Genetic Regulatory Hierarchy Research Articles

X-chromosome target specificity diverged between dosage compensation mechanisms of two closely related Caenorhabditis species.

An evolutionary perspective enhances our understanding of biological mechanisms. Comparison of sex determination and X-chromosome dosage compensation mechanisms between the closely related nematode species Caenorhabditis briggsae (Cbr) and Caenorhabditis elegans (Cel) revealed that the genetic regulatory hierarchy controlling both processes is conserved, but the X-chromosome target specificity and mode of binding for the specialized condensin dosage compensation complex (DCC) controlling X expression have diverged. We identified two motifs within Cbr DCC recruitment sites that are highly enriched on X: 13 bp MEX and 30 bp MEX II. Mutating either MEX or MEX II in an endogenous recruitment site with multiple copies of one or both motifs reduced binding, but only removing all motifs eliminated binding in vivo. Hence, DCC binding to Cbr recruitment sites appears additive. In contrast, DCC binding to Cel recruitment sites is synergistic: mutating even one motif in vivo eliminated binding. Although all X-chromosome motifs share the sequence CAGGG, they have otherwise diverged so that a motif from one species cannot function in the other. Functional divergence was demonstrated in vivo and in vitro. A single nucleotide position in Cbr MEX can determine whether Cel DCC binds. This rapid divergence of DCC target specificity could have been an important factor in establishing reproductive isolation between nematode species and contrasts dramatically with the conservation of target specificity for X-chromosome dosage compensation across Drosophila species and for transcription factors controlling developmental processes such as body-plan specification from fruit flies to mice.

Engineered Maize Hybrids with Diverse Carotenoid Profiles and Potential Applications in Animal Feeding.

Multi-gene transformation methods need to be able to introduce multiple transgenes into plants in order to reconstitute a transgenic locus where the introduced genes express in a coordinated manner and do not segregate in subsequent generations. This simultaneous multiple gene transfer enables the study and modulation of the entire metabolic pathways and the elucidation of complex genetic control circuits and regulatory hierarchies. We used combinatorial nuclear transformation to produce multiplex-transgenic maize plants. In proof of principle experiments, we co-expressed five carotenogenic genes in maize endosperm. The resulting combinatorial transgenic maize plant population, equivalent to a "mutant series," allowed us to identify and complement rate-limiting steps in the extended endosperm carotenoid pathway and to recover corn plants with extraordinary levels of β-carotene and other nutritionally important carotenoids. We then introgressed the induced (transgenic) carotenoid pathway in a transgenic line accumulating high levels of nutritionally important carotenoids into a wild-type yellow-endosperm variety with a high β:ε ratio. Novel hybrids accumulated zeaxanthin at unprecedented amounts. We introgressed the same pathway into a different yellow corn line with a low β:ε ratio. The resulting hybrids, in this case, had a very different carotenoid profile. The role of genetic background in determining carotenoid profiles in corn was elucidated, and further rate-limiting steps in the pathway were identified and resolved in hybrids. Astaxanthin accumulation was engineered by overexpression of a β-carotene ketolase in maize endosperm. In early experiments, limited astaxanthin accumulation in transgenic maize plants was attributed to a bottleneck in the conversion of adonixanthin (4-ketozeaxanthin) to astaxanthin. More recent experiments showed that a synthetic β-carotene ketolase with a superior β-carotene/zeaxanthin ketolase activity is critical for the high-yield production of astaxanthin in maize endosperm. Engineered lines were used in animal feeding experiments which demonstrated not only the safety of the engineered lines but also their efficacy in a range of different animal production applications.

Transcription factor TEAD1 is essential for vascular development by promoting vascular smooth muscle differentiation.

TEAD1 (TEA domain transcription factor 1), a transcription factor known for the functional output of Hippo signaling, is important for tumorigenesis. However, the role of TEAD1 in the development of vascular smooth muscle cell (VSMC) is unknown. To investigate cell-specific role of Tead1, we generated cardiomyocyte (CMC) and VSMC-specific Tead1 knockout mice. We found CMC/VSMC-specific deletion of Tead1 led to embryonic lethality by E14.5 in mice due to hypoplastic cardiac and vascular walls, as a result of impaired CMC and VSMC proliferation. Whole transcriptome analysis revealed that deletion of Tead1 in CMCs/VSMCs downregulated expression of muscle contractile genes and key transcription factors including Pitx2c and myocardin. In vitro studies demonstrated that PITX2c and myocardin rescued TEAD1-dependent defects in VSMC differentiation. We further identified Pitx2c as a novel transcriptional target of TEAD1, and PITX2c exhibited functional synergy with myocardin by directly interacting with myocardin, leading to augment the differentiation of VSMC. In summary, our study reveals a critical role of Tead1 in cardiovascular development in mice, but also identifies a novel regulatory mechanism, whereby Tead1 functions upstream of the genetic regulatory hierarchy for establishing smooth muscle contractile phenotype.

