**4. Toward a functional definition of** *Dmgce*

A functional characterization of *gce*, named for its expression in a subset of embryonic germ cells (Moore *et al.,* 2000), is in its infancy. Column pulldown assays showed MET, in addition to forming homodimers, forms heterodimers with GCE, and addition of JH or either of two JHAs significantly impaired these interactions (Godlewski *et al*., 2006). It is unknown whether GCE forms homodimers, like MET, or whether GCE can bind JH or its analogs. *GAL4*/*UAS*-driven (Brand & Perrimon, 1993) overexpression of *Met+* from *actin* or *tubulin* promoters results in larval lethality in the absence of methoprene (Barry *et al.,* 2008), perhaps by upsetting the stoichiometry of MET and GCE dimers, favoring MET homodimerization at inappropriate times or in inappropriate tissues. Recently, JH was shown to inhibit MET and GCE in *D. melanogaster* by preventing caspase-driven programmed cell death (PCD) and histolysis of the larval fat body. DRONC and DRICE, evolutionarily conserved caspase genes involved in this physiology at the onset of metamorphosis, were shown to be downregulated in *Met* and *gce* deficient flies (Liu *et al.,* 2009). Similarly, methoprene interferes with caspase-driven midgut remodeling in *A. aegypti* (Nishiura *et al*., 2003; Wu *et al*., 2006) and *T. castaneum* (Parthasarathy *et al*., 2008; Parthsarathy *et al*., 2009), showing that this mechanism of JH action is evolutionarily conserved. It is noteworthy that recombinant MET can repress reporter gene expression in the absence of JH (presumably, MET forms homodimers in this system; Miura *et al.,* 2005); transcriptional repression has previously been reported in other PAS proteins (Dolwick *et al.,* 1993). Therefore, the JH-dependent, stage-specific formation of alternative MET/GCE dimers may have unique regulatory consequences on distinct suites of target genes.

### **4.1** *Dmgce* **substitution for** *DmMet*

To evaluate the notion that *gce* might confer viability to *Met* null flies, we manipulated *gce*  expression using a binary *UAS/GAL4* system to drive either a *gce* cDNA or an RNAi construct designed to target *gce* transcript. We carried these experiments out in a variety of genotypic contexts in order to examine the effect of *gce* transcript abundance on several methoprene conditional and non-conditional phenotypes (Baumann *et al*., 2010b). First, we explored the effect of *gce* over- and under-expression on a *Met*-specific non-conditional phenotype that manifests as a variable number of grossly malformed posterior facets of the compound eye (Figure 3). This phenotype is visible in *Met27* and *Metw3* flies, and is enhanced in the latter genotype. In our experiments, we found that *gce* overexpression in a *Metw3* genetic background can rescue the *Met*-specific eye phenotype, suggesting functional overlap of *gce* and *Met*. Notably, when *gce* was overexpressed in a *Met27* background from the *GawB}dan[AC116]* promoter, targeting transgene expression to the compound eye, the eye phenotype was completely rescued (Baumann *et al*., 2010b).

The *Met27* phenotype mimics a set of defects resulting from genetic ablation of the JHproducing corpus allatum (CAX), including a heterochronic shift in EcR-B1 expression in the optic lobe (Riddiford *et.al.*, 2010). Exogenous JH application rescues the entire suite of defects in CAX prepupae, while JH provision to *Met27* flies rescues only a subset of these defects, suggesting an alternate mechanism of JH signal transduction (Riddiford *et al*., 2010). Based on our findings that *gce* can substitute for *Met* in the compound eye, further study of GCE involvement in eye development may provide a link between these phenomena. For instance, GCE may partially substitute for MET as a ligand binder to mediate JH signaling when this hormone is supplied in excess.

Molecular Evolution of Juvenile Hormone Signaling 341

1956), suggesting a role in male reproductive biology. In *D. melanogaster*, JH controls MAG protein accumulation (Yamamoto *et al.,* 1988) and male *apterous* (*ap*) mutants court females less vigorously than wild-type flies (Tompkins, 1990). In females, the activity of these counteracting hormones is critical for ovarian development and oocyte maturation. Development of the *D. melanogaster* oocyte is under the control of JH through previtellogenic stages 8-9. Female *D. melanogaster apterous4* mutants are sterile owing to reduced levels of JH synthesis (Bownes, 1989); provision of exogenous JH rescues vitellogenic oocyte development in *ap* females (Postlethwait & Weiser, 1973). In *A. aegypti*, JH also controls previtellogenic ovarian development (Clements, 1992). In this case, JH signaling is necessary to promote 20E competence in the fat body, the site of post-blood meal vitellogenin synthesis. In contrast, vitellogenesis is retarded by JH treatment in the gypsy moth, *Lymantria dyspar* (Davis *et al.,* 1990). Thus, there is variation in hormonal

