Alvaro Sagasti - US grants

DESCRIPTION (provided by applicant): This proposal focuses on the mechanisms by which somatosensory neurons choose their peripheral arbor territories. Touch sensation in the face is accomplished by trigeminal sensory neurons. Each trigeminal neuron projects one peripheral axon that elaborates an intricately branched arbor in a discrete portion of the head. Together, the arbors of all trigeminal neurons blanket the entire epidermis with a network of sensory fibers. In previous work, we used the zebrafish trigeminal system as a model to investigate how trigeminal neurons coordinate with one another to create their even and comprehensive arbor arrangement. Using imaging and embryological approaches, we demonstrated that repulsive interactions between growing arbors limit their territories and ensure comprehensive innervation of the head epidermis. We showed with a behavioral experiment that removing these repulsive constraints disrupts the arbor territory map and impairs the ability of animals to locate stimuli in the environment. In humans, defects in the arrangement of trigeminal innervation territories that occur developmentally or as a result of injury can also lead to defects in touch sensation, including painful neuropathies. We propose here to take further advantage of the clarity, accessibility and molecular tractability of the zebrafish trigeminal system to address three related questions about how trigeminal neurons choose their peripheral territories during development and after injury, emphasizing the role of the repulsive interactions that shape arbor territories. First, we will explore whether subtypes of sensory neurons use independent repulsive systems to choose their territories and whether there is a correlation between neuronal shape and function. We will accomplish this by creating transgenes for independently monitoring the development and morphologies of each trigeminal sensory subtype in live embryos. Second, we will characterize the cellular and molecular strategies employed by trigeminal neurons during the re-establishment of territories after peripheral injury. For these experiments, we will employ a new method we have developed to inflict precise damage on trigeminal neurons with a laser. The clarity and accessibility of zebrafish trigeminal neurons allows us to follow the regeneration of single damaged trigeminal arbors at high-resolution in real time. Third, we will exploit the molecular and genomic tools available in zebrafish to identify the molecules that mediate mutual repulsion among trigeminal neurons. We will perform a comprehensive expression screen to determine which genes are the best candidates for participating in this process, and a loss-of-function approach to test their functions. With these studies, we hope not only to provide a description of the basic mechanisms used by neurons to coordinately choose their innervation territories, but also to shed light on the causes of trigeminal innervation defects that can contribute to facial neuropathies in humans. PUBLIC HEALTH RELEVANCE: Developmental defects in peripheral touch-sensing neurons, or damage to them later in life, can trigger painful neuropathies. Peripheral neuropathies are a common and heterogeneous collection of diseases, affecting some 20 million Americans, that can result in heightened and often persistent pain sensation, but very little is understood about them at the cellular and molecular level. The studies in this proposal aim at understanding the basic mechanisms that control the development and repair of touch-sensing neurons, which we believe will ultimately lead to better ways for treating patients who suffer from peripheral neuropathies.

Touch sensation in the skin is accomplished by a heterogeneous variety of nerve cell subtypes that are each specialized to sense different kinds of touch stimuli, including temperature, pressure, and chemicals. Activation of each cell type elicits distinct perceptions and behavioral responses that are appropriate for the particular stimulus. This observation implies that each touch cell subtype connects to a distinct neural circuit in the brain. This project will investigate how particular features of touch-sensing neurons are specialized for their sensory function. To accomplish this, they are using zebrafish larvae as a model because they have a simplified nervous system, their optical clarity allows direct imaging of cellular morphology, and they are amenable to a wide variety of molecular and cellular manipulations. The PI has identified a unique class of touch-sensing cells in zebrafish that connect to a distinct part of the brain; whereas most touch-sensing nerve cells connect to circuits in the hindbrain, this particular subtype connects to spinal cord circuits. The project will use live imaging techniques to determine whether these cells possess other distinctive morphological features, to use molecular techniques to determine whether they express a distinct set of genes, to identify the stimuli that they sense using live physiological techniques, and to determine what behaviors they elicit by specifically activating them and monitoring the animal's behavior. Together these experiments will provide insight into how a sensory cell's particular morphological and molecular features are optimized for their functions. This project will be headed by a graduate student. Several undergraduates will also participate on different aspects of the project, providing them with a rare opportunity for exposure to basic neurobiological research.