Metabolic engineering of fatty acid biosynthetic pathway in sesame (Sesamum indicum L.): assembling tools to develop nutritionally desirable sesame seed oil

Vegetable oils are an essential component of human diet, in terms of their health beneficial roles. Despite their importance, the fatty acid profile of most commonly used edible oil seed crop plants are imbalanced; this skewed ratio of fatty acids in the diet has been shown to be a major reason for the occurrence of cardiovascular and autoimmune diseases. Until recently, it was not possible to exert significant control over the fatty acid composition of vegetable oils derived from different plants. However, the advent of metabolic engineering, knowledge of the genetic networks and regulatory hierarchies in plants have offered novel opportunities to tailor-made the composition of vegetable oils for their optimization in regard to food functionality and dietary requirements. Sesame (Sesamum indicum L.) is one of the ancient oilseed crop in Indian subcontinent but its seed oil is devoid of balanced proportion of ω-6:ω-3 fatty acids. A recent study by our group has shed new lights on metabolic engineering strategies for the purpose of nutritional improvement of sesame seed oil to divert the carbon flux from the production of linoleic acid (C18:2) to α-linolenic acid (C18:3). Apart from that, this review evaluates current understanding of regulation of fatty acid biosynthetic pathways in sesame and attempts to identify the major options of metabolic engineering to produce superior sesame seed oil.

The epidermal growth factor receptor critically regulates endometrial function during early pregnancy.

Infertility and adverse gynecological outcomes such as preeclampsia and miscarriage represent significant female reproductive health concerns. The spatiotemporal expression of growth factors indicates that they play an important role in pregnancy. The goal of this study is to define the role of the ERBB family of growth factor receptors in endometrial function. Using conditional ablation in mice and siRNA in primary human endometrial stromal cells, we identified the epidermal growth factor receptor (Egfr) to be critical for endometrial function during early pregnancy. While ablation of Her2 or Erbb3 led to only a modest reduction in litter size, mice lacking Egfr expression are severely subfertile. Pregnancy demise occurred shortly after blastocyst implantation due to defects in decidualization including decreased proliferation, cell survival, differentiation and target gene expression. To place Egfr in a genetic regulatory hierarchy, transcriptome analyses was used to compare the gene signatures from mice with conditional ablation of Egfr, wingless-related MMTV integration site 4 (Wnt4) or boneless morphogenic protein 2 (Bmp2); revealing that not only are Bmp2 and Wnt4 key downstream effectors of Egfr, but they also regulate distinct physiological functions. In primary human endometrial stromal cells, marker gene expression, a novel high content image-based approach and phosphokinase array analysis were used to demonstrate that EGFR is a critical regulator of human decidualization. Furthermore, inhibition of EGFR signaling intermediaries WNK1 and AKT1S1, members identified in the kinase array and previously unreported to play a role in the endometrium, also attenuate decidualization. These results demonstrate that EGFR plays an integral role in establishing the cellular context necessary for successful pregnancy via the activation of intricate signaling and transcriptional networks, thereby providing valuable insight into potential therapeutic targets.

Aggf1 acts at the top of the genetic regulatory hierarchy in specification of hemangioblasts in zebrafish.

The hemangioblast is a multipotential progenitor, which is derived from the mesoderm and can further differentiate into hematopoietic and endothelial lineages. The molecular mechanism governing the specification of hemangioblasts is fundamental to regenerative medicine based on embryonic stem cells for the treatment of various hematologic and vascular diseases. Here we show that aggf1 acts at the top of the genetic regulatory hierarchy in the specification of hemangioblasts in zebrafish. Knockdown of aggf1 expression decreases expression of endothelial cell-specific markers (cdh5, admr) and disrupts primitive hematopoiesis as shown by a decreased number of erythroid cells and reduced expression of gata1 (marker for erythroid progenitors) and pu.1 (myeloid progenitors). Aggf1 knockdown also decreases expression of runx1 and c-myb, indicating that it is required for specification of hematopoietic stem cells (definitive hematopoiesis). Aggf1 knockdown led to dramatically reduced expression of hemangioblast markers fli1, etsrp, lmo2, and scl, and hematopoietic/endothelial defects in aggf1 morphants were rescued by messenger RNA for scl, fli-vp16, or etsrp. Taken together, these data indicate that aggf1 is involved in differentiation of both hematopoietic and endothelial lineages and that aggf1 acts upstream of scl, fli1, and etsrp in specification of hemangioblasts.