GCE clearly compensates for MET deficiency in preadult development (Baumann *et al*., 2010b). In our experiments, over-expressed *gce* failed to rescue both the documented behavior of reduced courtship in *Met27; UAS-gce*/*tubulin-GAL4* males and the reduction in oocyte development and oviposition in these females. Therefore, it appears that excess *gce*  cannot compensate for *Met*-induced reduction of reproductive capacity. This result suggests that the functional roles for MET and GCE are incompletely partitioned between preadult

In *A. aegypti*, Aa*Met* regulates the transcription of several JH target genes in newly eclosed, previtellogenic adult females (Zhu *et al*., 2010). Presumably, the MET-like gene product in lower Diptera serves an analogous function both MET and GCE in JH signaling, but through the action of a single gene. This is perhaps accomplished by virtue of its modular architecture of *DmMet-* and *Dmgce-*specific domains. Higher sequence identity exists between the bHLH and PAS B of *Dmgce* and more primitive holometabolous *Met-*like genes, while the PAS A and PAC domains share higher sequence identity with *DmMet*. These domains may confer a discriminating *Met*-like function that may partially underlie the

Overepxression studies demonstrated that *gce* can substitute for *Met* in a tissue specific manner to rescue several preadult *Met* mutant phenotypes. Hence, our results empirically support the notion of functional redundancy between *Met* and its paralog *gce*. To further explore the relationship between *Met* and *gce* in JH signaling, we carried out underexpression studies in *Met+* and *Met* mutant backgrounds by driving the expression of

First, we examined the consequence of *gce* deficiency in a *Met* mutant background under the justification that, if *gce* is responsible for *Met27* viability, then concomitant reduction of *Met* and *gce* could result in lethality. Interestingly, *Met27*; *UAS-gce-dsRNA*/*tubulin-GAL4* flies died as early pupae (0-2 days), whereas expression of the dsRNA construct from an *actin-GAL4* promoter caused lethality in the pharate adult stage. Next, we assessed the effects of *gce* reduction in *Met+* flies. Surprisingly, *Met+*; *UAS-gce-dsRNA*/*tubulin-GAL4* flies died as pharate adults, indicating that even in the presence of functional MET, *gce* is a vital gene. Driving the transgene from an *actin-GAL4* promoter allowed some degree of adult survival, but these adults were clearly affected by insufficient *gce*, dying within two to three days.

control in insects.

development and reproduction in adults.

**4.3** *Dmgce* **is a vital gene** 

a *gce* RNAi construct.

functional divergence of *Met* and *gce* in higher Diptera.

Fig. 3. Left: malformed facets in the posterior compound eye of *Metw3* flies appear dark under light microscopy. Right: EMS-induced production of an unidentified enhancer gene dramatically intensifies the *Metw3* phenotype (T.G.W., unpublished).

We also explored the effect of *gce* overexpression on several methoprene-conditional phenotypes. Overexpressed *gce* rescued both the diagnostic malrotation of male genitalia and sensitivity to the toxic effects of methoprene exposure. Sublethal doses of methoprene can induce malrotation of the male genital disc in *D. melanogaster*, resulting in terminalia that are improperly oriented for copulation (Bouchard & Wilson, 1987). *Met27* males are resistant to this phenotype. We found that global *gce* overexpression in a *Met27* background rescues blockage of the malrotation phenotype in *Met27; UAS-gce/ tubulin-GAL4* flies. When these flies were exposed to methoprene, we observed malrotation close to levels seen in *Met+* flies (Baumann *et al.,* 2010b).

*Met* and *gce* are generally co-expressed in JH target tissues, but we detected insignificant amounts of *gce* transcript in late third instar larval fat body. When *gce* was expressed from a construct targeting expression to this tissue, partial rescue of JH-induced pupal lethality was achived, perhaps as a result of supplying *gce* to a tissue in which its expression is normally depressed at this time in development. *gce* expression in the larval fat body was unable to rescue either the eye phenotype or to prevent methoprene-induced malrotation of the male genitalia, indicating that *gce* substitution for *Met* is tissue specific (Baumann *et al*., 2010b).

