A comparative analysis of gene and protein expression in chronic and acute models of photoreceptor degeneration in adult zebrafish.

Background: Adult zebrafish are capable of photoreceptor (PR) regeneration following acute phototoxic lesion (AL). We developed a chronic low light (CLL) exposure model that more accurately reflects chronic PR degeneration observed in many human retinal diseases. Methods: Here, we characterize the morphological and transcriptomic changes associated with acute and chronic models of PR degeneration at 8 time-points over a 28-day window using immunohistochemistry and 3'mRNA-seq. Results: We first observed a differential sensitivity of rod and cone PRs to CLL. Next, we found no evidence for Müller glia (MG) gliosis or regenerative cell-cycle re-entry in the CLL model, which is in contrast to the robust gliosis and proliferative response from resident MG in the AL model. Differential responses of microglia between the models was also observed. Transcriptomic comparisons between the models revealed gene-specific networks of PR regeneration and degeneration, including genes that are activated under conditions of chronic PR stress. Finally, we showed that CLL is at least partially reversible, allowing for rod and cone outer segment outgrowth and replacement of rod cell nuclei via an apparent upregulation of the existing rod neurogenesis mechanism. Discussion: Collectively, these data provide a direct comparison of the morphological and transcriptomic PR degeneration and regeneration models in zebrafish.

Probing the conserved roles of cut in the development and function of optically different insect compound eyes.

Astonishing functional diversity exists among arthropod eyes, yet eye development relies on deeply conserved genes. This phenomenon is best understood for early events, whereas fewer investigations have focused on the influence of later transcriptional regulators on diverse eye organizations and the contribution of critical support cells, such as Semper cells (SCs). As SCs in Drosophila melanogaster secrete the lens and function as glia, they are critical components of ommatidia. Here, we perform RNAi-based knockdowns of the transcription factor cut (CUX in vertebrates), a marker of SCs, the function of which has remained untested in these cell types. To probe for the conserved roles of cut, we investigate two optically different compound eyes: the apposition optics of D. melanogaster and the superposition optics of the diving beetle Thermonectus marmoratus. In both cases, we find that multiple aspects of ocular formation are disrupted, including lens facet organization and optics as well as photoreceptor morphogenesis. Together, our findings support the possibility of a generalized role for SCs in arthropod ommatidial form and function and introduces Cut as a central player in mediating this role.

Global Gene Expression Analysis Reveals Complex Cuticle Organization of the Tribolium Compound Eye.

The red flour beetle Tribolium castaneum is a resource-rich model for genomic and developmental studies. To extend previous studies on Tribolium eye development, we produced transcriptomes for normal-eyed and eye-depleted heads of pupae and adults to identify differentially transcript-enriched (DE) genes in the visual system. Unexpectedly, cuticle-related genes were the largest functional class in the pupal compound eye DE gene population, indicating differential enrichment in three distinct cuticle components: clear lens facet cuticle, highly melanized cuticle of the ocular diaphragm, which surrounds the Tribolium compound eye for internal fortification, and newly identified facet margins of the tanned cuticle, possibly enhancing external fortification. Phylogenetic, linkage, and high-throughput gene knockdown data suggest that most cuticle proteins (CPs) expressed in the Tribolium compound eye stem from the deployment of ancient CP genes. Consistent with this, TcasCPR15, which we identified as the major lens CP gene in Tribolium, is a beetle-specific but pleiotropic paralog of the ancient CPR RR-2 CP gene family. The less abundant yet most likely even more lens-specific TcasCP63 is a member of a sprawling family of noncanonical CP genes, documenting a role of local gene family expansions in the emergence of the Tribolium compound eye CP repertoire. Comparisons with Drosophila and the mosquito Anopheles gambiae reveal a steady turnover of lens-enriched CP genes during insect evolution.

EyeVolve, a modular PYTHON based model for simulating developmental eye type diversification.

