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dpilgrim

David Pilgrim

Professor

Science

Biological Sciences

Research

There are three major projects underway in the Pilgrim laboratory, on developmental neurobiology, sex determination, and the cell biology of myosin assembly and stability. Our primary research organism is the free-living nematode Caenorhabditis elegans , although we also use the zebrafish as a model vertebrate to understand conserved cellular processes. Both model systems allow us to use genetic tools to complement molecular and cellular biology approaches toward the study of the mechanisms controlling developmental and differentiation decisions.

C. elegans offers several advantages as a model: 
- primarily, a diploid animal with a rapid generation time makes forward genetic screens feasible for the isolation of many mutations affecting particular morphological and developmental processes 
a small, completely sequenced genome, with tools for reverse genetic analysis (e.g. RNAi) where single proteins (identified from the genome sequence) can be selectively removed one at a time, and we can examine the effect of their removal. 
- mutations are already available in thousands of different genes, and ongoing projects will make null or point mutations available in all genes in the genome 
- the ability to store frozen stocks for decades in liquid nitrogen ensures the availability of isogenic stocks in all labs throughout the world

Similarly, zebrafish also have genetic tools to identify new genes controlling cellular and developmental processes, and single gene knockdowns are simple (using morpholino-oligonucleotides), while being vertebrate, and therefore more directly relevant to medical research.

Current Research Projects in the Pilgrim Lab

Evolution and function of a signal transduction pathway controlling sex determination

C. elegans has proven to be an excellent system to study the function and evolution of signal transduction pathways regulating developmental decisions. We are developing a molecular picture of the pathway regulating sex determination. Although sex is determined in C. elegans by the number of sex ( ) chromosomes, mutations have been isolated which cause the animal to completely ignore the chromosomal signal. One such gene is fem-2 , which acts at an important branch point in the pathway for the regulation of sex determination in both the soma and germ line. C. elegans are normally hermaphrodites or males, but fem-2 mutants develop as females, regardless of the number of chromosomes. FEM-2 is a protein phosphatase and is thought to be one member of a complex that signals between a cell surface receptor (TRA-2) and a nuclear transcription factor (TRA-1). We also know that some components remain unidentified, and the functions of several of the known factors is not clear. The molecular analysis of this pathway in C. elegans has been hindered by the finding that many of the proteins do not have clear sequence homologues in other systems, and cell biological function is not always easy to infer. FEM-2 is typical of sex determining genes in many systems in that it is evolving very rapidly. We are presently undertaking a molecular, genetic and cell biological characterization of the fem-2 gene and the interaction of its product with other genes in the pathway.

We and our collaborators have also begun to use other Caenorhabditis species to aid in this characterization, under the assumption that sequence homologues of these genes will allow assignment of functional domains based on sequence conservation (or lack thereof). We have begun the isolation of C. briggsae sex determination mutants in an attempt to test whether the observed sequence divergence between orthologues is underlying functional changes at the protein level. Two surprises have arisen from this analysis: first, the orthologues of the C. elegans sex determining proteins are rapidly diverging; and second, mutations in these genes produce phenotypes in other Caenorhabditis species that differ from expectations, raising the idea that the sex determination pathway is regulated differently in these different species. In particular, the evolution of a male/hermaphrodite reproductive mode from a male/female ancestor appears to have occurred independently in C. elegans and C. briggsae. Whilefem-2 is necessary for hermaphrodite spermatogenesis in C. elegans , it does not play the exact same role in C. briggsae . In addition, other C. briggsae tra-2 suppressors do not control the onset of spermatogenesis in hermaphrodites though they are needed for somatic feminization in these animals. The regulation of germline fate that ultimately produces similar hermaphroditic outcomes is accomplished through different molecular mechanisms in these two species. The sex determination pathway in Caenorhabditis may be particularly tolerant to genetic changes that modulate the fate of the germline.

Role of the UNC-119 protein in neurogenesis

Another part of our research effort concerns development of the nervous system. The ability of a cell to respond to positional and guidance clues is critical for many developmental processes. Two of these processes, cell migration and neuronal axon pathfinding, have been extensively studied in model systems, and while mechanistically distinct, have several features in common. One of these is the control of cell shape by the modulation of the actin cytoskeleton. While some of the molecules involved in control of this actin-dependent process (including receptors for extracellular cues, Src-related kinases (SRKs), Rac GTPases, NIK kinase, and Enabled) have been shown, in some cases, to be potent effectors of cell guidance, the signaling pathways that link these factors together are poorly understood. Our laboratory and others have recent evidence linking the UNC-119 protein family with Rac-mediated cytoskeletal processes in migrating cells and neurons, as well as Src-mediated signal transduction pathways, in both vertebrates and invertebrates. Work underway will directly test the hypothesis that UNC-119 family members participate in Rac signaling pathways as effectors of cell guidance signals in neurons and other migrating cells.