Advanced genetic tools for plant biotechnology

Basic research has provided a much better understanding of the genetic networks and regulatory hierarchies in plants. To meet the challenges of agriculture, we must be able to rapidly translate this knowledge into generating improved plants. Therefore, in this Review, we discuss advanced tools that are currently available for use in plant biotechnology to produce new products in plants and to generate plants with new functions. These tools include synthetic promoters, 'tunable' transcription factors, genome-editing tools and site-specific recombinases. We also review some tools with the potential to enable crop improvement, such as methods for the assembly and synthesis of large DNA molecules, plant transformation with linked multigenes and plant artificial chromosomes. These genetic technologies should be integrated to realize their potential for applications to pressing agricultural and environmental problems.

A simulation study of mutations in the genetic regulatory hierarchy for butterfly eyespot focus determination

The color patterns on the wings of butterflies have been an important model system in evolutionary developmental biology. A recent computational model tested genetic regulatory hierarchies hypothesized to underlie the formation of butterfly eyespot foci [Evans, T.M., Marcus, J.M., 2006. A simulation study of the genetic regulatory hierarchy for butterfly eyespot focus determination. Evol. Dev. 8, 273–283]. The computational model demonstrated that one proposed hierarchy was incapable of reproducing the known patterns of gene expression associated with eyespot focus determination in wild-type butterflies, but that two slightly modified alternative hierarchies were capable of reproducing all of the known gene expressions patterns. Here we extend the computational models previously implemented in Delphi 2.0 to two mutants derived from the squinting bush brown butterfly ( Bicyclus anynana). These two mutants, comet and Cyclops, have aberrantly shaped eyespot foci that are produced by different mechanisms. The comet mutation appears to produce a modified interaction between the wing margin and the eyespot focus that results in a series of comet-shaped eyespot foci. The Cyclops mutation causes the failure of wing vein formation between two adjacent wing-cells and the fusion of two adjacent eyespot foci to form a single large elongated focus in their place. The computational approach to modeling pattern formation in these mutants allows us to make predictions about patterns of gene expression, which are largely unstudied in butterfly mutants. It also suggests a critical experiment that will allow us to distinguish between two hypothesized genetic regulatory hierarchies that may underlie all butterfly eyespot foci.

Genetic control of dorsoventral patterning and neuroblast specification in the Drosophila Central Nervous System

The Drosophila embryonic Central Nervous System (CNS) develops from the ventrolateral region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most of our knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, we combined loss of function with ectopic activation of Egfr activity. We observe that ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. Through this work, we identified the genetic criteria required for the development of the medial and intermediate column cell fates. We also show that ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoventral axis of the CNS. Finally, we demonstrate that Egfr and the genes of the achaete-scute complex act in parallel to regulate the individual fate of neural stem cells.

A simulation study of the genetic regulatory hierarchy for butterfly eyespot focus determination

The color patterns on the wings of butterflies have been an important model system in evolutionary developmental biology. Two types of models have been used to study these patterns. The first type of model employs computational techniques and generalized mechanisms of pattern formation to make predictions about how color patterns will vary as parameters of the model are changed. These generalized mechanisms include diffusion gradient, reaction-diffusion, lateral inhibition, and threshold responses. The second type of model uses known genetic interactions from Drosophila melanogaster and patterns of candidate gene expression in one of several butterfly species (most often Junonia (Precis) coenia or Bicyclus anynana) to propose specific genetic regulatory hierarchies that appear to be involved in color pattern formation. This study combines these two approaches using computational techniques to test proposed genetic regulatory hierarchies for the determination of butterfly eyespot foci (also known as border ocelli foci). Two computer programs, STELLA 8.1 and Delphi 2.0, were used to simulate the determination of eyespot foci. Both programs revealed weaknesses in a genetic model previously proposed for eyespot focus determination. On the basis of these simulations, we propose two revised models for eyespot focus determination and identify components of the genetic regulatory hierarchy that are particularly sensitive to changes in model parameter values. These components may play a key role in the evolution of butterfly eyespots. Simulations like these may be useful tools for the study of other evolutionary developmental model systems and reveal similar sensitive components of the relevant genetic regulatory hierarchies.