Vision is among the oldest and arguably most important sensory modalities for animals to interact with their external environment. Although many different eye types exist within the animal kingdom, mounting evidence indicates that the genetic networks required for visual system formation and function are relatively well conserved between species. This raises the question as to how common developmental programs are modified in functionally different eye types. Here, we approached this issue through EyeVolve, an open-source PYTHON-based model that recapitulates eye development based on developmental principles originally identified in Drosophila melanogaster. Proof-of-principle experiments showed that this program's animated timeline successfully simulates early eye tissue expansion, neurogenesis, and pigment cell formation, sequentially transitioning from a disorganized pool of progenitor cells to a highly organized lattice of photoreceptor clusters wrapped with support cells. Further, tweaking just five parameters (precursor pool size, founder cell distance and placement from edge, photoreceptor subtype number, and cell death decisions) predicted a multitude of visual system layouts, reminiscent of the varied eye types found in larval and adult arthropods. This suggests that there are universal underlying mechanisms that can explain much of the existing arthropod eye diversity. Thus, EyeVolve sheds light on common principles of eye development and provides a new computational system for generating specific testable predictions about how development gives rise to diverse visual systems from a commonly specified neuroepithelial ground plan.

Semper's cells in the insect compound eye: Insights into ocular form and function.

The arthropod compound eye represents one of two major eye types in the animal kingdom and has served as an essential experimental paradigm for defining fundamental mechanisms underlying sensory organ formation, function, and maintenance. One of the most distinguishing features of the compound eye is the highly regular array of lens facets that define individual eye (ommatidial) units. These lens facets are produced by a deeply conserved quartet of cuticle-secreting cells, called Semper cells (SCs). Also widely known as cone cells, SCs were originally identified for their secretion of the dioptric system, i.e. the corneal lens and underlying crystalline cones. Additionally, SCs are now known to execute a diversity of patterning and glial functions in compound eye development and maintenance. Here, we present an integrated account of our current knowledge of SC multifunctionality in the Drosophila compound eye, highlighting emerging gene regulatory modules that may drive the diverse roles for these cells. Drawing comparisons with other deeply conserved retinal glia in the vertebrate single lens eye, this discussion speaks to glial cell origins and opens new avenues for understanding sensory system support programs.

Development and characterization of a chronic photoreceptor degeneration model in adult zebrafish that does not trigger a regenerative response.

Zebrafish (Danio rerio) have become a highly-utilized model system in the field of regenerative biology because of their endogenous ability to regenerate many tissues and organs, including the retina. The vast majority of previous research on retinal regeneration in adult zebrafish utilizes acute methodologies for retinal damage. Acute retinal cell death triggers a reactive gliosis response of Müller glia (MG), the resident macroglia of the retina. In addition, each activated MG undergoes asymmetric cell division to produce a neuronal progenitor, which continues to divide and ultimately gives rise to new retinal neurons. Studies using these approaches have uncovered many crucial mechanisms by which MG respond to acute damage. However, they may not adequately mimic the chronic neuronal degeneration observed in many human retinal degenerative diseases. The current study aimed to develop a new long-term, chronic photoreceptor damage and degeneration model in adult zebrafish. Comparing the subsequent cellular responses to that of the commonly-used acute high-intensity model, we found that low, continuous light exposure damaged the outer segments of both rod and cone photoreceptors, but did not result in significant apoptotic cell death, MG gliosis, or MG cell-cycle re-entry. Instead, chronic light nearly completely truncated photoreceptor outer segments and resulted in a recruitment of microglia to the area. Together, these studies present a chronic photoreceptor model that can be performed in a relatively short time frame (21 days), that may lend insight into the cellular events underlying non-regenerative photoreceptor degeneration observed in other model systems.

Opposing transcriptional and post-transcriptional roles for Scalloped in binary Hippo-dependent neural fate decisions.

The Hippo tumor suppressor pathway plays many fundamental cell biological roles during animal development. Two central players in controlling Hippo-dependent gene expression are the TEAD transcription factor Scalloped (Sd) and its transcriptional co-activator Yorkie (Yki). Hippo signaling phosphorylates Yki, thereby blocking Yki-dependent transcriptional control. In post-mitotic Drosophila photoreceptors, a bistable negative feedback loop forms between the Hippo-dependent kinase Warts/Lats and Yki to lock in green vs blue-sensitive neuronal subtype choices, respectively. Previous experiments indicate that sd and yki mutants phenocopy each other's functions, both being required for promoting the expression of the blue photoreceptor fate determinant melted (melt) and the blue-sensitive opsin Rh5. Here, we demonstrate that Sd ensures the robustness of this neuronal fate decision via multiple antagonistic gene regulatory roles. In Hippo-positive (green) photoreceptors, Sd directly represses both melt and Rh5 gene expression through defined TEAD binding sites, a mechanism that is antagonized by Yki in Hippo-negative (blue) cells. Additionally, in blue photoreceptors, Sd is required to promote the translation of the Rh5 protein through a 3'UTR-dependent and microRNA-mediated process. Together, these studies reveal that Sd can drive context-dependent cell fate decisions through opposing transcriptional and post-transcriptional mechanisms.

