Browsing by Subject "Morphogenesis"

Mutations in myosin VIIa (MyoVIIa), an unconventional myosin, have been shown to cause Usher Syndrome Type 1B in humans. Usher Syndrome Type 1B is characterized by congenital sensorineural deafness, vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. Mouse model studies show that sensorineural deafness and vestibular dysfunction in MyoVIIa mutants is caused by disruption in the structure of microvilli-like projections (stereocilia) of hair cells in the cochlea and vestibular organ. MyoVIIa has also been shown to affect adaptation of mechanoelectrical transduction channels in stereocilia.

In Drosophila melanogaster mutations in MyoVIIa encoded by crinkled (ck) cause defects in hair and bristle morphogenesis and deafness. Here we study the formation of bristles and hairs in Drosophila melanogaster to investigate the molecular basis of ck/MyoVIIa function and its regulation. We use live time-lapse confocal microscopy and genetic manipulations to investigate the requirement of ck/MyoVIIa function in various steps of morphogenesis of hairs and bristles. Here we show that null or near null mutations in ck/MyoVIIa lead to the formation of 8-10 short and thin hairs (split hairs) per epithelial cell that are likely the result of the failure of association of hair-actin bundles that in wild-type cells come together to form a single hair.

The myosin super family of motor proteins is divided into 17 classes by virtue of differences in the sequence of their motor domain, which presumably affect their physiological functions. In addition, substantial variety in the overall structure of their tail plays an important role in the differential regulation of myosin function. In this study we show that ck/MyoVIIa, that has two MyTH4 FERM domains in its tail separated by an SH3 domain, requires both MyTH4 FERM repeats for efficient association of hair-actin bundles to form hairs. We also show that the "multiple hair" phenotype of over-expression of ck/MyoVIIa requires both MyTH4 FERM domain function but not the tail-SH3 domain. We further demonstrate that the tail-SH3 domain of ck/MyoVIIa plays a role in keeping actin bundles, which run parallel to the length of the growing bristle, separate from each other. Our data also suggests that the tail-SH3 domain plays a role in the association of the actin filament bundles with the membrane and regulates F-actin levels in bristles.

We further demonstrate that over-expression of Quail (villin) can rescue the hair elongation defects seen in ck/MyoVIIa null or near null mutants but does not rescue the split hair defects. We show that over-expression of Alpha-actinin-GFP, another actin bundling protein, phenocopies the multiple hair phenotype of ck/MyoVIIa over-expression. Over-expression of Alpha-actinin-GFP in a ck/MyoVIIa null or near null background shows that Alpha-actinin-GFP cannot rescue the split or short hair phenotype of ck/MyoVIIa loss-of-function. However, cells over-expressing Alpha-actinin-GFP in a ck/MyoVIIa null or near null background have more than the normal 8-10 split hairs, suggesting that Alpha-actinin-GFP over-expression causes the formation of more than the normal complement of hair-actin bundles per cell, resulting in a multiple hair phenotype. We show that Twinfilin, an actin monomer sequestering protein implicated in negatively regulating F-actin bundle elongation in stereocilia in a MyoVIIa-dependent manner, is required for F-actin bundle stability.

In addition, we use yeast two-hybrid strategies to identify Slam as a protein that directly binds to ck/MyoVIIa. We show that Slam, a novel membrane-associated protein, likely functions to regulate ck/MyoVIIa function during hair and bristle morphogenesis. We show that over-expression of Slam and loss-of-function mutations in Slam phenocopy ck/MyoVIIa loss-of-function split and short hair phenotype. We also show that disruption of Slam and RhoGEF2 association causes split hair defects similar to ck/MyoVIIa loss-of-function phenotype suggesting that Slam probably regulates ck/MyoVIIa function via RhoGEF2.

Together our results show that ck/MyoVIIa plays an important role in regulating the actin cytoskeleton that underlies actin-based cellular protrusions like hairs and bristles.

The means by which cells, tissues, and organisms undergo morphogenesis are variable and highly regulated, and the mechanisms that govern cellular changes in response to signaling cues are poorly understood. This study seeks to address the role of a newly characterized protein in zebrafish in translating signaling cues into physical changes within a cell.