DESCRIPTION (provided by applicant): Sensory neurons that innervate the skin are easily damaged. When damage exceeds the capacity of these cells for repair, peripheral neuropathy (PN) manifests. Cancer patients who survive chemotherapy, or diabetics who struggle to control blood sugars, may be left with months to a lifetime of numbness or shooting pain. Free nerve endings in the skin mediate pain and temperature sensation, both of which are compromised early in most forms of PN. The repair mechanism of these sensory endings has not been elucidated, although it is the key to understanding the onset of PN and may also be critical for developing strategies to delay or cure PN. Axon regeneration uses a common set of machinery in all organisms and neurons in which it has been studied. This core machinery includes a MAP kinase cascade initiated by DLK and the activation of AP-1 transcription factors. This core pathway mediates regeneration when axons of sensory neurons in Drosophila are injured, but regeneration of peripheral sensory endings in the same cells is completely independent of this machinery. In Drosophila, sensory endings have been defined as dendrites by cell biology studies, suggesting that the DLK pathway may be specific to axon regeneration and not used for dendrite regeneration. Determining whether sensory endings in vertebrates are repaired using the conserved axon regeneration pathway or a completely unexplored dendrite regeneration pathway will be a critical foundation for understanding PN. In this exploratory work, two major lines of experimentation will be used to determine the repair pathway used to regenerate vertebrate sensory endings in the skin. The sensory neurons that innervate the skin in zebrafish will be used as a model for all of these experiments. Zebrafish larval sensory neurons known as Rohon-Beard (RB) neurons are accessible for imaging and manipulation with current methodology, so these cells will be used as a first model in which to study sensory ending cell biology and injury responses. Analogous methods will then be developed for dorsal root ganglion neurons (DRGs), as these are the most relevant for understanding PN. To determine whether vertebrate free sensory endings are dendrites as in Drosophila, or axons as they are historically classified in vertebrates, we will determine the polarity of their microtubules. All known dendrites contain at least 50% minus-end-out microtubules while axons are close to 100% plus-end-out. Microtubule polarity has never been assayed in vertebrate sensory endings, so this will be done in both RB and DRG cells. To determine whether injury to sensory endings triggers the identified axon regeneration pathway or a novel dendrite regeneration pathway, we will develop markers for DLK signaling and compare responses to central axon injury and peripheral sensory ending injury in zebrafish sensory neurons. These two lines of experimentation will form a foundation for understanding how sensory endings are repaired, and how this process fails in patients with PN.

DESCRIPTION (provided by applicant): Somatosensory neurons project peripheral axons to the skin early in development to detect touch stimuli. Although the cutaneous terminals of these axons are often a proportionally small component of the total peripheral axon length, they are critical for function, since they are the sites where touch stimuli are first detected. Cutaneous axon endings are particularly vulnerable to damage by injury, diabetes, and inherited syndromes, notably Charcot-Marie-Tooth diseases. All of these conditions cause debilitating peripheral neuropathies characterized by chronic pain or the inability to sense touch. Characterizing how cutaneous sensory endings are formed, maintained, and respond to injury is thus essential for understanding these conditions and developing effective treatments. We have developed a larval zebrafish model to study the development of somatosensory peripheral axons and the skin cells that they innervate. Because zebrafish larvae are fertilized externally, develop rapidly, and are optically clear, the zebrafish somatosensory system offers unparalleled experimental access to the early stages of skin innervation. By contrast, studying these cutaneous sensory terminals in mammals is challenging, since they develop in utero and their complete three-dimensional structures are difficult to visualize. Most anatomical and molecular features of somatosensory axon territories in the skin are well conserved from fish to mammals, making zebrafish a relevant model for uncovering potential disease mechanisms. Our studies of the past few years have revealed that skin cells play several critical roles in the development, repair and function of somatosensory axon terminals in the skin. The goal of this proposal is to identify and characterize the molecular dialogues between axons and skin cells that regulate the establishment and maintenance of somatosensory axon territories. Using a unique and powerful set of molecular techniques and transgenic tools that we have developed in recent years, we will investigate three questions about the nature of axon/skin interactions during specific stages of somatosensory neuron ontogeny. First, how are sensory axons guided to the skin? Second, once in the skin how do axons become structurally associated with skin cells? And third, how does the skin respond to axon damage and contribute to repair? We will address these questions with a powerful combination of live imaging, embryology and molecular perturbations. Collectively, these studies will provide the first molecular insight into the regulation of several newly discovered functions of skin cells in the development and maintenance of the somatosensory system.