### **4.2 Functional partitioning of** *DmMet* **and** *Dmgce* **in** *D. melanogaster* **reproduction**

Following metamorphosis, the interaction of 20E and JH is crucial in insect reproduction. JH was first isolated in large quantities from the MAG of *Hyalophora cecropia* (Williams,

Fig. 3. Left: malformed facets in the posterior compound eye of *Metw3* flies appear dark under light microscopy. Right: EMS-induced production of an unidentified enhancer gene

We also explored the effect of *gce* overexpression on several methoprene-conditional phenotypes. Overexpressed *gce* rescued both the diagnostic malrotation of male genitalia and sensitivity to the toxic effects of methoprene exposure. Sublethal doses of methoprene can induce malrotation of the male genital disc in *D. melanogaster*, resulting in terminalia that are improperly oriented for copulation (Bouchard & Wilson, 1987). *Met27* males are resistant to this phenotype. We found that global *gce* overexpression in a *Met27* background rescues blockage of the malrotation phenotype in *Met27; UAS-gce/ tubulin-GAL4* flies. When these flies were exposed to methoprene, we observed malrotation close to levels seen in

*Met* and *gce* are generally co-expressed in JH target tissues, but we detected insignificant amounts of *gce* transcript in late third instar larval fat body. When *gce* was expressed from a construct targeting expression to this tissue, partial rescue of JH-induced pupal lethality was achived, perhaps as a result of supplying *gce* to a tissue in which its expression is normally depressed at this time in development. *gce* expression in the larval fat body was unable to rescue either the eye phenotype or to prevent methoprene-induced malrotation of the male genitalia, indicating that *gce* substitution for *Met* is tissue specific (Baumann *et* 

**4.2 Functional partitioning of** *DmMet* **and** *Dmgce* **in** *D. melanogaster* **reproduction**  Following metamorphosis, the interaction of 20E and JH is crucial in insect reproduction. JH was first isolated in large quantities from the MAG of *Hyalophora cecropia* (Williams,

dramatically intensifies the *Metw3* phenotype (T.G.W., unpublished).

*Met+* flies (Baumann *et al.,* 2010b).

*al*., 2010b).

1956), suggesting a role in male reproductive biology. In *D. melanogaster*, JH controls MAG protein accumulation (Yamamoto *et al.,* 1988) and male *apterous* (*ap*) mutants court females less vigorously than wild-type flies (Tompkins, 1990). In females, the activity of these counteracting hormones is critical for ovarian development and oocyte maturation. Development of the *D. melanogaster* oocyte is under the control of JH through previtellogenic stages 8-9. Female *D. melanogaster apterous4* mutants are sterile owing to reduced levels of JH synthesis (Bownes, 1989); provision of exogenous JH rescues vitellogenic oocyte development in *ap* females (Postlethwait & Weiser, 1973). In *A. aegypti*, JH also controls previtellogenic ovarian development (Clements, 1992). In this case, JH signaling is necessary to promote 20E competence in the fat body, the site of post-blood meal vitellogenin synthesis. In contrast, vitellogenesis is retarded by JH treatment in the gypsy moth, *Lymantria dyspar* (Davis *et al.,* 1990). Thus, there is variation in hormonal control in insects.

GCE clearly compensates for MET deficiency in preadult development (Baumann *et al*., 2010b). In our experiments, over-expressed *gce* failed to rescue both the documented behavior of reduced courtship in *Met27; UAS-gce*/*tubulin-GAL4* males and the reduction in oocyte development and oviposition in these females. Therefore, it appears that excess *gce*  cannot compensate for *Met*-induced reduction of reproductive capacity. This result suggests that the functional roles for MET and GCE are incompletely partitioned between preadult development and reproduction in adults.

In *A. aegypti*, Aa*Met* regulates the transcription of several JH target genes in newly eclosed, previtellogenic adult females (Zhu *et al*., 2010). Presumably, the MET-like gene product in lower Diptera serves an analogous function both MET and GCE in JH signaling, but through the action of a single gene. This is perhaps accomplished by virtue of its modular architecture of *DmMet-* and *Dmgce-*specific domains. Higher sequence identity exists between the bHLH and PAS B of *Dmgce* and more primitive holometabolous *Met-*like genes, while the PAS A and PAC domains share higher sequence identity with *DmMet*. These domains may confer a discriminating *Met*-like function that may partially underlie the functional divergence of *Met* and *gce* in higher Diptera.

### **4.3** *Dmgce* **is a vital gene**

Overepxression studies demonstrated that *gce* can substitute for *Met* in a tissue specific manner to rescue several preadult *Met* mutant phenotypes. Hence, our results empirically support the notion of functional redundancy between *Met* and its paralog *gce*. To further explore the relationship between *Met* and *gce* in JH signaling, we carried out underexpression studies in *Met+* and *Met* mutant backgrounds by driving the expression of a *gce* RNAi construct.

First, we examined the consequence of *gce* deficiency in a *Met* mutant background under the justification that, if *gce* is responsible for *Met27* viability, then concomitant reduction of *Met* and *gce* could result in lethality. Interestingly, *Met27*; *UAS-gce-dsRNA*/*tubulin-GAL4* flies died as early pupae (0-2 days), whereas expression of the dsRNA construct from an *actin-GAL4* promoter caused lethality in the pharate adult stage. Next, we assessed the effects of *gce* reduction in *Met+* flies. Surprisingly, *Met+*; *UAS-gce-dsRNA*/*tubulin-GAL4* flies died as pharate adults, indicating that even in the presence of functional MET, *gce* is a vital gene. Driving the transgene from an *actin-GAL4* promoter allowed some degree of adult survival, but these adults were clearly affected by insufficient *gce*, dying within two to three days.