The Cordon–bleu (Cobl) gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. In vitro, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl in vivo has yet to be determined. We have identified the zebrafish cobl ortholog and have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. The utilization of antibodies developed against Cobl shows that the protein is concentrated along the apical domain of ciliated cells, in close proximity to the apical actin cap.

Reduction of cobl by antisense morpholinos reveals an essential role in embryonic morphogenesis and organ development. A requirement for Cobl was shown for the proper function of various and ciliated epithelial organs. Cobl appears to direct the elongation of motile cilia in organs such as Kupffer’s vesicle and the pronephros. In Kupffer’s vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Additionally, Cobl may play a role during gastrulation cell movements and convergence and extension morphogenesis during early embryonic development. Thus, Cobl may represent a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support elongation of motile cilia and tissue morphogenesis.

Cell sheet morphogenesis is essential for metazoan development and homeostasis, contributing to key developmental stages such as neural tube closure as well as tissue maintenance through wound healing. Dorsal closure, a well-characterized stage in Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis. Closure is a remarkably robust process where coordination of conserved gene expression and signaling cascades regulate cellular movements that drive closure. While well-characterized, new ‘dorsal closure genes’ continue to be discovered due to advances in microscopy and genetics. Here, we use live imaging and a set of large deletions, deficiencies (Dfs), that together remove 98.9% of the genes on 2L in order to identify regions of the genome required for normal closure. We successfully screened 96.1% of the genes on 2L and identified diverse dorsal closure defects in embryos homozygous for 47 Dfs, 26 of which have no known dorsal closure gene located within the Df region. We have already identified pimples, odd-skipped, paired, and sloppy-paired 1 as dorsal closure genes on the 2L affecting lateral epidermal cell shapes, and anticipate we will continue to identify novel ‘dorsal closure genes’ with further analysis. We also investigate the changes in dorsal closure dynamics and forces in the even-skipped (eve) mutant, which has aberrant cell shapes and behaviors as well as reduced actin and myosin at the purse string, but completes closure. We find that loss of wg/wnt-1 signaling in eve causes the observed defects in closure and that crumbs, a regulator of actin and myosin, is mis-expressed. Additionally, laser microsurgery demonstrates that the eve or wg mutant embryos are under a global tension in the anterior-posterior direction. Lastly, we identify a lesion in echinoid that is responsible for the jagged purse string and ectopic zipping dorsal closure phenotype previously thought to be due to a lesion in Zasp52.

Cell sheet morphogenesis characterizes key developmental transitions throughout phylogeny. As such, it plays a crucial role in developmental milestones in vertebrate morphogenesis including gastrulation, neural tube formation, and palate formation. It also plays important roles in wound healing. Dorsal closure, a process during Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis due to the ability to image embryos in-vivo the genetic tractability of Drosophila. While 140 genes are currently published to affect dorsal closure, new genes are identified each year. In addition our understanding of dorsal closure is far from complete with many questions remaining regarding the molecular mechanisms involved in this complex process. To identify a more complete list of genes involved in dorsal closure, we used a set of large deletions (deficiencies), which collectively remove 98.5% of the genes on the right arm of the 2nd chromosome. Through two crosses, we unambiguously identified homozygous deficiencies and imaged them for the duration of dorsal closure. Images were then analyzed for defects in the cell shapes and morphogenesis. 48 deficiencies were identified to have notable defects on dorsal closure. We anticipate these deficiencies will lead to the identification of at least 31 novel dorsal closure genes. We expect the large number of novel dorsal closure will identify links to pathways already known to coordinate various aspects of closure in addition to new processes and pathways that are currently unidentified as involved in closure.

Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism’s genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. Morphogenesis is a complex orchestration of movements by cells that are specified early in development.