The UNC-119 gene family is one of the more highly conserved across metazoans, yet up until recently, we had little understanding of the cell biological role of the protein. Vertebrates contain at least three related proteins (two closely related to C. elegans UNC-119, the other first identified as a phosphodiesterase-interacting protein, PDE6 d ), while only two exist in invertebrates (UNC-119 and PDL-1, which is similar to PDE6 d ). Mutations in C. elegansunc-119 lead to behavioural defects (in movement, mechanosensation and chemosensation), and cellular defects (extensive and aberrant axonal branching, ventral nerve cord defasciculation, neuronal cell body misplacement, aberrant ciliary endings in chemosensory neurons, and misdirection of neuronal processes). Defects in function of the motor neurons have been reported in unc-119 mutant C. elegans , and we have shown similar effects in vertebrates. UNC-119 and PDL-1 in C. elegans are neuronal, and human (as well as Drosophila ) UNC-119 can phenotypically rescue C. elegans unc-119 mutants, showing that the proteins are functionally as well as structurally conserved, at least for activity in C. elegans .

In vertebrates, however, other roles have been postulated. Both human UNC-119 and PDE6 d are enriched in adult retina, where a synaptic terminal role has been proposed. We have shown that both UNC-119 and the second vertebrate homologue UNC-119B are expressed outside of the retina during mouse embryogenesis, and are enriched in brain and other tissues. In zebrafish, both UNC-119 and UNC-119B knockdown experiments produce a mutant phenotype suggesting roles outside of the eye. Finally, UNC-119 interacts with SRKs in T-cells, and is necessary for eosinophil survival. However, we have shown that residues important for the T-cell function are dispensable for C. elegans activity. Clearly, either the protein has several biochemical functions, or there is a common cellular thread that we do not yet recognize.

UNC-45, a myosin interacting protein/cochaperone with distinct roles in muscle and cytokinesis

The UNC-45 family of proteins is a recently recognized class of myosin co-chaperones, and family members are disease candidates for skeletal muscle and heart developmental anomalies. unc-45 mutants which cause disorganized body wall muscle sarcomere organization were originally isolated in the nematode Caenorhabditis elegans . We have since shown that the protein is essential, with at least two distinct roles in the embryo. First, maternal UNC-45 interacts with a non-muscle myosin (NMY-2) to allow asymmetric division of the early embryonic blastomeres, and is required for cytokinesis. Second, zygotic UNC-45 is necessary for stable assembly of Myosin Heavy Chain B (MHC B) into thick filaments of body wall muscles. Control of UNC-45 through the ubiquitination pathway is critical for muscle development. However, unlike ‘classical' chaperones, UNC-45 is (i) specific to a single target (myosin), and (ii) remains associated with the target after protein folding and assembly. Our working model is that UNC-45 function is required for type II (and perhaps type V) myosin localized assembly, and that this is a critical mechanism for motor protein regulation.

UNC-45 related proteins have been found in fungi and metazoans. Fungal homologues are involved in cytokinesis and mRNA transport and interact with myosins. We have shown that the Drosophila homologue is essential, and that mice and zebrafish contain (at least) two homologues. One of the zebrafish homologues is expressed ubiquitously, the other shows embryonic expression in developing somites, and later expression in a variety of muscles, including the heart. Oligo-mediated knockdowns of expression of the latter result in paralyzed embryos, with defects in thick filament assembly in skeletal muscle, heart contraction and blood circulation, showing that UNC-45 may have a similar role in vertebrate muscle to worms, and that the two zfUNC-45 homologues may not be redundant. While our ultimate goal is to understand the relevance of UNC-45 to muscle function in vertebrate development and human disease, we first need to understand the fundamental cellular role of the protein. Since the cellular role of UNC-45 seem conserved from C. elegans to vertebrates, and the cellular, molecular and genetic tools available in C. elegans are so powerful, we will continue to elucidate the molecular role of UNC-45 in C. elegans cytokinesis and muscle development and function.