Andrew D. Chisholm - US grants

Our goal is to understand the mechanisms underlying pattern formation and morphogenesis in development. We are focusing on head region development in the nematode worm Caenorhabditis elegans. We have found that the gene vab-3 plays a key role in patterning the head region of the worm, and that vab-3 encodes a member of the Pax-6 family of paired domain containing transcriptional regulators. Pax-6 genes function in head and eye development in other species and are mutated in human eye disorders. Thus, C. elegans and vertebrates use similar genes to pattern their cephalic regions. Our first aim is to define the roles of the C elegans Pax-6 locus in the specification of the head region. We have found that the C. elegans Pax-6 locus is genetically complex and that it produces multiple protein products. Existing mutations affect subsets of functions of this complex locus. We will isolate mutations that abolish all products from this locus and thus determine the phenotype of null mutations in the C. elegans Pax-6 locus. The small cell number and defined anatomy makes it possible to analyze the requirements for Pax-6 function with a degree of precision not available in other systems. Our second aim is to characterize the genetic pathway in which Pax-6 genes function, as this is likely to have been conserved through evolution. We will use genetic screens for mutations that enhance weak vab-3 mutations or that suppress vab-3 gain-of-function mutations, with the aim of identifying genes that are regulated by Pax-6. We will also search for genes regulated by Pax-6 by identifying mRNAs that are differentially expressed between wild type and vab-3 mutants. Other genes in the Pax-6 pathway may mutate to similar phenotypes as vab-3, so we will therefore also analyze genetically other mutants with similar phenotypes to vab-3 mutants. Homologs of novel genes isolated by the above approaches will be sought in other organisms. Our third aim is to identify additional genes involved in head region development. We will perform a general screen for mutants with defects in head region development. This approach may identify components of the Pax-6 pathway and may also identify genes functioning in parallel pathways to control head development.

DESCRIPTION (provided by applicant): Epidermal morphogenesis underlies much of animal development, yet the molecular mechanisms of epithelial movements are still poorly understood. We study the epidermis of the nematode Caenorhabditis elegans as a simple model for many aspects of epithelial morphogenesis. Our long-term goal is to identify the proteins that regulate epidermal morphogenesis. We have identified genes that function in neuronal cell signaling to regulate C. elegans epidermal morphogenesis. These include an Eph receptor tyrosine kinase (VAB-1), an ephrin ligand 3. Our genetic analysis showed that Eph signaling forms part of a network of partly redundant for the VAB-1 receptor (EFN-l/VAB-2), and the LAR-type receptor tyrosine phosphatase PTP- signaling pathways that regulate C. elegans morphogenesis. We will analyze how Eph signaling functions to regulate cell behaviors in morphogenesis, both to better understand the mechanisms of Eph signaling pathways and to elucidate their in vivo function in morphogenesis. We have three specific aims. (1) We will characterize a divergent C. elegans ephrin, EFN-4. Mutations in EFN-4 affect morphogenesis. We will test whether EFN-4 is a ligand with the VAB-1 receptor and whether it modulates effects of other C. elegans ephrins. We will also search for novel EFN-4 binding proteins. (2) Our earlier work showed that Eph signaling function is partly redundant with two other pathways. In screens to identify new components of such pathways, we have identified a locus that shows synthetic-lethal interactions with vab-1. To better understand the pathways that interact with Eph signaling we will characterize this gene and identify other vab-1-interacting genes. (3) Suppressor analysis can identify components of signaling pathways that may not be found by direct genetic screens. We have identified suppressors of vab-1 loss-of-function mutations and will analyze one of these suppressors genetically and by molecular cloning. We will also generate gain-of-function (constitutively activated) versions of VAB-1 and use these in new suppressor screens. Eph signaling plays many roles in neural and vascular development and has been implicated in tumorigenesis. These aims take advantage of genetic approaches available in C. elegans to study Eph signaling and its roles in neural and epidermal morphogenesis.