A Complex Lens for a Complex Eye.

A key innovation for high resolution eyes is a sophisticated lens that precisely focuses light onto photoreceptors. The eyes of holometabolous larvae range from very simple eyes that merely detect light to eyes that are capable of high spatial resolution. Particularly interesting are the bifocal lenses of Thermonectus marmoratus larvae, which differentially focus light on spectrally-distinct retinas. While functional aspects of insect lenses have been relatively well studied, little work has explored their molecular makeup, especially in regard to more complex eye types. To investigate this question, we took a transcriptomic and proteomic approach to identify the major proteins contributing to the principal bifocal lenses of T. marmoratus larvae. Mass spectrometry revealed 10 major lens proteins. Six of these share sequence homology with cuticular proteins, a large class of proteins that are also major components of corneal lenses from adult compound eyes of Drosophila melanogaster and Anopheles gambiae. Two proteins were identified as house-keeping genes and the final two lack any sequence homologies to known genes. Overall the composition seems to follow a pattern of co-opting transparent and optically dense proteins, similar to what has been described for other animal lenses. To identify cells responsible for the secretion of specific lens proteins, we performed in situ hybridization studies and found some expression differences between distal and proximal corneagenous cells. Since the distal cells likely give rise to the periphery and the proximal cells to the center of the lens, our findings highlight a possible mechanism for establishing structural differences that are in line with the bifocal nature of these lenses. A better understanding of lens composition provides insights into the evolution of proper focusing, which is an important step in the transition between low-resolution and high-resolution eyes.

Multifunctional glial support by Semper cells in the Drosophila retina.

Glial cells play structural and functional roles central to the formation, activity and integrity of neurons throughout the nervous system. In the retina of vertebrates, the high energetic demand of photoreceptors is sustained in part by Müller glia, an intrinsic, atypical radial glia with features common to many glial subtypes. Accessory and support glial cells also exist in invertebrates, but which cells play this function in the insect retina is largely undefined. Using cell-restricted transcriptome analysis, here we show that the ommatidial cone cells (aka Semper cells) in the Drosophila compound eye are enriched for glial regulators and effectors, including signature characteristics of the vertebrate visual system. In addition, cone cell-targeted gene knockdowns demonstrate that such glia-associated factors are required to support the structural and functional integrity of neighboring photoreceptors. Specifically, we show that distinct support functions (neuronal activity, structural integrity and sustained neurotransmission) can be genetically separated in cone cells by down-regulating transcription factors associated with vertebrate gliogenesis (pros/Prox1, Pax2/5/8, and Oli/Olig1,2, respectively). Further, we find that specific factors critical for glial function in other species are also critical in cone cells to support Drosophila photoreceptor activity. These include ion-transport proteins (Na/K+-ATPase, Eaat1, and Kir4.1-related channels) and metabolic homeostatic factors (dLDH and Glut1). These data define genetically distinct glial signatures in cone/Semper cells that regulate their structural, functional and homeostatic interactions with photoreceptor neurons in the compound eye of Drosophila. In addition to providing a new high-throughput model to study neuron-glia interactions, the fly eye will further help elucidate glial conserved "support networks" between invertebrates and vertebrates.

The cuticular nature of corneal lenses in Drosophila melanogaster.

The dioptric visual system relies on precisely focusing lenses that project light onto a neural retina. While the proteins that constitute the lenses of many vertebrates are relatively well characterized, less is known about the proteins that constitute invertebrate lenses, especially the lens facets in insect compound eyes. To address this question, we used mass spectrophotometry to define the major proteins that comprise the corneal lenses from the adult Drosophila melanogaster compound eye. This led to the identification of four cuticular proteins: two previously identified lens proteins, drosocrystallin and retinin, and two newly identified proteins, Cpr66D and Cpr72Ec. To determine which ommatidial cells contribute each of these proteins to the lens, we conducted in situ hybridization at 50% pupal development, a key age for lens secretion. Our results confirm previous reports that drosocrystallin and retinin are expressed in the two primary corneagenous cells-cone cells and primary pigment cells. Cpr72Ec and Cpr66D, on the other hand, are more highly expressed in higher order interommatidial pigment cells. These data suggest that the complementary expression of cuticular proteins give rise to the center vs periphery of the corneal lens facet, possibly facilitating a refractive gradient that is known to reduce spherical aberration. Moreover, these studies provide a framework for future studies aimed at understanding the cuticular basis of corneal lens function in holometabolous insect eyes.