The activation of an upstream GRN is crucial in order to orchestrate downstream morphogenetic events. In the sea urchin, activation of the endomesoderm GRN occurs after the asymmetric 4th cleavage. Embryonic asymmetric cell divisions often are accompanied by differential segregation of fate-determinants into one of two daughter cells. That asymmetric cleavage of the sea urchin micromeres leads to a differential animal-vegetal (A/V) nuclear accumulation of cell fate determinants, β-Catenin and SoxB1. Β-Catenin protein is localized into the nuclei of micromeres and activates the endomesoderm gene regulatory network, while SoxB1 is excluded from micromeres and enters the nucleus of the macromeres, the large progeny of the unequal 4th cleavage. Although nuclear localization of β-Catenin and SoxB1 shows dependence on the asymmetric cleavage, the mechanics behind the asymmetrical division has not been demonstrated. In Chapter 3, we show that micromere formation requires the small RhoGTPase, Cdc42 by directing the apical/basal orientation of the mitotic spindle at the apical cortex. By attenuating or augmenting sea urchin Cdc42 function, micromere divisions became defective and failed to correctly localize asymmetrically distributed determinants. As a consequence, cell fates were altered and multiple A/V axes were produced resulting in a “Siamese-twinning” phenotype that occurred with increasing frequency depending on the quantitative level of perturbation. Our findings show that Cdc42 plays a pivotal role in the asymmetric division of the micromeres, endomesoderm fate-determinant segregation, and A/V axis formation.

This dissertation also characterizes, at high resolution, the repertoire of cellular movements contributing to three different morphogenetic processes of sea urchin development: the elongation of gut, the formation of the primary mouth, and the migration of the small micromeres (the presumptive primordial germ cells) in the sea urchin, Lytechinus variegatus. Descriptive studies of the cellular processes during the different morphogenetic movements allow us to begin investigating their molecular control.

In Chapter 4, we dissected the series of complex events that coordinate gut and mouth morphogenesis. Until now, it was thought that lateral rearrangement of endoderm cells by convergent extension was the main contributor to sea urchin archenteron elongation and that cell divisions were minimal during elongation. We performed cell transplantations to live image and analyze a subset of labeled endoderm cells at high-resolution in the optically clear sea urchin embryo. We found that the endomesoderm cells that initially invaginate into the sea urchin blastocoel remained contiguous throughout extension, so that, if convergent extension were present, it was not a major contributor to elongation. We also found a prevalence of cell divisions throughout archenteron elongation that increased the number of cells within the gut linearly over time; however, we showed that the proliferation did not contribute to growth, and their spindle orientations were randomized during divisions and therefore did not selectively contribute to the final gut length. When cell divisions were inhibited, we saw no difference in the ability of the cells within the gut to migrate in order to elongate. Also in Chapter 4, we describe our observations of the cell biological processes underlying primary mouth formation at the end of gastrulation. Using time-lapse microscopy, photo-convertible Kaede, and an assay of the basement membrane remodeling, we describe a sequential orchestration of events that leads to the fusion of the oral ectoderm and the foregut endoderm. Our work characterizes, at higher resolution than previously recorded, the temporal sequence and repertoire of the cellular movements contributing to the length of the sea urchin larval gut and tissue fusion with the larval primary mouth.

In Chapter 5, the migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a “coherent feed-forward” transcriptional subcircuit composed of Pax6, Six3, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.

The sea urchin gene regulatory network (GRN) describes the cell fate specification of the developing embryo; however, the GRN does not describe specific cell biological events driving the three distinct sequences of cell movements. Our ability to connect the GRN to the morphogenetic events of gastrulation, primary mouth formation, and small micromere migration will provide a framework for characterizing these remarkable sequences of cell movements in the simplest of deuterostome models at an unprecedented scale.

The brain, the source of human cognition, is composed of numerous cell types and a staggering number (over 1 quadrillion) of specialized connections called synapses. Each central nervous system (CNS) synapse is a complex entity organized as a presynaptic axon terminal and a postsynaptic dendritic structure, which pass chemical signals from one neuron to the next. How does each synapse form and how do synapses assemble into organized networks that permit cognition?

Research from the past two decades have identified that glial cells, predominantly astrocytes, regulate the formation and function of CNS synapses. Astrocytes are morphologically complex cells that interact with and ensheathe synapses through fine perisynaptic astrocyte processes. However, the molecular mechanisms of astrocyte development lay largely unknown. Furthermore, it is unclear how the morphology of the astrocyte is linked to its function as a regulator of synapse formation and function. Notwithstanding, more than a dozen astrocyte-secreted factors have been discovered that govern synaptogenesis, including proteins such as thrombospondins, glypicans, and hevin.