DESCRIPTION (provided by applicant): Funding is requested for the Santa Cruz Meetings on Developmental Biology, to be held in 2004, 2006, and 2008. The Santa Cruz Meetings on Developmental Biology (SCDB meetings) provide a stimulating and focused forum for discussion of current research by developmental biologists. SCDB meetings are international in scope and are organized to allow extensive participation by junior faculty, postdoctoral fellows, and graduate students. Topics are covered in a thematic format, and emphasize mechanistic and experimental analysis of development. The topics for the 2004 meeting include: pattern formation; morphogenesis and cell migration; evolution and development; stem cells; non-coding RNAs; cellular asymmetry; organogenesis; development of the nervous system, and disease. Speakers are invited from both plant and animal development fields. SCDB meetings last five days and have 40 to 50 platform speakers, of which five to ten are chosen from abstracts submitted to the meeting organizers. The platform sessions are organized to allow ample time for discussions; the extensive discussion periods have been cited as one of the best features of previous SCDB meetings. Posters are continuously displayed and several poster sessions are scheduled in the meeting, as well as time for informal discussions. The 2004, 2006, and 2008 SCDB meetings will build on the success of previous meetings in this series (held in 1992, 1994, 1996, 2000, and 2002) and will take place in even-numbered years (alternating with the Gordon Research Conference on Developmental Biology). The University of California, Santa Cruz (UCSC) hosts the SCDB meetings. The conference site is arranged so that platform sessions, posters, accommodation, and dining facilities are in close proximity; access for disabled participants is ensured by UCSC. The UCSC setting is cost effective. Conference logistics are provided by UCSC Conference Services. The UCSC campus is within an hour's drive of the San Francisco Bay Area; travel from nearby airports is simple, but the site is sufficiently isolated and pleasant that speakers and participants tend to stay in residence for the entire meeting. Thus, the SCDB meeting, although modeled on a Gordon Research Conference, has several additional advantages that have made it a popular venue for the Developmental Biology community.

A major challenge in developmental biology is to understand the intricate cellular interactions between tissues that underlie morphogenesis of complex organs. To dissect the molecular basis of morphogenesis it s advantageous to study simple model tissues or organs. The long term objective of this proposal is a molecular and cellular explanation of embryonic morphogenesis of epidermis of the nematode C. elegans. In embryonic development the epidermis spreads over substrate cells, the ventral neuroblasts. Our previous work showed that signaling via the Eph receptor tyrosine kinase and its ephrin ligands is required for movements of substrate ventral neuroblasts. A specific form of Eph signaling involving both receptor and igand activation (bidirectional signaling) likely promotes adhesion or attraction among ventral neuroblasts. Here we propose to investigate how Eph signaling and parallel pathways regulate neuroblast adhesion and movements. 1. We will analyze the cellular basis of ventral neuroblast motility by a combination of confocal timelapse analysis, genetics, pharmacological interventions, and laser microsurgery. The results will allow us to interpret how the signaling pathways affect motility. 2. Reverse signaling by GPI-linked ephrins is poorly understood. In RNAi enhancer screens we identified a G protein subunit as a potential component of ephrin reverse signaling. We will define the role of this G protein in ephrin signaling using genetic and biochemical tests. 3. Eph signaling functions partly redundantly with several other pathways that provide cell adhesion in the embryo and in axon guidance. To address the nature of this redundancy we will analyze one such parallel pathway involving KAL-1, the C. elegans ortholog of anosmin-1. We have shown KAL-1 interacts with the cell surface heparan sulfated proteoglycans syndecan and glypican. We will define the regulatory pathway of KAL-1 and HSPGs and whether they are required in the same cells. HSPGs are essential for C. elegans embryonic morphogenesis. We will identify the other core proteins that account for this essential function by a combination of candidate gene testing and biochemical purification. 4. Epidermal cells migrate over the ventral neuroblast substrate. The molecules involved in epidermal- substrate adhesion are not known. Our preliminary data suggest netrin signaling has functions both in neuroblast migration and in epidermal substrate attachment. We will define the roles of netrin signaling in morphogenesis and test the hypothesis that netrin acts over short range in epidermal substrate adhesion. Relevance: An understanding of morphogenesis is relevant to treatment of human birth defects, to the development of artificial organs and tissue repair, and to tumor development. Eph signaling in particular has been implicated in human genetic disease, cancer, and in maintenance of neural stem cell niches.