Rhomboid Enhancer Activity Defines a Subset of Drosophila Neural Precursors Required for Proper Feeding, Growth and Viability.

Organismal growth regulation requires the interaction of multiple metabolic, hormonal and neuronal pathways. While the molecular basis for many of these are well characterized, less is known about the developmental origins of growth regulatory structures and the mechanisms governing control of feeding and satiety. For these reasons, new tools and approaches are needed to link the specification and maturation of discrete cell populations with their subsequent regulatory roles. In this study, we characterize a rhomboid enhancer element that selectively labels four Drosophila embryonic neural precursors. These precursors give rise to the hypopharyngeal sensory organ of the peripheral nervous system and a subset of neurons in the deutocerebral region of the embryonic central nervous system. Post embryogenesis, the rhomboid enhancer is active in a subset of cells within the larval pharyngeal epithelium. Enhancer-targeted toxin expression alters the morphology of the sense organ and results in impaired larval growth, developmental delay, defective anterior spiracle eversion and lethality. Limiting the duration of toxin expression reveals differences in the critical periods for these effects. Embryonic expression causes developmental defects and partially penetrant pre-pupal lethality. Survivors of embryonic expression, however, ultimately become viable adults. In contrast, post-embryonic toxin expression results in fully penetrant lethality. To better define the larval growth defect, we used a variety of assays to demonstrate that toxin-targeted larvae are capable of locating, ingesting and clearing food and they exhibit normal food search behaviors. Strikingly, however, following food exposure these larvae show a rapid decrease in consumption suggesting a satiety-like phenomenon that correlates with the period of impaired larval growth. Together, these data suggest a critical role for these enhancer-defined lineages in regulating feeding, growth and viability.

Hypo- or hyper-hippo: a balancing act with bHLH transcription factors.

Balancing cell growth with differentiation is essential for tissue integrity. In this issue of Developmental Cell, Wang and Baker (2015) demonstrate unsuspected cross-talk between bHLH transcription factors, important regulators of organogenesis, with the Hippo tumor suppressor pathway to ensure that inappropriately differentiating cells are eliminated during development.

Integration of an abdominal Hox complex with Pax2 yields cell-specific EGF secretion from Drosophila sensory precursor cells.

Cis-regulatory modules (CRMs) ensure specific developmental outcomes by mediating both proper spatiotemporal gene expression patterns and appropriate transcriptional levels. In Drosophila, the precise transcriptional control of the serine protease rhomboid regulates EGF signaling to specify distinct cell types. Recently, we identified a CRM that activates rhomboid expression and thereby EGF secretion from a subset of abdominal sensory organ precursor cells (SOPs) to induce an appropriate number of lipid-processing cells called oenocytes. Here, we use scanning mutagenesis coupled with reporter assays, biochemistry and genetics to dissect the transcriptional mechanisms regulating SOP-specific rhomboid activation. Our results show that proper spatial activity of the rhomboid CRM is dependent upon direct integration of the abdomen-specific Hox factor Abdominal-A and the SOP-restricted Pax2 factor. In addition, we show that the Extradenticle and Homothorax Hox co-factors are differentially integrated on the rhomboid CRM by abdominal versus thoracic Hox proteins in the presence of Pax2. Last, we show that Abdominal-A uses both Pax2-dependent and Pax2-independent mechanisms to stimulate rhomboid CRM activity to induce proper oenocyte numbers. Thus, these data demonstrate how a CRM integrates Hox and neural transcriptional inputs to regulate the appropriate spatial pattern and levels of EGF secretion to specify an essential cell fate.

The lens in focus: a comparison of lens development in Drosophila and vertebrates.