Neuronal defects in the hindbrain of Hoxa1, Hoxb1 and Hoxb2 mutants reflect regulatory interactions among these Hox genes.

Hox genes are instrumental in assigning segmental identity in the developing hindbrain. Auto-, cross- and para-regulatory interactions help establish and maintain their expression. To understand to what extent such regulatory interactions shape neuronal patterning in the hindbrain, we analysed neurogenesis, neuronal differentiation and motoneuron migration in Hoxa1, Hoxb1 and Hoxb2 mutant mice. This comparison revealed that neurogenesis and differentiation of specific neuronal subpopulations in r4 was impaired in a similar fashion in all three mutants, but with different degrees of severity. In the Hoxb1 mutants, neurons derived from the presumptive r4 territory were re-specified towards an r2-like identity. Motoneurons derived from that territory resembled trigeminal motoneurons in both their migration patterns and the expression of molecular markers. Both migrating motoneurons and the resident territory underwent changes consistent with a switch from an r4 to r2 identity. Abnormally migrating motoneurons initially formed ectopic nuclei that were subsequently cleared. Their survival could be prolonged through the introduction of a block in the apoptotic pathway. The Hoxa1 mutant phenotype is consistent with a partial misspecification of the presumptive r4 territory that results from partial Hoxb1 activation. The Hoxb2 mutant phenotype is a hypomorph of the Hoxb1 mutant phenotype, consistent with the overlapping roles of these genes in facial motoneuron specification. Therefore, we have delineated the functional requirements in hindbrain neuronal patterning that follow the establishment of the genetic regulatory hierarchy between Hoxa1, Hoxb1 and Hoxb2.

Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes.

We have cloned a chick homologue of Drosophila dachshund (dac), termed Dach1. Dach1 is the orthologue of mouse and human Dac/Dach (hereafter referred to as Dach1). We show that chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless (ey, the Pax6 orthologue), eyes absent (eya), and dac operate together to regulate Drosophila eye development, and that ey regulates the expression of eya and dac. We find that in the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homologue). Both Pax2 and Eya1 are required for normal ear development. Our expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, we have examined the expression of mouse Dach1 in Pax6, Pax2 and Eya1 mutant backgrounds. Our results indicate that Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy.

The hindsight Gene Is Required for Epithelial Maintenance and Differentiation of the Tracheal System in Drosophila

During animal development, morphogenesis of tissues and organs requires dynamic cell shape changes and movements that are accomplished without loss of epithelial integrity. Data from vertebrate and invertebrate systems have implicated several cell surface and cytoskeleton-associated molecules in the establishment and maintenance of epithelial architecture, but there has been little analysis of the genetic regulatory hierarchies that control epithelial morphogenesis in specific tissues. Here we show that the Drosophila Hindsight nuclear zinc-finger protein is required during tracheal morphogenesis for the maintenance of epithelial integrity and assembly of apical extracellular structures known as taenidia. In hindsight (hnt) mutants tracheal placodes form, invaginate, and undergo primary branching as well as early fusion events. Starting at midembryogenesis, however, the tracheal epithelium collapses or expands to give rise to sacs of tissue. While a subset of hnt mutant tracheal cells enters the apoptotic pathway, genetic suppression of apoptosis indicates that this is not the cause of the epithelial defects. Surviving hnt mutant tracheal cells retain cell–cell junctions and a normal subcellular distribution of apical markers such as Crumbs and DE-Cadherin. However, taenidia do not form on the lumenal surface of tracheal cells. While loss of epithelial integrity is a common feature of crumbs, stardust, and hnt mutants, defective assembly of taenidia is unique to hnt mutants. These data suggest that HNT is a tissue-specific factor that regulates maintenance of the tracheal epithelium as well as differentiation of taenidia.

Expression of the early-late gene encoding the nuclear receptor HR3 suggests its involvement in regulating the vitellogenic response to ecdysone in the adult mosquito