Project Summary / Abstract Description: Few studies have exploited the power of genetics and functional genomics to understand the mechanisms of regrowth of axons following injury. We have developed femtosecond laser axotomy to cut single axons in intact living C. elegans animals. Severed axons of several C. elegans cell types show robust regrowth and functional recovery. We have shown that several factors, including cell type, position of axotomy and life stage, can regulate whether axons regrow after injury. Conserved signaling pathways, including cyclic AMP signaling and ephrin signaling, regulate regenerative growth of axons. We also found an unexpected role for synaptic branches in regulating axon regrowth. The tractable genetic and genomic tools available in C. elegans facilitate large scale screens for new regeneration genes. A pilot screen has uncovered several new genes that promote or repress regenerative growth. Our three specific Aims build on these preliminary results: First, we will dissect the mechanism by which the synaptic branch regulates regeneration in mechanosensory neurons. We hypothesize that the synaptic branch point contains a sorting area that regulates membrane and organelle traffic after injury. We will analyze the transport of motors and cargoes required for regrowth and will specifically test the role of the Liprin pathway in promoting regrowth. Second we will define how cAMP signaling promotes C. elegans neuronal regeneration. We will test whether cAMP or its effectors are required for regrowth. We will examine the effects of axotomy on cAMP dynamics in vivo. We will test the role of a putative cAMP-regulated transcription factor that we have found is essential for regeneration. Third, we will perform a large scale functional genomic screen to identify new genes with roles in regenerative axon growth. The mechanisms of genes with strong pro- or anti-regeneration roles will be studied in detail. Relevance: This work will yield a systematic understanding of the pathways that regulate axon regeneration after injury in a simple model system. Knowledge of the conserved mechanisms controlling axon regeneration will allow their manipulation in therapies for nervous system disease and injury.

DESCRIPTION (provided by applicant): The overall goal of this project is to use the genetically tractable model organism C. elegans to dissect the molecular basis of axon regeneration after injury. The small size, transparent body and simple anatomy of C. elegans allows single axons to be severed in vivo and their regrowth studied in depth. In the prior funding period we used large- scale genetic screens in C. elegans to discover conserved genes and pathways that play regrowth-promoting or regrowth-inhibiting roles in vivo. Many of these pathways are distinct from those involved in developmental axon outgrowth. Our large scale screens and analysis of genetic interactions have led to models for the function of these regrowth factors that we will test mechanistically in this proposal. We will define the roles of ne genes that affect regrowth via axonal microtubule dynamics. We will investigate the role of membrane trafficking regulators in axon regrowth. Results from this work will elucidate intrinsic mechanisms that allow mature axons to regrow after damage. In vertebrates, peripheral nerves are capable of regrowth, yet recovery after peripheral nerve trauma is often incomplete. Improved knowledge of regrowth mechanisms could also inform our understanding of why other neurons do not regrow. The mammalian CNS is only minimally capable of regeneration after injury, reflecting the combined effects of an inhibitory environment and of reduced intrinsic regrowth capacity. Our work addresses intrinsic mechanisms that promote or inhibit axon regrowth, a high priority for this field. Some signaling pathways have conserved roles in axon regrowth, suggesting analysis of C. elegans axon regrowth has implications for understanding axon repair mechanisms in medically relevant situations.

? DESCRIPTION (provided by applicant): The overall goal of this project is to use the genetically tractable model organism C. elegans to dissect the molecular basis of axon regeneration after injury. The small size, transparent body, and simple anatomy of C. elegans allows single axons to be severed in vivo and their regrowth studied in depth. In the prior funding period we used large- scale genetic screens in C. elegans to discover conserved genes and pathways that play regrowth-promoting or regrowth-inhibiting roles in vivo. Many of these pathways are distinct from those involved in developmental axon outgrowth. Our large scale screens and analyses of genetic interactions have led to models for the function of these regrowth factors that we will test mechanistically in this proposal. We will dissect a signaling pathway that inhibits axon regrowth via axonal microtubule dynamics. We will investigate the role of membrane trafficking regulators in axon extension. Results from this work will elucidate intrinsic mechanisms that allow mature axons to regrow after damage. In vertebrates, peripheral nerves are capable of regrowth, yet recovery after peripheral nerve trauma is often slow and incomplete. The mammalian CNS undergoes minimal regeneration after injury, reflecting the combined effects of an inhibitory environment and of reduced intrinsic regrowth capacity. Improved knowledge of regrowth mechanisms will also inform our understanding of why CNS neurons do not regrow. Our work addresses intrinsic mechanisms that promote or inhibit axon regrowth, a high priority for this field. Many signaling pathways have conserved roles in axon regrowth, suggesting analysis of C. elegans axon regrowth has implications for understanding axon repair mechanisms in medically relevant situations.