Here, I examine the molecular mechanism of astrocyte-synapse interactions and how excitatory synapses are assembled in the CNS using the mouse as a model system. First, I investigated the mechanism of hevin-induced thalamocortical excitatory synapse formation. I find that hevin trans-synaptically binds to two neuronal cell adhesion molecules, neurexin-1 and neuroligin-1B and that this molecular bridge is critical for the formation and plasticity of thalamocortical synapses in the cerebral cortex. Second, I uncover that astrocytes express a family of cell adhesion molecules, the neuroligins, which are critical for governing the developmental morphogenesis of astrocytes. Furthermore, I discover that astrocytic neuroligins are essential to establish the proper balance between synaptic excitation and inhibition in the brain.

Taken together, the data presented here detail two mechanisms of how astrocytes control the assembly of CNS synapses. Moreover, they highlight how bidirectional signals between astrocytes and neurons properly form the brain and its specialized connections. The research performed herein is of high importance to the clinical neuroscience community, because the genes investigated (neuroligins, neurexins, and hevin) are linked to neurological disorders such as autism and schizophrenia. By understanding of how these cells communicate in development and disease, the neuroscience field will hopefully be able to design therapeutics aimed at restoring cognition for patients afflicted with neurological impairments.

Cryptococcus neoformans pathogenesis results from the proliferation of yeast-phase fungal cells within the human host. The Ras1 signal transduction cascade is a major regulator of C. neoformans yeast and hyphal-phase morphogenesis, thermotolerance, and pathogenesis. Previous work identified the conserved Rho-GTPases Cdc42 and Rac1 as potential downstream targets of Ras1. In this work, we identify the duplicate Cdc42 and Rac paralogs, Cdc42 and Cdc420, and Rac1 and Rac2, as major effectors of Ras1-mediated thermotolerance and polarized growth, respectively. Using genetic and molecular biology techniques, including mutant analyses and over-expression studies, we determine the separate and overlapping roles of the four Rho-GTPases in Ras1-mediated morphogenesis. The Cdc42 paralogs are non-essential but are required for thermotolerance and pathogenesis. Ras1 acts through the Cdc42 paralogs to regulate cytokinesis via the organization of septin proteins. The major paralog, Cdc42, and the minor paralog, Cdc420, exhibit functional differences that are primarily dictated by transcriptional regulation. Additionally, CDC42 transcription is induced by exposure to temperature stress conditions. In contrast, Ras1 acts through the equivalently transcribed RAC paralogs to regulate polarized growth during both yeast and hyphal-phase morphogenesis. Rac1 and Rac2 are individually dispensable and appear to be functionally redundant but are synthetically required for yeast phase growth and spore development. The sub-cellular localization of the Rac paralogs is dependent on both Ras1 and post-translational modification by prenyl transferases. The identification and characterization of the conserved elements of the Ras1 signal transduction cascade presented here constitute an important contribution towards the design of anti-fungal agents that are based on existing Ras-pathway inhibitors.

Tip growth in fungi involves highly polarized secretion and modification of the cell wall at the growing tip. The genetic requirements for initiating polarized growth are perhaps best understood for the model budding yeast, Saccharomyces cerevisiae. Once the cell is committed to enter the cell cycle by activation of G1 cyclin/cyclin-dependent kinase (CDK) complexes, the polarity regulator Cdc42 becomes concentrated at the presumptive bud site, actin cables are oriented towards that site, and septin filaments assemble into a ring around the polarity site. Several minutes later, the bud emerges. Here, we investigated the mechanisms that regulate the timing of these events at the single cell level and the role of polarisome during pheromone-induced polarized growth. We employed genetics and live cell microscopy to characterize cellular events. Septin recruitment was delayed relative to polarity establishment, and our findings suggest that a CDK-dependent septin “priming” facilitates septin recruitment by Cdc42. Bud emergence was delayed relative to the initiation of polarized secretion, and our findings suggest that the delay reflects the time needed to weaken the cell wall sufficiently to bud. Rho1 activation by Rom2 occurred at around the time of bud emergence, perhaps in response to local cell wall weakening. This report reveals regulatory mechanisms underlying the morphogenetic events in the budding yeast.