DESCRIPTION (provided by applicant): Mucosal epithelia form the external interface of many sensitive tissues. The outer surface of cells in these epithelia displays a glycoprotein calyx and adsorbs mucins to create a mucus layer that protects those tissues from abrasion and maintains their hydration. In the cornea, for example, the mucus layer retains the tear film that keeps our eyes wet. Defects in the mucosal layer of the human cornea cause dry eye diseases (DED). These conditions are common, painful, and progress with age. DED is often accompanied by dry mouth, suggesting that its underlying causes affect properties common to mucosal epithelia. One such property is the presence of elaborate actin-based structures on the surface of these epithelial cells, known as microplicae and microridges. These structures have been little studied, but likely make a vital contribution to the functional properties of mucosal epithelia by increasin the surface area of the glycocalyx, thus maximizing their ability to hydrate tissues. Most studies of DED pathology have focused on tear production, but given the critical role of mucosal epithelia in maintaining the mucus layer and tear film, it is likely that defects in epithelial morphogenesis also contribute to these conditions. One of the main obstacles to studying the morphogenesis of microplicae and microridges has been the lack of an accessible model system. We have developed the larval zebrafish skin as a model for studying mucosal epithelial development. The entire surface of zebrafish larvae is wrapped in a single-layered mucosal epithelium known as the periderm. The apical surface of periderm cells is covered my microplicae and microridges that remarkably resemble structures on the surface of the human cornea. These cells are exceptionally accessible to transgenic labeling and confocal imaging, making it possible to visualize the formation of ridges in living animals. Moreover, the amenability of the zebrafish system to sophisticated genetic and transgenic manipulations will make it possible to uncover the underlying molecular mechanisms of microridge formation. In this proposal we combine descriptive, hypothesis-driven, and discovery-based approaches to dissect the process of ridge morphogenesis. Specifically, in Aim 1 we will use live imaging to describe the initial formation of microridges and their re-organization during cellular contraction In Aim 2 we will use molecular and imaging approaches to test the hypothesis that phosphoinositide microdomains orchestrate the formation of microridges. Finally, in Aim 3 we will use RNA-Seq to identify BAR domain proteins and actin regulators enriched in periderm and test whether select candidate proteins play roles in microridge development. Together these studies will provide the first insights into ridge morphogenesis and establish the zebrafish model as a system for understanding not only ridge formation in normal cells, but also the causes of their pathology in diseases, such as DED.