The evolution of the eye has been a major subject of study dating back centuries. The advent of molecular genetics offered the surprising finding that morphologically distinct eyes rely on conserved regulatory gene networks for their formation. While many of these advances often stemmed from studies of the compound eye of the fruit fly, Drosophila melanogaster, and later translated to discoveries in vertebrate systems, studies on vertebrate lens development far outnumber those in Drosophila. This may be largely historical, since Spemann and Mangold's paradigm of tissue induction was discovered in the amphibian lens. Recent studies on lens development in Drosophila have begun to define molecular commonalities with the vertebrate lens. Here, we provide an overview of Drosophila lens development, discussing intrinsic and extrinsic factors controlling lens cell specification and differentiation. We then summarize key morphological and molecular events in vertebrate lens development, emphasizing regulatory factors and networks strongly associated with both systems. Finally, we provide a comparative analysis that highlights areas of research that would help further clarify the degree of conservation between the formation of dioptric systems in invertebrates and vertebrates.

Molecular evidence for color discrimination in the Atlantic sand fiddler crab, Uca pugilator.

Fiddler crabs are intertidal brachyuran crabs that belong to the genus Uca. Approximately 97 different species have been identified, and several of these live sympatrically. Many have species-specific body color patterns that may act as signals for intra- and interspecific communication. To understand the behavioral and ecological role of this coloration we must know whether fiddler crabs have the physiological capacity to perceive color cues. Using a molecular approach, we identified the opsin-encoding genes and determined their expression patterns across the eye of the sand fiddler crab, Uca pugilator. We identified three different opsin-encoding genes (UpRh1, UpRh2 and UpRh3). UpRh1 and UpRh2 are highly related and have similarities in their amino acid sequences to other arthropod long- and medium-wavelength-sensitive opsins, whereas UpRh3 is similar to other arthropod UV-sensitive opsins. All three opsins are expressed in each ommatidium, in an opsin-specific pattern. UpRh3 is present only in the R8 photoreceptor cell, whereas UpRh1 and UpRh2 are present in the R1-7 cells, with UpRh1 expression restricted to five cells and UpRh2 expression present in three cells. Thus, one photoreceptor in every ommatidium expresses both UpRh1 and UpRh2, providing another example of sensory receptor coexpression. These results show that U. pugilator has the basic molecular machinery for color perception, perhaps even trichromatic vision.

Building a fly eye: terminal differentiation events of the retina, corneal lens, and pigmented epithelia.

In the past, vast differences in ocular structure, development, and physiology throughout the animal kingdom led to the widely accepted notion that eyes are polyphyletic, that is, they have independently arisen multiple times during evolution. Despite the dissimilarity between vertebrate and invertebrate eyes, it is becoming increasingly evident that the development of the eye in both groups shares more similarity at the genetic level than was previously assumed, forcing a reexamination of eye evolution. Understanding the molecular underpinnings of cell type specification during Drosophila eye development has been a focus of research for many labs over the past 25 years, and many of these findings are nicely reviewed in Chapters 1 and 4. A somewhat less explored area of research, however, considers how these cells, once specified, develop into functional ocular structures. This review aims to summarize the current knowledge related to the terminal differentiation events of the retina, corneal lens, and pigmented epithelia in the fly eye. In addition, we discuss emerging evidence that the different functional components of the fly eye share developmental pathways and functions with the vertebrate eye.

Proneural and abdominal Hox inputs synergize to promote sensory organ formation in the Drosophila abdomen.

The atonal (ato) proneural gene specifies a stereotypic number of sensory organ precursors (SOP) within each body segment of the Drosophila ectoderm. Surprisingly, the broad expression of Ato within the ectoderm results in only a modest increase in SOP formation, suggesting many cells are incompetent to become SOPs. Here, we show that the SOP promoting activity of Ato can be greatly enhanced by three factors: the Senseless (Sens) zinc finger protein, the Abdominal-A (Abd-A) Hox factor, and the epidermal growth factor (EGF) pathway. First, we show that expression of either Ato alone or with Sens induces twice as many SOPs in the abdomen as in the thorax, and do so at the expense of an abdomen-specific cell fate: the larval oenocytes. Second, we demonstrate that Ato stimulates abdominal SOP formation by synergizing with Abd-A to promote EGF ligand (Spitz) secretion and secondary SOP recruitment. However, we also found that Ato and Sens selectively enhance abdominal SOP development in a Spitz-independent manner, suggesting additional genetic interactions between this proneural pathway and Abd-A. Altogether, these experiments reveal that genetic interactions between EGF-signaling, Abd-A, and Sens enhance the SOP-promoting activity of Ato to stimulate region-specific neurogenesis in the Drosophila abdomen.