Molecular Evolution of Juvenile Hormone Signaling 343

*castaneum*. These beetles are both amenable to genetic manipulation and gene knockdown owing to the dramatic effects of systemic RNAi, and the larvae of this species are very sensitive to JH, unlike *D. melanogaster* larvae. Exposure to JH or a number of its chemical analogs precipitates supernumerary larval instars, similar to the effects of JH on the model lepidopteran, *Manduca sexta* (Parthasarathy & Palli, 2009). *T. castaneum*, like mosquitoes, has a single *Met-like* gene. In their seminal paper, Konopova and Jindra (2007) demonstrated that RNAi-mediated knockdown of *TcMet* results not only in a methoprene resistance phenotype, but also in the precocious metamorphosis of early instar larvae. A long soughtafter result, the genetic reduction of *TcMet* provided the phenotype frustratingly absent in *D. melanogaster*: metamorphic disruption. Reproductive roles for *TcMet* have also been shown; *TcMet* knockdown results in a substantial decrease in vitellogenin transcription, (Parthasarathy, *et al.*, 2010) consistent with *Met* deficiency in *D. melanogaster* females (Wilson & Ashok, 1998). These results demonstrate that the single *Met-*like genes in primitive Holometabola function in both development (metamorphosis) and reproduction. Further functional characterization of *TcMe*t (and the single *Met*-like gene of lower Diptera) could lead to a better understanding of how *DmMet* has apparently co-opted reproductive

The molecular networks that link JH and 20E signaling pathways form the foundation of multiple aspects of insect physiology, as evidenced by the criticality of both hormones in development, reproduction, and diapause (Zhou & Riddiford, 2002; Soller *et al.,* 1999; Denlinger, 1985). *Broad Complex* (*Broad* or *BR-C*) is an early gene in the 20E cascade that encodes a family of alternatively spliced zinc finger transcription factors (four in *D. melanogaster*, Z1-Z4) fused to a common core protein. Certain *Broad* alleles phenocopy the morphogenetic defects incurred by methoprene exposure in *D. melanogaster*. Wilson *et al* (2006) showed phenotypic synergism in *Met* and *broad* double mutants, demonstrating JHsensitive MET and BROAD interaction (BROAD protein accumulation is comparable to that of wild type flies, suggesting physical interaction with, rather than transcriptional regulation

In a hemimetabolous insect, *Oncopeltus fasciatus*, continuous *Broad* expression directs progressive development through nymphal instars (Erezyilmaz *et al.,* 2006). In Holometabola, *Broad* expression is confined to the prepupal stage, acting as a pupal specifier (Zhou & Riddiford, 2002). Loss of *Broad* expression, characteristic of the *npr1* mutant (*nonpupariating*; a deletion of the entire complementation group), results in the namesake phenotype of failure to enter the pupal program. Consequently, a restriction of *Broad*  expression during this developmental stage may have contributed to the evolution of complete metamorphosis. During larval development in *D. melanogaster*, JH represses *broad*. At pupariation, exogenous JH induces a second wave of *broad* expression in the abdominal epidermis, resulting in the deposition of a second pupal cuticle (Zhou & Riddiford, 2002), demonstrating that the networks underlying these signaling mechanisms are complex. In *T. castaneum*, methoprene exposure induces *Broad* expression and this upregulation is ablated upon *TcMet* knockdown. Therefore, *TcMet* is upstream of *Broad* in JH signaling in these beetles (Konopova & Jindra, 2008). *Krüppel homolog 1* (*Kr-h1*) is upstream of *Broad* in *D. melanogaster* JH signaling, where its expression in abdominal epidermis produces sternal bristle disruption similar to that seen following low dose JHA exposure (Minakuchi *et al*.,

by *Met*), and providing a link between JH and 20E signaling (Wilson *et al*., 2006).

functional roles from a *gce*-like ancestor in higher Diptera

**5.1 JH regulation of the E-20 transcriptional cascade** 

Differential intensity of transgene expression from *actin* and *tubulin* promoters was previously reported in our lab (Barry *et al*., 2008).

*gce* underexpression had no observable effect on embryonic development, a stage during which no role for JH has been demonstrated. We have shown that *gce* transcription begins after about eight hours in early embryos, in contrast to *Met*, which is supplied as a maternal message (Baumann *et al*., 2010a). The importance of such divergence in temporal expression profiles is unclear.
