Huntingtin Plays a Role in the Physiological Response to Ethanol in Drosophila.

BACKGROUND: Huntingtin (htt) protein is an essential regulator of nervous system function through its various neuroprotective and pro-survival functions, and loss of wild-type htt function is implicated in the etiology of Huntington's disease. While its pathological role is typically understood as a toxic gain-of-function, some neuronal phenotypes also result from htt loss. Therefore, it is important to understand possible roles for htt in other physiological circumstances. OBJECTIVE: To elucidate the role of htt in the context of ethanol exposure, we investigated how loss of htt impacts behavioral and physiological responses to ethanol in Drosophila. METHODS: We tested flies lacking htt for ethanol sensitivity and tolerance, preference for ethanol using capillary feeder assays, and recovery of mobility after intoxication. Levels of dopamine neurotransmitter and numbers of dopaminergic cells in brains lacking dhtt were also measured. RESULTS: We found that dhtt-null flies are both less sensitive and more tolerant to ethanol exposure in adulthood. Moreover, flies lacking dhtt are more averse to alcohol than controls, and they recover mobility faster following acute ethanol intoxication. We showed that dhtt mediates these effects at least in part through the dopaminergic system, as dhtt is required to maintain normal levels of dopamine in the brain and normal numbers of dopaminergic cells in the adult protocerebrum. CONCLUSIONS: Our results demonstrate that htt regulates the physiological response to ethanol and indicate a novel neuroprotective role for htt in the dopaminergic system, raising the possibility that it may be involved more generally in the response to toxic stimuli.

Correction: Dilp-2-mediated PI3-kinase activation coordinates reactivation of quiescent neuroblasts with growth of their glial stem cell niche.

[This corrects the article DOI: 10.1371/journal.pbio.3000721.].

Dilp-2-mediated PI3-kinase activation coordinates reactivation of quiescent neuroblasts with growth of their glial stem cell niche.

Dietary nutrients provide macromolecules necessary for organism growth and development. In response to animal feeding, evolutionarily conserved growth signaling pathways are activated, leading to increased rates of cell proliferation and tissue growth. It remains unclear how different cell types within developing tissues coordinate growth in response to dietary nutrients and whether coordinated growth of different cell types is necessary for proper tissue function. Using the early Drosophila larval brain, we asked whether nutrient-dependent growth of neural stem cells (neuroblasts), glia, and trachea is coordinated and whether coordinated growth among these major brain cell types is required for neural development. It is known that in response to dietary nutrients and PI3-kinase activation, brain and ventral nerve cord neuroblasts reactivate from quiescence and ventral nerve cord glia expand their membranes. Here, we assay growth in a cell-type specific manner at short time intervals in the brain and determine that growth is coordinated among different cell types and that coordinated growth is mediated in part through activation of PI3-kinase signaling. Of the 7 Drosophila insulin-like peptides (Dilps), we find that Dilp-2 is required for PI3-kinase activation and growth coordination between neuroblasts and glia in the brain. Dilp-2 induces brain cortex glia to initiate membrane growth and make first contact with quiescent neuroblasts. Once reactivated, neuroblasts promote cortex glia growth to ultimately form a selective membrane barrier. Our results highlight the importance of bidirectional growth signaling between neural stem cells and surrounding cell types in the brain in response to nutrition and demonstrate how coordinated growth among different cell types drives tissue morphogenesis and function.

Eyeless uncouples mushroom body neuroblast proliferation from dietary amino acids in Drosophila.

