Eugenia C. Olesnicky

A caudal mRNA gradient controls posterior development in the wasp Nasonia

One of the earliest steps of embryonic development is the establishment of polarity along the anteroposterior axis. Extensive studies of Drosophila embryonic development have elucidated mechanisms for establishing polarity, while studies with other model systems have found that many of these molecular components are conserved through evolution. One exception is Bicoid, the master organizer of anterior development in Drosophila and higher dipterans, which is not conserved. Thus, the study of anteroposterior patterning in insects that lack Bicoid can provide insight into the evolution of the diversity of body plan patterning networks. To this end, we have established the long germ parasitic wasp Nasonia vitripennis as a model for comparative studies with Drosophila. Here we report that, in Nasonia, a gradient of localized caudal mRNA directs posterior patterning, whereas, in Drosophila, the gradient of maternal Caudal protein is established through translational repression by Bicoid of homogeneous caudal mRNA. Loss of caudal function in Nasonia results in severe segmentation defects. We show that Nasonia caudal is an activator of gap gene expression that acts far towards the anterior of the embryo, placing it atop a cascade of early patterning. By contrast, activation of gap genes in flies relies on redundant functions of Bicoid and Caudal, leading to a lack of dramatic action on gap gene expression: caudal instead plays a limited role as an activator of pair-rule gene expression. These studies, together with studies in short germ insects, suggest that caudal is an ancestral master organizer of patterning, and that its role has been reduced in higher dipterans such as Drosophila.

Regulation and function of tailless in the long germ wasp Nasonia vitripennis

In the long germ insect Drosophila, the gene tailless acts to pattern the terminal regions of the embryo. Loss of function of this gene results in the deletion of the anterior and posterior terminal structures and the eighth abdominal segment. Drosophila tailless is activated by the maternal terminal system through Torso signaling at both poles of the embryo, with additional activation by Bicoid at the anterior. Here, we describe the expression and function of tailless in a long germ Hymenoptera, the wasp Nasonia vitripennis. Despite the morphological similarities in the mode of development of these two insects, we find major differences in the regulation and function of tailless between Nasonia and Drosophila. In contrast to the fly, Nasonia tll appears to rely on otd for its activation at both poles. In addition, the anterior domain of Nasonia tll appears to have little or no segmental patterning function, while the posterior tll domain has a much more extensive patterning role than its Drosophila counterpart.

Connecting genotypes, phenotypes and fitness: harnessing the power of CRISPR/Cas9 genome editing

One of the fundamental goals in evolution and ecology is to identify the genetic basis of adaptive phenotypes. Unfortunately, progress towards this goal has been hampered by a lack of genetic tools available for nonmodel organisms. The exciting new development of the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR‐associated nuclease 9) genome‐editing system now promises to transform the field of molecular ecology by providing a versatile toolkit for manipulating the genome of a wide variety of organisms. Here, we review the numerous applications of this groundbreaking technology and provide a practical guide to the creation of genetic knockouts, transgenics and other related forms of gene manipulation in nonmodel organisms. We also specifically discuss the potential uses of the CRISPR/Cas9 system in ecological and evolutionary studies, which will further advance the field towards the long‐standing goal of connecting genotypes, phenotypes and fitness.

Regional Modulation of a Stochastically Expressed Factor Determines Photoreceptor Subtypes in the Drosophila Retina

Stochastic mechanisms are sometimes utilized to diversify cell fates, especially in nervous systems. In the Drosophila retina, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) controls photoreceptor subtype choice. In one randomly distributed subset of R7 photoreceptors, Ss activates Rhodopsin4 (Rh4) and represses Rhodopsin3 (Rh3); counterparts lacking Ss express Rh3 and repress Rh4. In the dorsal third region of the retina, the Iroquois Complex transcription factors induce Rh3 in Rh4-expressing R7s. Here, we show that Ss levels are controlled in a binary on/off manner throughout the retina yet are attenuated in the dorsal third region to allow Rh3 coexpression with Rh4. Whereas the sensitivity of rh3 repression to differences in Ss levels generates stochastic and regionalized patterns, the robustness of rh4 activation ensures its stochastic expression throughout the retina. Our findings show how stochastic and regional inputs are integrated to control photoreceptor subtype specification in the Drosophila retina.

Combinatorial use of translational co-factors for cell type-specific regulation during neuronal morphogenesis in Drosophila

The translational regulators Nanos (Nos) and Pumilio (Pum) work together to regulate the morphogenesis of dendritic arborization (da) neurons of the Drosophila larval peripheral nervous system. In contrast, Nos and Pum function in opposition to one another in the neuromuscular junction to regulate the morphogenesis and the electrophysiological properties of synaptic boutons. Neither the cellular functions of Nos and Pum nor their regulatory targets in neuronal morphogenesis are known. Here we show that Nos and Pum are required to maintain the dendritic complexity of da neurons during larval growth by promoting the outgrowth of new dendritic branches and the stabilization of existing dendritic branches, in part by regulating the expression of cut and head involution defective. Through an RNA interference screen we uncover a role for the translational co-factor Brain Tumor (Brat) in dendrite morphogenesis of da neurons and demonstrate that Nos, Pum, and Brat interact genetically to regulate dendrite morphogenesis. In the neuromuscular junction, Brat function is most likely specific for Pum in the presynaptic regulation of bouton morphogenesis. Our results reveal how the combinatorial use of co-regulators like Nos, Pum and Brat can diversify their roles in post-transcriptional regulation of gene expression for neuronal morphogenesis.

