the larva (Schroeder et al. 2011). However, the expression pattern of pair- rule genes is both dynamic and transitory. Recent work has finely dissected the phases of pair-rule gene expression and suggests that as the only pair-rule gene not expressed in stripes, opa plays a unique role in regulating a shift in the frequency of stripe patterns of pair-rule genes. This shift causes a concomitant shift in regulatory interactions that set up the activation of segment polarity genes (Clark and Akam 2016). This study notes that other pair-rule genes have small transcription units (<3.5 kb), while opa’s transcription unit is much larger, due to the presence of the 14 kb intron. This could result in the opa protein being fully translated later than other pair-rule genes, allowing it to serve as a timer for this shift. It is clear from these analyses that opa’s role in embryonic segmentation is critical, and has yet to be fully elucidated.
3.4.2 Embryonic Midgut Formation
The internal mesodermal and endodermal structures of the Drosophila embryo are formed after gastrulation and the elongation and subsequent retraction of the germ band. The embryonic midgut is formed when a continuous sheet of visceral muscu- lature encircles migrating endoderm. This visceral musculature is responsible for the formation of three constrictions around the endoderm at invariant locations, resulting in the stereotypical four-lobed midgut structure. Four specialized out- growths called gastric caeca evaginate from the anterior-most portion of this gut structure.
opa is expressed in founder cells of the midgut visceral mesoderm starting at stage 11 (Cimbora and Sakonju 1995; Bilder and Scott 1998; Reim et al. 2017).
Genes expressed in the midgut largely follow a parasegmental pattern of expression.
opa expression is seen in PS 3–5 (weakly in PS 3) and PS 9–12 (Fig. 3.4a) and per-
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sists through gut development (Cimbora and Sakonju 1995; Schaub and Frasch 2013). Loss of function (LOF) opa mutations fail to make the three characteristic midgut constrictions (Cimbora and Sakonju 1995). The positions of these midgut constrictions and gastric caeca are determined by a regulatory network of transcription factors and signaling proteins expressed in tissue- and parasegment- specific domains of the visceral mesoderm (Fig. 3.4b). Visceral mesoderm-specific transcription factors, such as the Fox-F protein binou, the Drosophila T-box factor optomotor- blind-related-gene 1 (org-1), and the TALE homeobox factors extraden- ticle (exd) and homothorax (hth), provide tissue-specific gene activation, while the homeotic transcription factors Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominal-A (abd-A) are organized in specific domains
Fig. 3.4 Larval midgut gene regulation. (a) A stage 15 embryo shows the expression domains of opa in the developing midgut by immunohistochemistry. Anterior is oriented to the left. (b) A schematic diagram of midgut morphology depicts the gastric caeca (GC) and three constrictions:
anterior (A), central (C), and posterior (P). Directly below the midgut diagram are the correspond- ing parasegments (PS) and expression domains of various genes involved in midget formation including opa. These genes are regulated by a complex network of interactions indicated by arrows (←) for a positive interaction and bars ( ) to indicate negative regulation
3 Odd-Paired: The Drosophila Zic Gene
along the anterior-posterior axis of the visceral mesoderm and provide parasegment- specific transcriptional regulation. In addition, wg and the Drosophila bone mor- phogenetic protein (BMP) ortholog, decapentaplegic (dpp), also impart parasegment-specific regulatory inputs. opa transcription in the visceral mesoderm requires org-1, exd, and hth (Schaub and Frasch 2013). In the anterior paraseg- ments, opa also requires Antp for activation, while in the posterior parasegments, abd-A plays that role (Cimbora and Sakonju 1995). opa is confined to its paraseg- mental domains by repression by dpp, which is itself regulated by hth, exd, bin, Ubx, and wg (Cimbora and Sakonju 1995; Graba et al. 1997; Bilder et al. 1998;
Zaffran et al. 2001; Stultz et al. 2006a). The position of opa in PS 3–5 and PS 9–12 suggests that opa has a direct role in the formation of the first and third midgut con- strictions formed in those anterior and posterior regions. However, the loss of the second midgut constriction in opa mutants implies that opa may affect gene expres- sion indirectly in PS 6–8. The NK class homeobox transcription factor bagpipe (bap) is misexpressed in opa mutations, as is the expression of PS 7-specific Ubx (Cimbora and Sakonju 1995; Azpiazu et al. 1996), itself required for dpp expres- sion. These results suggest that opa may provide some feedback regulation to other transcription factors in the visceral mesoderm.