Cell proliferation is coupled with nutrient availability. If nutrients become limited, proliferation ceases, because growth factor and/or PI3-kinase activity levels become attenuated. Here, we report an exception to this generality within a subpopulation of Drosophila neural stem cells (neuroblasts). We find that most neuroblasts enter and exit cell cycle in a nutrient-dependent manner that is reversible and regulated by PI3-kinase. However, a small subset, the mushroom body neuroblasts, which generate neurons important for memory and learning, divide independent of dietary nutrient conditions and PI3-kinase activity. This nutrient-independent proliferation is regulated by Eyeless, a Pax-6 orthologue, expressed in mushroom body neuroblasts. When Eyeless is knocked down, mushroom body neuroblasts exit cell cycle when nutrients are withdrawn. Conversely, when Eyeless is ectopically expressed, some non-mushroom body neuroblasts divide independent of dietary nutrient conditions. Therefore, Eyeless uncouples MB neuroblast proliferation from nutrient availability, allowing preferential neurogenesis in brain subregions during nutrient poor conditions.

Developmental regulation of planar cell polarity and hair-bundle morphogenesis in auditory hair cells: lessons from human and mouse genetics.

Hearing loss is the most common and costly sensory defect in humans and genetic causes underlie a significant proportion of affected individuals. In mammals, sound is detected by hair cells (HCs) housed in the cochlea of the inner ear, whose function depends on a highly specialized mechanotransduction organelle, the hair bundle. Understanding the factors that regulate the development and functional maturation of the hair bundle is crucial for understanding the pathophysiology of human deafness. Genetic analysis of deafness genes in animal models, together with complementary forward genetic screens and conditional knock-out mutations in essential genes, have provided great insights into the molecular machinery underpinning hair-bundle development and function. In this review, we highlight recent advances in our understanding of hair-bundle morphogenesis, with an emphasis on the molecular pathways governing hair-bundle polarity and orientation. We next discuss the proteins and structural elements important for hair-cell mechanotransduction as well as hair-bundle cohesion and maintenance. In addition, developmental signals thought to regulate tonotopic features of HCs are introduced. Finally, novel approaches that complement classic genetics for studying the molecular etiology of human deafness are presented. WIREs Dev Biol 2016, 5:85-101. doi: 10.1002/wdev.202 For further resources related to this article, please visit the WIREs website.

Lis1 mediates planar polarity of auditory hair cells through regulation of microtubule organization.

The V-shaped hair bundles atop auditory hair cells and their uniform orientation are manifestations of epithelial planar cell polarity (PCP) required for proper perception of sound. PCP is regulated at the tissue level by a conserved core Wnt/PCP pathway. However, the hair cell-intrinsic polarity machinery is poorly understood. Recent findings implicate hair cell microtubules in planar polarization of hair cells. To elucidate the microtubule-mediated polarity pathway, we analyzed Lis1 function in the auditory sensory epithelium in the mouse. We show that conditional deletion of Lis1 in developing hair cells causes defects in cytoplasmic dynein and microtubule organization, resulting in planar polarity defects without overt effects on the core PCP pathway. Lis1 ablation during embryonic development results in defects in hair bundle morphology and orientation, cellular organization and junctional nectin localization. We present evidence that Lis1 regulates localized Rac-PAK signaling in embryonic hair cells, probably through microtubule-associated Tiam1, a guanine nucleotide exchange factor for Rac. Lis1 ablation in postnatal hair cells significantly disrupts centrosome anchoring and the normal V-shape of hair bundles, accompanied by defects in the pericentriolar matrix and microtubule organization. Lis1 is also required for proper positioning of the Golgi complex and mitochondria as well as for hair cell survival. Together, our results demonstrate that Lis1 mediates the planar polarity of hair cells through regulation of microtubule organization downstream of the tissue polarity pathway.

PTK7 regulates myosin II activity to orient planar polarity in the mammalian auditory epithelium.