Separable transcriptional regulatory domains within Otd control photoreceptor terminal differentiation events.

Orthodenticle (Otd)-related transcription factors are essential for anterior patterning and brain morphogenesis from Cnidaria to Mammals, and genetically underlie several human retinal pathologies. Despite their key developmental functions, relatively little is known regarding the molecular basis of how these factors regulate downstream effectors in a cell- or tissue-specific manner. Many invertebrate and vertebrate species encode two to three Otd proteins, whereas Drosophila encodes a single Otd protein. In the fly retina, Otd controls rhabdomere morphogenesis of all photoreceptors and regulates distinct Rhodopsin-encoding genes in a photoreceptor subtype-specific manner. Here, we performed a structure-function analysis of Otd during Drosophila eye development using in vivo rescue experiments and in vitro transcriptional regulatory assays. Our findings indicate that Otd requires at least three distinct transcriptional regulatory domains to control photoreceptor-specific rhodopsin gene expression and photoreceptor morphogenesis. Our results also uncover a previously unknown role for Otd in preventing co-expression of sensory receptors in blue vs. green-sensitive R8 photoreceptors. Sequence analysis indicates that many of the transcriptional regulatory domains identified here are conserved in multiple Diptera Otd-related proteins. Thus, these studies provide a basis for identifying shared molecular pathways involved in a wide range of developmental processes.

Pph13 and orthodenticle define a dual regulatory pathway for photoreceptor cell morphogenesis and function.

The function and integrity of photoreceptor cells are dependent upon the creation and maintenance of specialized apical structures: membrane discs/outer segments in vertebrates and rhabdomeres in insects. We performed a molecular and morphological comparison of Drosophila Pph13 and orthodenticle (otd) mutants to investigate the transcriptional network controlling the late stages of rhabdomeric photoreceptor cell development and function. Although Otd and Pph13 have been implicated in rhabdomere morphogenesis, we demonstrate that it is necessary to remove both factors to completely eliminate rhabdomere formation. Rhabdomere absence is not the result of degeneration or a failure of initiation, but rather the inability of the apical membrane to transform and elaborate into a rhabdomere. Transcriptional profiling revealed that Pph13 plays an integral role in promoting rhabdomeric photoreceptor cell function. Pph13 regulates Rh2 and Rh6, and other phototransduction genes, demonstrating that Pph13 and Otd control a distinct subset of Rhodopsin-encoding genes in adult visual systems. Bioinformatic, DNA binding and transcriptional reporter assays showed that Pph13 can bind and activate transcription via a perfect Pax6 homeodomain palindromic binding site and the Rhodopsin core sequence I (RCSI) found upstream of Drosophila Rhodopsin genes. In vivo studies indicate that Pph13 is necessary and sufficient to mediate the expression of a multimerized RCSI reporter, a marker of photoreceptor cell specificity previously suggested to be regulated by Pax6. Our studies define a key transcriptional regulatory pathway that is necessary for late Drosophila photoreceptor development and will serve as a basis for better understanding rhabdomeric photoreceptor cell development and function.

Atonal, Senseless, and Abdominal-A regulate rhomboid enhancer activity in abdominal sensory organ precursors.

The atonal (ato) proneural gene specifies different numbers of sensory organ precursor (SOP) cells within distinct regions of the Drosophila embryo in an epidermal growth factor-dependent manner through the activation of the rhomboid (rho) protease. How ato activates rho, and why it does so in only a limited number of sensory cells remains unclear. We previously identified a rho enhancer (RhoBAD) that is active within a subset of abdominal SOP cells to induce larval oenocytes and showed that RhoBAD is regulated by an Abdominal-A (Abd-A) Hox complex and the Senseless (Sens) transcription factor. Here, we show that ato is also required for proper RhoBAD activity and oenocyte formation. Transgenic reporter assays reveal RhoBAD contains two conserved regions that drive SOP gene expression: RhoD mediates low levels of expression in both thoracic and abdominal SOP cells, whereas RhoA drives strong expression within abdominal SOP cells. Ato indirectly stimulates both elements and enhances RhoA reporter activity by interfering with the ability of the Sens repressor to bind DNA. As RhoA is also directly regulated by Abd-A, we propose a model for how the Ato and Sens proneural factors are integrated with an abdominal Hox factor to regulate region-specific SOP gene expression.