3.4.3 Adult Ventral Head Morphogenesis
Mutations in opa were recovered in a screen for genes involved in Drosophila adult head formation, revealing a postembryonic role for the gene (Lee et al. 2007). All external adult structures in the fly are formed from saclike primordia called imagi- nal discs. The adult head is constructed from three pairs of these discs, the labial, clypeo-labral, and eye-antennal discs (Fig. 3.5a). Of these, the eye-antennal discs form the majority of head tissue. The adult Drosophila head is formed at metamor- phosis with the fusion of the separate imaginal discs into the complete head struc- ture. The paired labial, clypeo-labral, and eye-antennal discs fuse along the left-right midline to form the anterior portion, or “face,” of the fly head.
The Drosophila BMP, dpp, plays a role in the morphogenesis of the adult ventral head via its expression in the eye-antennal discs (Stultz et al. 2006b). This dpp expression persists into the pupal period and is found aligning along the edges of the anterior domain of fusion (Fig. 3.5a). Failure to express dpp in the ventral head leads to apoptotic cell death and loss of head tissue, including sensory structures (Fig. 3.5b, c).
dpp head mutations can have incomplete inheritance and variable expressivity (Stultz et al. 2005) and are highly sensitive to the dose of BMP signaling. For instance, the effects of a single copy of a dpp head defect mutation can be enhanced by single copy LOF mutations in other members of the BMP pathway, such as the BMP type 1 receptor thickveins. This two-gene contribution to a phenotype is known as digenic inheritance. A genome-wide screen for additional genes that dis- played this same type of dominant genetic interactions with dpp head mutations
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recovered LOF mutations in opa. These genetic interactions of dpp and opa are identical to those seen with dpp mutations and BMP receptors, i.e., one copy each of recessive opa and dpp mutations produces flies with adult head defects (Lee et al.
2007). A temperature-sensitive opa mutation, when shifted to nonpermissive tem- perature after embryogenesis, also displays the same ventral head defect observed in crosses of opa LOF mutations to dpp mutations. These data demonstrate that opa plays a role with dpp in ventral adult head formation.
The dpp expression responsible for head formation is controlled by a specific transcriptional enhancer in the 5′ end of the dpp gene; dpp head mutations delete this enhancer without disrupting the dpp coding region (Stultz et al. 2006b). Analysis of the behavior of this enhancer using reporter constructs in transgenic flies indi- cates that opa is required cell autonomously for its expression (Lee et al. 2007) and suggests that opa is acting upstream of dpp as a transcription factor to activate the head-specific enhancer. Sequences specifically bound by the opa zinc finger protein domain were determined by systematic evolution of ligands by exponential enrich- ment (SELEX), and a preferred binding site identified. However, similar sequences are not found in the dpp head enhancer nor does an expressed opa zinc finger pro- tein domain interact with enhancer DNA in electrophoretic mobility shift assays (EMSA) (Sen et al. 2010). Thus while opa seems to be necessary to activate tran- scription from this enhancer, it appears to do this indirectly, without requiring direct DNA binding.
BMP pathway and Zic family members also interact genetically in vertebrates suggesting that this genetic network might be conserved evolutionarily. Mutations in BMP pathway members and Zic genes are associated with vertebrate head abnor- malities, in particular holoprosencephaly (Brown et al. 1998; Fernandes et al. 2007).