BACKGROUND: Planar cell polarity (PCP) signaling is a key regulator of epithelial morphogenesis, including neural tube closure and the orientation of inner ear sensory hair cells, and is mediated by a conserved noncanonical Wnt pathway. Ptk7 is a novel vertebrate-specific regulator of PCP, yet the mechanisms by which Ptk7 regulates mammalian epithelial PCP remain poorly understood. RESULTS: Here we show that, in the mammalian auditory epithelium, Ptk7 is not required for membrane recruitment of Dishevelled 2; Ptk7 and Frizzled3/Frizzled6 receptors act in parallel and have opposing effects on hair cell PCP. Mosaic analysis identified a requirement of Ptk7 in neighboring supporting cells for hair cell PCP. Ptk7 and the noncanonical Wnt pathway differentially regulate a contractile myosin II network near the apical surface of supporting cells. We provide evidence that this apical myosin II network exerts polarized contractile tension on hair cells to align their PCP, as revealed by asymmetric junctional recruitment of vinculin, a tension-sensitive actin binding protein. In Ptk7 mutants, compromised myosin II activity resulted in loss of planar asymmetry and reduced junctional localization of vinculin. By contrast, vinculin planar asymmetry and stereociliary bundle orientation were restored in Fz3(-/-);Ptk7(-/-) double mutants. CONCLUSIONS: These findings suggest that PTK7 acts in conjunction with the noncanonical Wnt pathway to orient epithelial PCP through modulation of myosin II-based contractile tension between supporting cells and hair cells.

Redundant functions of Rac GTPases in inner ear morphogenesis.

Development of the mammalian inner ear requires coordination of cell proliferation, cell fate determination and morphogenetic movements. While significant progress has been made in identifying developmental signals required for inner ear formation, less is known about how distinct signals are coordinated by their downstream mediators. Members of the Rac family of small GTPases are known regulators of cytoskeletal remodeling and numerous other cellular processes. However, the function of Rac GTPases in otic development is largely unexplored. Here, we show that Rac1 and Rac3 redundantly regulate many aspects of inner ear morphogenesis. While no morphological defects were observed in Rac3(-/-) mice, Rac1(CKO); Rac3(-/-) double mutants displayed enhanced vestibular and cochlear malformations compared to Rac1(CKO) single mutants. Moreover, in Rac1(CKO); Rac3(-/-) mutants, we observed compromised E-cadherin-mediated cell adhesion, reduced cell proliferation and increased cell death in the early developing otocyst, leading to a decreased size and malformation of the membranous labyrinth. Finally, cochlear extension was severely disrupted in Rac1(CKO); Rac3(-/-) mutants, accompanied by a loss of epithelial cohesion and formation of ectopic sensory patches underneath the cochlear duct. The compartmentalized expression of otic patterning genes within the Rac1(CKO); Rac3(-/-) mutant otocyst was largely normal, however, indicating that Rac proteins regulate inner ear morphogenesis without affecting cell fate specification. Taken together, our results reveal an essential role for Rac GTPases in coordinating cell adhesion, cell proliferation, cell death and cell movements during otic development.

Kif3a regulates planar polarization of auditory hair cells through both ciliary and non-ciliary mechanisms.

Auditory hair cells represent one of the most prominent examples of epithelial planar polarity. In the auditory sensory epithelium, planar polarity of individual hair cells is defined by their V-shaped hair bundle, the mechanotransduction organelle located on the apical surface. At the tissue level, all hair cells display uniform planar polarity across the epithelium. Although it is known that tissue planar polarity is controlled by non-canonical Wnt/planar cell polarity (PCP) signaling, the hair cell-intrinsic polarity machinery that establishes the V-shape of the hair bundle is poorly understood. Here, we show that the microtubule motor subunit Kif3a regulates hair cell polarization through both ciliary and non-ciliary mechanisms. Disruption of Kif3a in the inner ear led to absence of the kinocilium, a shortened cochlear duct and flattened hair bundle morphology. Moreover, basal bodies are mispositioned along both the apicobasal and planar polarity axes of mutant hair cells, and hair bundle orientation was uncoupled from the basal body position. We show that a non-ciliary function of Kif3a regulates localized cortical activity of p21-activated kinases (PAK), which in turn controls basal body positioning in hair cells. Our results demonstrate that Kif3a-PAK signaling coordinates planar polarization of the hair bundle and the basal body in hair cells, and establish Kif3a as a key component of the hair cell-intrinsic polarity machinery, which acts in concert with the tissue polarity pathway.