The mouse gonad begins its development as a bipotential primordia, capable of developing into a testis or ovary depending on the presence of the sex-determining gene, Sry. In the XY gonad, opposing pro-testis and pro-ovary pathways compete in gonadal supporting cells. While the individual cellular decision process is well understood, the higher-level process of coordination of cell fates across the gonad remains to be explained. The testis and ovary exhibit distinct patterns of differentiation, suggesting that either development of each organ requires a particular organizing principle or bipotentiality requires regional separation for fate specification or stabilization. The overall goal of this work is to improve characterizations of the spatiotemporal features of sex determination and gonadogenesis, including cell fate organization, morphogenic processes, and system context.Though several hypotheses have connected gonad morphogenesis to sex determination, the morphogenic processes that occur in the gonad have not been sufficiently characterized for formulating testable hypotheses. To capture and analyze the complexity of genital ridge morphogenesis, we generated a 3-dimensional time course of gonad development in native form and context using whole embryo tissue clearing and light sheet microscopy. Analysis revealed that the early gonad exhibits anterior-to-posterior patterns as well as increased rates of growth, rotation, and separation in the central domain. In extending characterization to the neighboring nephric ducts, we found a close alignment of gonad and mesonephric duct movements as well as delayed duct development in Cbx2 mutants, which undergo XY sex reversal and gonad dysgenesis. These data support mechanical integration of gonad and mesonephric duct morphogenesis. In investigating the mechanisms underlying the center-to-pole pattern of testis differentiation, we performed anteroposterior axis analyses and ex vivo gonad reconstruction cultures. These experiments allowed us to rule out two commonly accepted theories in the field: paracrine relay and center-first Sry expression. After searching for patterns in other cellular processes during gonadogenesis, including cell cycle arrest and coelomic epithelium proliferation, we uncovered a center-biased pattern of supporting cell precursor ingression. The updated model indicates that differences between the patterns of differentiation in the testis and ovary are due to features of their respective regulatory networks connecting their fate dynamics to different general gonadal organizing principles acting upstream of supporting cell differentiation. Following recent work on the rete testis and rete ovarii suggesting these structures contribute to gonadal supporting cell populations, we characterized early development of the rete and adjacent tissues in both sexes. Comparison of the GATA4+/PAX8+ presumptive rete with mesonephric and gonadal cells led to the identification of undescribed patterns in mesonephros development which may play a role in sexual dimorphism of the rete. Cells of the rete may derive from mesonephric condensates in a process similar to kidney nephron development. Cell cycle analysis revealed the mesonephric tubules and early rete to be a largely non-proliferating population of cells, suggesting expansion through recruitment of new cells. These results were used to establish preliminary theories for lineage relationships in early urogenital development. Initial attempts at lineage tracing to test the theory were unsuccessful. The findings presented here contribute to a more comprehensive and systems level understanding of sex determination and gonad development. In particular, the incorporation of high-resolution spatial information into theories of sex determination serves to connect individual cell fate decisions to organ level patterns of differentiation in space and time. These results will be useful for novel hypothesis generation as well as for designing more detailed models and simulations of sex determination and gonadogenesis.

Organismal form emerges from the relative growth of the body and its parts. In this dissertation, I address how developmental processes produce the size relationships between body parts that shape the characteristic morphologies of animals and plants. Specifically, I study the nutrition-dependent growth of wings in butterflies and moths to determine the developmental physiological mechanisms underlying morphological scaling relationships. Additionally, I dive into how form within a tissue is established by studying the mechanism that patterns growth of butterfly wings. Using an extensive data set from rats fed on a low protein diet, I analyze how different bones respond to nutritional variation and its effect on morphological integration. Finally, I end with a theoretical chapter discussing the ways in which development can be plastic and how that shapes both genetic and phenotypic evolution. I find that developmental plasticity emerges from changes in the concentration of systemic hormones in response to environmental stimuli. Hormones, in turn, interact with every developing part. This interaction is characterized by character-specific gene regulatory networks that affect their sensitivity to hormones. Thus, developmental plasticity emerges from the interplay between the organism sensing the environment that tunes the strength of a systemic signal that moderates development, and character-specific use of molecular networks that defines the range of character states in response to signal variation.