Fig. 3.5 A Drosophila model for midline defects. (a) A diagram of the Drosophila adult head shows the expression domain of dpp along the regions where the six imaginal discs that form the adult head fuse together (shaded area). (b) A wild-type adult head is compared to a (c) mutant adult head resulting from dpp head mutation. opaLOF/dpphead mutations have identical mutant phenotypes, as do opa temperature-sensitive alleles moved to nonpermissive temperature after second larval instar. Note the disruption of ventral head structures including a smaller eye, loss of cuticle tissue, disordered sensory vibrissae, and missing maxillary palps. Solid arrow indicates wild-type vibris- sae and solid arrowhead indicates wild-type palp. Open arrow indicates mutant vibrissae, and open arrowhead indicates missing palp
3 Odd-Paired: The Drosophila Zic Gene
Holoprosencephaly is a craniofacial abnormality of vertebrates characterized by defects in forebrain and midline facial structures (Muenke and Beachy 2000;
Dubourg et al. 2007; Petryk et al. 2015). Holoprosencephaly is also characterized by digenic inheritance, incomplete penetrance, and variable expressivity (Ming and Muenke 2002; Petryk et al. 2015). Modifier genes and multiple interacting loci can also influence the penetrance and severity of these defects (Fernandes and Hebert 2008). While arthropod and vertebrate heads are constructed by different embryo- logical mechanisms, it is intriguing to speculate that aspects of the genetic regula- tory network that specifies midline morphogenesis may have been conserved through metazoan evolution.
3.4.4 opa’s Role in the Drosophila Nervous System
Zic proteins play numerous roles in vertebrate neural development (Aruga 2004), and this role appears to be ancestral for the Zic family (Layden et al. 2010). opa is differentially expressed during the formation of neural progenitors (Eroglu et al.
2014), and it seems likely that it plays a role in neuronal development. In Drosophila, data from in situ analysis, tissue-specific arrays, and whole genome RNA-seq of isolated cells, as outlined above, indicates that opa mRNA is robustly expressed in both the embryonic and adult nervous system. For example, expression is found in mushroom bodies, which control memory and learning, and the subesophageal gan- glia, which receive gustatory and neurosecretory inputs. Interestingly, a recent genome-wide association study identified opa as one of a set of genes with poly- morphisms related to variations in olfactory perception (Arya et al. 2015), suggest- ing that opa plays a role in the synaptic connectivity related to odor perception.
However, no developmental genetic studies have as yet demonstrated opa’s function in neural development, despite its expression in many parts of the developing fly nervous system. Additional work is necessary to elucidate the role of opa in neuro- genesis and determine if those roles correspond to observed functions of vertebrate Zic genes.
3.4.5 opa Is Part of the Circadian Clock
Fruit flies have been an important model for the study of circadian rhythms, since the identification of the first circadian rhythm gene, the Drosophila period (per) (Konopka and Benzer 1971). The genes involved in circadian rhythms in Drosophila have been studied extensively (Hardin 2011). The circadian pacemaker is initiated when two E-box transcription factors, clock (clk) and cycle (cyc), form a heterodi- mer to induce transcription of the genes per and timeless (tim). Per and Tim in turn inhibit Clk/Cyc activity via phosphorylation. Per and Tim degrade over the day, which allows resetting of the pacemaker through resumption of activity of Clk and
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Cyc. This regular oscillation in gene expression allows the expression of both behavioral and metabolic genes to be linked to the circadian clock (Tataroglu and Emery 2014). opa may be involved in providing tissue specificity to the clock. In a genome-wide ChIP-seq analysis for targets of Clk and Cyc, Stark and colleagues found that opa is additionally required for expression of circadian transcriptional targets in the head (Meireles-Filho et al. 2014). Another GATA family transcription factor, serpent, serves a similar purpose in the body of the fly. Further analysis of targets that require Opa/Clk/Cyc for transcription in the fly head will likely reveal additional complexity to the role of opa in the circadian clock of Drosophila.