The small GTPase Rac1 regulates auditory hair cell morphogenesis.

Morphogenesis of sensory hair cells, in particular their mechanotransduction organelle, the stereociliary bundle, requires highly organized remodeling of the actin cytoskeleton. The roles of Rho family small GTPases during this process remain unknown. Here we show that deletion of Rac1 in the otic epithelium resulted in severe defects in cochlear epithelial morphogenesis. The mutant cochlea was severely shortened with a reduced number of auditory hair cells and cellular organization of the auditory sensory epithelium was abnormal. Rac1 mutant hair cells also displayed defects in planar cell polarity and morphogenesis of the stereociliary bundle, including bundle fragmentation or deformation, and mispositioning or absence of the kinocilium. We further demonstrate that a Rac-PAK (p21-activated kinase) signaling pathway mediates kinocilium-stereocilia interactions and is required for cohesion of the stereociliary bundle. Together, these results reveal a critical function of Rac1 in morphogenesis of the auditory sensory epithelium and stereociliary bundle.

The use of microarray technology in nonmammalian vertebrate systems.

Among vertebrates, the mammalian systems that are frequently used to investigate questions related to human health have gained the most benefit from microarray technology to date. However, it is clear that biological investigations and the generalized conclusions drawn from them, can only be enhanced by including organisms in which specific processes can be readily studied because of their genetic, physiological, or developmental disposition. As a result, the field of functional genomics has recently begun to embrace a number of other vertebrate species. This review summarizes the current state of microarray technology in a subset of these vertebrate organisms, including Xenopus, Rana, zebrafish, killifish (Fundulus sp.), medaka (Oryzias latipes), Atlantic salmon, and rainbow trout. A summary of various applications of microarray technology and a brief introduction to the steps involved in carrying out a microarray experiment are also presented.

In silico gene selection for custom oligonucleotide microarray design.

A method for systematically selecting the large number of sequences needed to custom design an oligonucleotide microarray was presented. This approach uses a Perl script to query sequence databases with gene lists obtained from previously designed (and publicly available) microarrays. Homologous sequences passing a user-defined threshold are returned and stored in a candidate gene database. Using this versatile technique, microarrays can be designed for any organism having sequence data. In addition, the ability to select specific input gene lists allows the design of microarrays tailored to address questions pertaining to specific pathways or processes. Given recent concerns about the accuracy of annotation in public sequence databases, it is also necessary to confirm the correct orientation of candidate sequences. This step is performed by a second Perl script that extracts protein similarity information from individual Unigene records, checks for consistency of features, and adds this information to the candidate gene database. Discrepancies between the orientations determined using protein similarities and that predicted by a given sequence's assigned orientation are readily apparent by querying the candidate gene database.

The role of early lineage in GABAergic and glutamatergic cell fate determination in Xenopus laevis.

Proper functioning of the adult nervous system is critically dependent on neurons adopting the correct neurotransmitter phenotype during early development. Whereas the importance of cell-cell communication in fate determination is well documented for a number of neurotransmitter phenotypes, the contributions made by early lineage to this process remain less clear. This is particularly true for gamma-aminobutyric acid (GABA)ergic and glutamatergic neurons, which are present as the most abundant inhibitory and excitatory neurons, respectively, in the central nervous system of all vertebrates. In the present study, we have investigated the role of early lineage in the determination of these two neurotransmitter phenotypes by constructing a fate map of GABAergic and glutamatergic neurons for the 32-cell stage Xenopus embryo with the goal of determining whether early lineage influences the acquisition of these two neurotransmitter phenotypes. To examine these phenotypes, we have cloned xGAT-1, a molecular marker for the GABAergic phenotype in Xenopus, and described its expression pattern over the course of development. Although we have identified isolated examples of a blastomere imparting a statistically significant bias, when taken together, our results suggest that blastomere lineage does not impart a widespread bias for subsequent GABAergic or glutamatergic fate determination. In addition, the fate map presented here suggests a general dorsal-anterior to ventral-posterior patterning progression of the nervous system for the 32-cell stage Xenopus embryo.