The insect steroid hormone, 20-hydroxyecdysone (20E), is a key factor controlling critical developmental events of embryogenesis, larval molting, metamorphosis, and, in some insects, reproduction. We are interested in understanding the molecular basis of the steroid hormone ecdysone action in insect egg development. The yellow fever mosquito, Aedes aegypti, in addition to being an important vector of human diseases, represents an outstanding model for studying molecular mechanisms underlying egg maturation due to stringently controlled, blood meal-activated reproductive events in this insect. To elucidate the genetic regulatory hierarchy controlling the reproductive ecdysone response, we have investigated ecdysone-regulated gene expression in vitellogenic mosquito ovaries and fat bodies. We have previously demonstrated the conservation of a primary ecdysone-triggered regulatory hierarchy, implicated in development of immature stages of Drosophila, represented by the ecdysone receptor/Ultraspiracle complex and an early gene E75 during the reproductive ecdysone response (Wang, S.-F., Miura, K., Miksicek, R.J., Segraves, W.A., Raikhel, A.S., 1998. DNA binding and transactivation characteristics of the mosquito ecdysone receptor — Ultraspiracle complex. J. Biol. Chem. 273, 27531–27540; Pierceall, W.E., Li, C., Biran, A., Miura, K., Raikhel, A.S., Segraves, W.A., 1999. E75 expression in mosquito ovary and fat body suggests reiterative use of ecdysone-regulated hierarchies in development and reproduction. Mol. Cell. Endocrinol. 150, 73–89). The present paper demonstrates that conservation of the factors involved in the ecdysone-responsive genetic hierarchy regulating female reproduction extends beyond the early genes. Here, we identify AHR3, a highly conserved homologue of the Drosophila HR3 early-late ecdysone-inducible gene in the mosquito. We show that AHR3 is expressed in both vitellogenic tissues of the female mosquito, the fat body and the ovary. The expression of AHR3 correlates with the ecdysteroid titer, reaching a peak at 24 h after a blood meal. Moreover, in vitro fat body culture experiments demonstrate that the kinetics and dose response of AHR3 to 20-hydroxyecdysone (20E), an active ecdysteroid in the mosquito, is similar to those of the late vitellogenic genes rather than the early E75 gene. However, as shown for other early and early-late genes, the 20E activation of AHR3 is not inhibited by the presence of cycloheximide, a protein synthesis inhibitor. Taken together, these findings strongly suggest AHR3 involvement in regulating the vitellogenic response to ecdysone in the adult mosquito.

DHR3 Is Required for the Prepupal–Pupal Transition and Differentiation of Adult Structures during Drosophila Metamorphosis

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. Here we use both a heat-inducible DHR3 rescue construct and clonal analysis to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, and no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and βFTZ-1. Reductions in EcR and βFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. These studies demonstrate that DHR3 is an essential regulator of the βFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis.

E75 expression in mosquito ovary and fat body suggests reiterative use of ecdysone-regulated hierarchies in development and reproduction

The steroid hormone ecdysone controls genetic regulatory hierarchies underlying insect molting, metamorphosis and, in some insects, reproduction. Cytogenetic and molecular analysis of ecdysone response in Drosophila larval salivary glands has revealed regulatory hierarchies including early genes which encode transcription factors controlling late ecdysone response. In order to determine whether similar hierarchies control reproductive ecdysone response, we have investigated ecdysone-regulated gene expression in vitellogenic mosquito ovaries and fat bodies. Here, we identify the homologue of the Drosophila E75 early ecdysone inducible gene in the yellow fever mosquito Aedes aegypti, and show that, as in Drosophila, the mosquito homologue, AaE75, consists of three overlapping transcription units with three mRNA isoforms, AaE75A, AaE75B, and AaE75C, originating as a result of alternative splicing. All three AaE75 isoforms are induced at the onset of vitellogenesis by a blood meal-activated hormonal cascade, and highly expressed in the mosquito ovary and fat body, suggesting their involvement in the regulation of oogenesis and vitellogenesis, respectively. Furthermore, in vitro fat body culture experiments demonstrate that AaE75 isoforms are induced by 20-hydroxyecdysone, an active ecdysteroid in the mosquito. These findings suggest that related ecdysone-triggered regulatory hierarchies may be used reiteratively during developmental and reproductive ecdysone responses.

The DHR78 Nuclear Receptor Is Required for Ecdysteroid Signaling during the Onset of Drosophila Metamorphosis

Pulses of ecdysteroids direct Drosophila through its life cycle by activating stage- and tissue-specific genetic regulatory hierarchies. Here we show that an orphan nuclear receptor, DHR78, functions at the top of the ecdysteroid regulatory hierarchies. Null mutations in DHR78 lead to lethality during the third larval instar with defects in ecdysteroid-triggered developmental responses. Consistent with these phenotypes, DHR78 mutants fail to activate the mid-third instar regulatory hierarchy that prepares the animal for metamorphosis. DHR78 protein is bound to many ecdysteroid-regulated puff loci, suggesting that DHR78 directly regulates puff gene expression. In addition, ectopic expression of DHR78 has no effects on development, indicating that its activity is regulated posttranslationally. We propose that DHR78 is a ligand-activated receptor that plays a central role in directing the onset of Drosophila metamorphosis.