PROJECT SUMMARY To adopt forms optimized for their functions, individual cells sometimes project remarkably elaborate membrane protrusions, and even arrange them in complex patterns on their surfaces. To create and support membrane structures, the underlying cortical cytoskeleton must be arranged in specific conformations. The particular complement of cytoskeletal associated proteins, and the local regulation of their activity, thus determines the shape and pattern of cell membrane protrusions. To understand how cytoskeletal proteins together create unique cellular structures, we will study the formation of stunning actin-based structures on the surface of zebrafish skin cells called microridges. Microridges (or similar structures called microplicae) are found on most mucosal epithelial cells, which not only form the outer layer of fish skin, but also many of our own tissues, including the cornea, mouth, and parts of our gut. The microridge-covered surfaces of these cells display a glycoprotein calyx and adsorb mucins, suggesting that the unique structure of microridges is optimized for mucus retention. Mucus protects vulnerable epithelial tissues from abrasion and drying out, so understanding how microridges form could provide insight into the etiology of diseases affecting mucosal tissues, such as dry eye and dry mouth conditions. This proposal builds on successful descriptive and discovery-based studies supported by an NIH R21 grant that led to the identification of several new proteins in microridges. The experiments proposed here investigate mechanisms by which these specific proteins contribute to microridge morphogenesis, and, from a broader perspective, how they function as an ensemble to create the unique shapes and properties of microridges. In Aim 1 we will test the hypothesis that two proteins, Ezrin and Drebrin-like, initiate the first step of microridge morphogenesis, the formation of microvilli-like microridge precursors. Aim 2 investigates the interactions between F-actin and intermediate filaments (IFs) in microridges by testing if F-actin patterning determines IF patterning, and by testing the hypothesis that two candidate proteins, Envoplakin and Periplakin, link these cytoskeletal elements together. Finally, in Aim 3, live imaging, pharmacology, and molecular approaches will be used to characterize how myosin-based contraction and Rho GTPase signaling contribute to microridge morphogenesis. Collectively these studies will provide mechanistic insights into microridge morphogenesis, illuminate how cytoskeletal proteins together create elaborate cellular structures, and potentially point to how defects in epithelial morphogenesis contribute to diseases afflicting mucosal epithelia.

PROJECT SUMMARY Selective interactions between neurons and non-neuronal cells are crucial for the development and function of neural circuits. Pain-sensing somatosensory neurons project peripheral axons to the skin, where they branch extensively amongst epithelial epidermal cells. Although these sensory terminals are called ?free endings?, recent studies have revealed that they are often wrapped by epidermal cells, which enclose them into ensheathment channels reminiscent of those formed by non-myelinating Remak Schwann cells. Although underappreciated, epidermal ensheathment channels have been observed in worms, flies, fish, and mammals, indicating that ensheathment is a conserved feature of epidermal sensory endings, and thus likely plays critical roles in the development and function of nociceptive axons. Little is known about the morphogenetic process of axon ensheathment by epidermal cells, and nothing is known about how these structures contribute to sensory function or disease in vertebrate animals. This proposal investigates the morphogenetic mechanisms that create epidermal ensheathment channels and how they contribute to sensory function in zebrafish larvae. Their external development and the availability of unique transgenic tools make zebrafish an ideal model for studying this dynamic morphogenetic process. Preliminary work using live fluorescent reporters for subcellular structures in zebrafish skin cells identified a sequence of events leading up to ensheathment and suggested a step-wise morphogenetic process. First, axons growing into this epidermis induce the formation of specialized lipid microdomains at skin cell-axon contact sites. Second, F-actin is recruited to these microdomains, likely promoting membrane invagination to initiate the ensheathment process. Finally, adherens junctions and desmosomes form at ?necks? of ensheathment channels to tightly seal the channels. The first two aims of this proposal use innovative microscopy approaches for high spatial and temporal resolution live imaging, cell-specific molecular manipulations, and CRISPR/Cas9-mediated mutagenesis to determine how axons and skin cells establish selective interactions and execute the ensheathment process. These studies will illuminate morphogenetic mechanisms relevant not just to axon ensheathment by epidermal and glial cells, but also to basic cellular processes, like the formation of membrane signaling domains and junction assembly. The third aim combines imaging and behavioral assays to reveal how axon ensheathment impacts neuronal structure and function. These studies have the potential to uncover a critical feature of the touch-sensing apparatus and suggest how ensheathment contributes to disease conditions affecting pain and touch sensation.