Extensive Use of RNA Binding Proteins in Drosophila Sensory Neuron Dendrite Morphogenesis

The large number of RNA-binding proteins and translation factors encoded in the Drosophila and other metazoan genomes predicts widespread use of post-transcriptional regulation in cellular and developmental processes. Previous studies identified roles for several RNA-binding proteins in dendrite branching morphogenesis of Drosophila larval sensory neurons. To determine the larger contribution of post-transcriptional gene regulation to neuronal morphogenesis, we conducted an RNA interference screen to identify additional Drosophila proteins annotated as either RNA-binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 genes encoding such proteins whose knockdown resulted in aberrant dendritic morphology, including alterations in dendritic branch number, branch length, field size, and patterning of the dendritic tree. In particular, splicing and translation initiation factors were associated with distinct and characteristic phenotypes, suggesting that different morphogenetic events are best controlled at specific steps in post-transcriptional messenger RNA metabolism. Many of the factors identified in the screen have been implicated in controlling the subcellular distributions and translation of maternal messenger RNAs; thus, common post-transcriptional regulatory strategies may be used in neurogenesis and in the generation of asymmetry in the female germline and embryo.

Conserved RNA-Binding Proteins Required for Dendrite Morphogenesis in Caenorhabditis elegans Sensory Neurons

The regulation of dendritic branching is critical for sensory reception, cell−cell communication within the nervous system, learning, memory, and behavior. Defects in dendrite morphology are associated with several neurologic disorders; thus, an understanding of the molecular mechanisms that govern dendrite morphogenesis is important. Recent investigations of dendrite morphogenesis have highlighted the importance of gene regulation at the posttranscriptional level. Because RNA-binding proteins mediate many posttranscriptional mechanisms, we decided to investigate the extent to which conserved RNA-binding proteins contribute to dendrite morphogenesis across phyla. Here we identify a core set of RNA-binding proteins that are important for dendrite morphogenesis in the PVD multidendritic sensory neuron in Caenorhabditis elegans. Homologs of each of these genes were previously identified as important in the Drosophila melanogaster dendritic arborization sensory neurons. Our results suggest that RNA processing, mRNA localization, mRNA stability, and translational control are all important mechanisms that contribute to dendrite morphogenesis, and we present a conserved set of RNA-binding proteins that regulate these processes in diverse animal species. Furthermore, homologs of these genes are expressed in the human brain, suggesting that these RNA-binding proteins are candidate regulators of dendrite development in humans.

Drosophila Shep and C. elegans SUP-26 are RNA-binding proteins that play diverse roles in nervous system development

The Caenorhabditis elegans gene sup-26 encodes a well-conserved RNA-recognition motif-containing RNA-binding protein (RBP) that functions in dendrite morphogenesis of the PVD sensory neuron. The Drosophila ortholog of sup-26, alan shepard (shep), is expressed throughout the nervous system and has been shown to regulate neuronal remodeling during metamorphosis. Here, we extend these studies to show that sup-26 and shep are required for the development of diverse cell types within the nematode and fly nervous systems during embryonic and larval stages. We ascribe roles for sup-26 in regulating dendrite number and the expression of genes involved in mechanosensation within the nematode peripheral nervous system. We also find that in Drosophila, shep regulates dendrite length and branch order of nociceptive neurons, regulates the organization of neuronal clusters of the peripheral nervous system and the organization of axons within the ventral nerve cord. Taken together, our results suggest that shep/sup-26 orthologs play diverse roles in neural development across animal species. Moreover, we discuss potential roles for shep/sup-26 orthologs in the human nervous system.

The RNA-binding protein Caper is required for sensory neuron development in Drosophila melanogaster

Background: Alternative splicing mediated by RNA‐binding proteins (RBPs) is emerging as a fundamental mechanism for the regulation of gene expression. Alternative splicing has been shown to be a widespread phenomenon that facilitates the diversification of gene products in a tissue‐specific manner. Although defects in alternative splicing are rooted in many neurological disorders, only a small fraction of splicing factors have been investigated in detail. Results: We find that the splicing factor Caper is required for the development of multiple different mechanosensory neuron subtypes at multiple life stages in Drosophila melanogaster. Disruption of Caper function causes defects in dendrite morphogenesis of larval dendrite arborization neurons and neuronal positioning of embryonic proprioceptors, as well as the development and maintenance of adult mechanosensory bristles. Additionally, we find that Caper dysfunction results in aberrant locomotor behavior in adult flies. Transcriptome‐wide analyses further support a role for Caper in alternative isoform regulation of genes that function in neurogenesis. Conclusions: Our results provide the first evidence for a fundamental and broad requirement for the highly conserved splicing factor Caper in the development and maintenance of the nervous system and provide a framework for future studies on the detailed mechanism of Caper‐mediated RNA regulation. Developmental Dynamics 246:610–624, 2017.

Shep interacts with posttranscriptional regulators to control dendrite morphogenesis in sensory neurons

RNA binding proteins (RBPs) mediate posttranscriptional gene regulatory events throughout development. During neurogenesis, many RBPs are required for proper dendrite morphogenesis within Drosophila sensory neurons. Despite their fundamental role in neuronal morphogenesis, little is known about the molecular mechanisms in which most RBPs participate during neurogenesis. In Drosophila, alan shepard (shep) encodes a highly conserved RBP that regulates dendrite morphogenesis in sensory neurons. Moreover, the C. elegans ortholog sup-26 has also been implicated in sensory neuron dendrite morphogenesis. Nonetheless, the molecular mechanism by which Shep/SUP-26 regulate dendrite development is not understood. Here we show that Shep interacts with the RBPs Trailer Hitch (Tral), Ypsilon schachtel (Yps), Belle (Bel), and Poly(A)-Binding Protein (PABP), to direct dendrite morphogenesis in Drosophila sensory neurons. Moreover, we identify a conserved set of Shep/SUP-26 target RNAs that include regulators of cell signaling, posttranscriptional gene regulators, and known regulators of dendrite development.