DHR3, an ecdysone-inducible early-late gene encoding a Drosophila nuclear receptor, is required for embryogenesis.

Response to the steroid hormone ecdysone in Drosophila is controlled by genetic regulatory hierarchies that include eight members of the nuclear receptor protein family. The DHR3 gene, located within the 46F early-late ecdysone-inducible chromosome puff, encodes an orphan nuclear receptor that recently has been shown to exert both positive and negative regulatory effects in the ecdysone-induced genetic hierarchies at metamorphosis. We used a reverse genetics approach to identify 11 DHR3 mutants from a pool of lethal mutations in the 46F region on the second chromosome. Two DHR3 mutations result in amino acid substitutions within the conserved DNA binding domain. Analysis of DHR3 mutants reveals that DHR3 function is required to complete embryogenesis. All DHR3 alleles examined result in nervous system defects in the embryo.

A steroid-triggered switch in E74 transcription factor isoforms regulates the timing of secondary-response gene expression.

The steroid hormone 20-hydroxyecdysone (referred to here as ecdysone) directs Drosophila metamorphosis by activating a series of genetic regulatory hierarchies. ETS domain transcription factors encoded by the ecdysone-inducible E74 early gene, E74A and E74B, act at the top of these hierarchies to coordinate the induction of target genes. We have ectopically expressed these E74 isoforms to understand their regulatory functions during the onset of metamorphosis. We show that E74 can regulate its own transcription, most likely through binding sites within its gene. Ectopic expression of E74B can partially repress the E78B and DHR3 orphan receptor genes, suggesting a role for E74 in the appropriate timing of early-late gene expression. Furthermore, E74A is both necessary and sufficient for E78B induction, implicating E74A as a key regulator of E78B expression. We also show, consistent with our studies of E74 loss-of-function mutations, that E74B is a potent repressor of late gene transcription and E74A is sufficient to prematurely induce the L71-1 late gene. However, ectopic expression of both Broad-Complex and E74A activators in an E74B mutant background is not sufficient to prematurely induce all late genes, indicating that other factors contribute to this regulatory circuit. These observations demonstrate that the steroid-triggered switch in E74 transcription factor isoforms plays a central role in the proper timing of secondary-response gene expression.

The Brn-3 family of POU-domain factors: primary structure, binding specificity, and expression in subsets of retinal ganglion cells and somatosensory neurons

A search for POU domain sequences expressed in the human retina has led to the identification of three closely related genes: Brn-3a, Brn-3b, and Brn-3c. The structure and expression pattern of Brn-3b was reported earlier (Xiang et al., 1993); we report here the structures and expression patterns of Brn-3a and Brn-3c. Antibodies specific for each Brn-3 protein were generated and shown to label only ganglion cells in a variety of vertebrate retinas. A complex pattern of strongly and weakly immunolabeled ganglion cells was observed in mouse, cat, and monkey retinae. In mouse and cat retinae, Brn-3a and Brn-3b proteins are found in a large fraction of ganglion cells, whereas Brn-3c is present in fewer ganglion cells. In the cat retina, anti-Brn-3a immunoreactivity was strong in the small ganglion cells (gamma cells) and weak in the remaining ganglion cells (alpha and beta cells); anti-Brn-3b immunoreactivity was present in all ganglion cells; and anti-Brn3c immunoreactivity was confined to the small ganglion cells. Immunolabeling of macaque retinae following retrograde labeling from the lateral geniculate nucleus revealed strong anti-Brn-3a immunoreactivity in a minority of retrogradely labeled P-type ganglion cells, and weak Brn-3a immunoreactivity in all of the remaining P- and M-type ganglion cells. In the same retinae, strong anti-Brn-3b immunoreactivity was seen in nearly all P-type ganglion cells and weak immunoreactivity in nearly all M-type ganglion cells. Each of the Brn-3-specific antibodies also labeled subsets of neurons in the dorsal root and trigeminal ganglia, suggesting that primary somatosensory neurons and retinal ganglion cells share genetic regulatory hierarchies. In vitro selection of an optimal DNA binding site using the Brn-3b POU domain has revealed a consensus [(A/G)CTCATTAA(T/C)] that is recognized by each of the Brn-3 POU domains and is distinct from binding sites previously described for other POU domain proteins.