The goal of this work is to define how axons regrow and reconnect after injury, focusing on molecular regulators acting within individual axons. Our model system is the simple animal C. elegans, in which single axons can be severed and regrow in vivo in a generally permissive environment. We have used large-scale genetic screens to discover conserved genes that promote or repress axon regrowth, most of which are not involved in developmental axon outgrowth. We propose to examine in depth the roles and interactions of three new regrowth-inhibiting pathways revealed from screening. First, we will dissect the roles of a conserved regulator of axonal sprouting that may regulate neuronal lipid metabolism. Second, we will examine how a highly conserved kinase pathway inhibits axon regrowth. Finally, we will elucidate the role of mRNA decay regulators in axonal regrowth and their potential link to mitochondrial function. Results from this work will elucidate intrinsic mechanisms that allow mature axons to respond to injury and regrow after damage. In vertebrates, peripheral nerves are capable of regrowth, yet recovery after peripheral nerve trauma is often slow and incomplete. The human CNS undergoes minimal regeneration after injury, reflecting the combined effects of an inhibitory environment and of reduced intrinsic regrowth capacity. Improved knowledge of regrowth mechanisms in organisms with high intrinsic regrowth capacity will also inform our understanding of why CNS neurons do not regrow. Many C. elegans pathways have been found to have conserved roles in axon regrowth, indicating the mechanisms underlying C. elegans axon regrowth will continue to yield insights into general principles of neuronal repair.

Summary The skin is the primary interface between animals and their external environment. The skin maintains its structural integrity throughout the life of an organism while providing passive and active defenses against external challenges. While understanding of skin development has advanced rapidly in recent years, less is known of how the mature skin is maintained in adult life, including how it heals wounds. More broadly, the mechanisms that maintain mature tissues are still relatively poorly understood. The overall goal of this research program is to understand at the molecular level how a simple skin layer is repaired and maintained during adult life. The system of choice is the skin of the nematode C. elegans, a relatively simple and experimentally tractable model for skin layers. The C. elegans skin comprises a simple epidermal epithelium and associated basal and apical extracellular matrices, the basement membrane and the cuticle. Previous studies have how the mature skin responds to injury and repairs itself. This work led to the discovery of genetic regulators of adult skin maintenance; loss of function in such genes does not affect skin development but results in progressive adult-onset degeneration of the adult skin. Future studies will extend these approaches to dissect how the mature epidermis is maintained in normal and perturbed conditions. The epidermis and extracellular matrices of the skin function as an integrated unit in biogenesis, maintenance and repair. The proposed work will also investigate how the epidermis and extracellular matrix communicate in tissue maintenance and wound repair. These studies will also address the roles of an extracellular matrix lipid layer in formation and repair of the skin permeability barrier. The resulting insights should advance knowledge of skin biology relevant to several aspects of human health, including normal wound healing, chronic wounds, and skin fibrosis in hypertrophic scarring. Page 6 Project Summary/Abstract

Summary The skin is the primary interface between animals and their external environment. The skin maintains its structural integrity throughout the life of an organism while providing passive and active defenses against external challenges. While understanding of skin development has advanced rapidly in recent years, less is known of how the mature skin is maintained in adult life, including how it heals wounds. More broadly, the mechanisms that maintain mature tissues are still relatively poorly understood. The overall goal of this research program is to understand at the molecular level how a simple skin layer is repaired and maintained during adult life. The system of choice is the skin of the nematode C. elegans, a relatively simple and experimentally tractable model for skin layers. The C. elegans skin comprises a simple epidermal epithelium and associated basal and apical extracellular matrices, the basement membrane and the cuticle. Previous studies have how the mature skin responds to injury and repairs itself. This work led to the discovery of genetic regulators of adult skin maintenance; loss of function in such genes does not affect skin development but results in progressive adult-onset degeneration of the adult skin. Future studies will extend these approaches to dissect how the mature epidermis is maintained in normal and perturbed conditions. The epidermis and extracellular matrices of the skin function as an integrated unit in biogenesis, maintenance and repair. The proposed work will also investigate how the epidermis and extracellular matrix communicate in tissue maintenance and wound repair. These studies will also address the roles of an extracellular matrix lipid layer in formation and repair of the skin permeability barrier. The resulting insights should advance knowledge of skin biology relevant to several aspects of human health, including normal wound healing, chronic wounds, and skin fibrosis in hypertrophic scarring.