In silico gene selection strategy for custom microarray design.

Microarray technology has become an important tool for studying large-scale gene expression for a diversity of biological applications. However, there are a number of experimental settings for which commercial arrays are either unsuitable or unavailable despite the existence of sequence information. With the increasing availability of custom array manufacturing services, it is now feasible to design high-density arrays for any organism having sequence data. However, there have been relatively few reports discussing gene selection, an important first step in array design. Here we propose an in silico strategy for custom microarray gene selection that is applicable to a wide range of organisms, based on utilizing public domain microarray information to interrogate existing sequence data and to identify a set of homologous genes in any organism of interest. We demonstrate the utility of this approach by applying it to the selection of candidate genes for a custom Xenopus laevis microarray. A significant finding of this study is that 3%-4% of Xenopus expressed sequence tags (ESTs) are in an orientation contrary to that indicated in the public database entry (http://mssaha.people.wm.edu/suppMSS.html).

Mechanisms regulating the origins of the vertebrate vascular system.

In order to sustain growth, differentiation, and organogenesis, vertebrate embryos must form a functional vascular system early in embryonic development. Intrinsic interest in this process as well as the promise of potential clinical applications has led to significant progress in understanding the mechanisms governing the formation of the vascular system, however the earliest stages of vascular development--the emergence of committed endothelial precursors from the mesoderm--remain unclear. A review of the current literature reveals an unexpected diversity and heterogeneity with respect to where vascular endothelial cells originate in the embryo, when they become committed and the mechanisms governing how endothelial cells acquire their identity. Spatially, a widespread region of the early mesoderm possesses the ability to give rise to vascular endothelial cells; temporally the process is not limited to a small window during embryogenesis, but rather, may continue throughout the lifespan of the organism. On the molecular level, recent findings point to several determinative pathways that regulate, modulate, and extend the scope of the Flk1/VEGF signaling system. An expanding array of novel gene products implicated in endothelial cell type determination appear to act synergistically, with different combinations of factors leading to diverse cellular responses, varying patterns of differentiation, and considerable heterogeneity of endothelial cell types during embryogenesis.

Short upstream region drives dynamic expression of hypoxia-inducible factor 1alpha during Xenopus development.

Hypoxia-inducible factor 1alpha (HIF-1alpha) plays a central role in regulating oxygen-dependent gene expression and is involved in a range of pathways implicated in cellular survival, proliferation, and development. While the posttranslational regulation of HIF-1alpha is well characterized, the relative importance of its control at the transcriptional level during development remains less clear. Although the mouse and human promoter regions have been analyzed in vitro, to date, there has been no in vivo analysis of any vertebrate HIF-1alpha promoter. To investigate the transcriptional regulation of HIF-1alpha during development of the amphibian Xenopus laevis, we have described the gene's expression pattern and isolated the xHIF-1alpha upstream regulatory regions. We show xHIF-1alpha mRNA to be constitutively expressed at low levels throughout embryogenesis, but with significant up-regulation during gastrula stages, and subsequently, in specific regions of the central nervous system and axial tissues. Our functional analysis using a series of truncated xHIF-1alpha promoter constructs demonstrates that a 173-bp region of the proximal promoter, which is 100% conserved among five allelic variants, is sufficient to drive correct expression in transgenic embryos. Although these results are corroborated by a parallel set of in vitro transfection experiments in a Xenopus cell line, some key differences suggest the importance of using transgenic methods in conjunction with in vitro assays.