Fungal cells sense the amount and quality of external nutrients through multiple interconnected signaling networks, which allow them to adjust their metabolism, transcriptional profiles and developmental programs to adapt readily and appropriately to changing nutritional states. In organisms ranging from yeasts to humans, the Tor signaling pathway responds to nutrient-derived signals and orchestrates cell growth. While in the baker's yeast Saccharomyces cerevisiae Tor responds to nutrient-derived signals and orchestrates cell growth and proliferation, in Schizosaccharomyces pombe Tor signaling modulates sexual differentiation in response to nutritional cues. Thus, these differences provide a framework to consider the roles of Tor in other fungal organisms, in particular those that are pathogens of humans.

In this dissertation, I demonstrate that in the human fungal pathogen Candida albicans, Tor signaling also functions to promote growth. This study also uncovered a novel role for the Tor molecular pathway in promoting hyphal growth of C. albicans on semi-solid surfaces and in controlling cell-cell adherence. Gene expression analysis and genetic manipulations identified several transcriptional regulators (Bcr1, Efg1, Nrg1, and Tup1) that together with Tor compose a regulatory network governing adhesin gene expression and cellular adhesion. While the Tor kinases are broadly conserved, these studies further demonstrate the contrasting strategies employed by fungal organism in utilizing the Tor signaling cascade.

While extensive studies have focused on elucidating functions for the Tor signaling cascades among ascomycetes, little is known about the pathway in basal fungal lineages, in particular among zygomycetes and chytrids. Moreover, given that the Tor pathway is the target of several small molecule inhibitors including rapamycin, a versatile pharmacological drug used in medicine, there is considerable interest in Tor signaling pathways and their function. Capitalizing on emerging genome sequences now available for several basal fungal species, we show a remarkable pattern of conservation, duplication, and loss of the Tor signaling cascade among basal fungal lineages. Targeting the pathway with rapamycin results in growth arrest of several zygomycete species, indicating a conserved role for this pathway in regulating fungal growth. In addition, we show a potential therapeutic advantage of using rapamycin in a heterologous model of zygomycosis. Taken together, the Tor signaling cascade and its inhibitors provide robust platforms from which to develop novel antimicrobial therapies, which may include less immunosuppressive rapamycin analogs.

During development, morphogenetic processes require the integration of numerous signals to properly shape cells and tissues. These signals are interpreted by cells to induce the precise transcriptional circuitry that controls morphogenesis. In the fly embryonic epidermis, the fly Wnt, wingless (wg), generates naked cuticle zones that separate the ventral denticle belts by repressing expression of shavenbaby (svb), which encodes a transcription factor required for denticle formation. What is not known is how Wg and Svb interact to produce the stereotyped diversity of denticle shapes within each belt. One possibility is that graded levels of Svb, responding to graded levels of Wg signaling, determine denticle shape. Indeed, we found that the svb promoter responds differentially to altered Wg activity levels. However, artificially increasing the levels of ectopic svb does not produce morphologically distinct denticles, suggesting that additional factors are involved. We have discovered that a second Wg-responsive transcription factor, encoded by SoxNeuro (SoxN), cooperates with Svb to shape the denticles. Co-expressing SoxN with svb is sufficient to rescue the morphology of denticles in an ectopic location. Furthermore, embryos that lack Svb activity retain the ability to produce a small number of rounded, reduced ventral denticles, due to SoxN activity. We found that svb;SoxN double mutant embryos secrete cuticles that completely lack ventral denticles and dorsal hairs. We also found that a group of known Svb target genes belonging to the zona pellucida family of proteins are differentially activated by SoxN. Finally, we discovered two novel target genes of SoxN, CG16885 and CG30101, which are expressed in denticle-producing cells and which are regulated independently of Svb. SoxN was shown previously to down-regulate Wg signaling and to promote expression of svb. Here, we propose that SoxN acts with Svb, in an additional, direct role, to promote denticle morphogenesis.