Gian Garriga - US grants

DESCRIPTION (provided by applicant): A fundamental question in development is how cells are specified. Elucidating mechanisms of cell-type specification is essential to the understanding and eventually treating human diseases, such as cancer, where this process is altered. One mechanism that specifies cell type is Asymmetric Cell Division, where a cell divides to produce two daughter cells that adopt distinct fates. Both asymmetrically distributed molecules and cell signaling appear to polarize dividing cells, but a connection between these two mechanisms has been lacking. A physical interaction between HAM-l, an asymmetrically distributed molecule, and DSH-2, a conserved Wnt signaling component, provides a link between asymmetric molecules and signaling. The overall goal of the proposed research is to define the roles of DSH-2 and HAM-1 in asymmetric cell division. The proposal has four specific aims. 1. To explore the interaction between DSH-2 and HAM-1. Immunoprecipitation experiments will be conducted to confirm a DSH-2/HAM-1 complex. Molecular genetic experiments will probe whether the interaction between the proteins is necessary for their function in asymmetric divisions and whether one protein is necessary for the others distribution. 2. To test whether the asymmetric distributions of DSH-2 and HAM-1 are necessary for their roles in asymmetric cell division. The localization domains of both proteins will be defined, and mislocalization experiments will test whether asymmetric distribution of two proteins is important to specify daughter cell fate. 3. To determine which proteins act upstream of DSH-2 and HAM-1 to control their asymmetric distribution. Using genetic and immunocytochemical approaches, Wnts and Frizzled receptors will be tested for roles in the distribution of DSH-2 and HAM-1. These molecules will then be misexpressed to test whether asymmetric signaling regulates DSH-2 and HAM-1 distribution. 4. To determine how DSH-2 and HAM-1 signal to control asymmetric cell division. Using reverse genetic approaches, candidate genes will be tested for roles in asymmetric divisions that require DSH-2 and HAM-1. In addition, genes identified in genetic screens will be tested for a role the HAM-1 /DSH-2 pathway. Genes that act in the pathway will be characterized molecularly.

DESCRIPTION (provided by applicant): Neuronal cell body and growth cone migrations shape the overall pattern and connectivity of nervous systems. The objective of the proposed research is to investigate the mechanisms that control neuronal migrations. Understanding these basic mechanisms could lead to insights into how damaged nervous systems might be repaired. The application has four specific aims. To determine whether EGL-20 Wnt acts as a guidance cue for the HSN. The migrations of the HSN motor neuron require the Wnt homolog EGL-20, and preliminary experiments suggest that EGL-20 acts as a guidance cue, a novel activity for a Wnt. To determine whether EGL-20 guides the HSNs to their destinations, EGL-20 will be misexpressed. Downstream components in EGL-20 Wnt will be identified to determine whether they act in the HSN. 1. To determine how the CAM-1 Ror kinase antagonizes EGL-20 in HSN migration. Genetic interactions between the egl-20 and cam-1 mutations indicate that these genes antagonize each other in HSN migration. CAM-1 could alter the distribution of EGL-20 by directly binding to EGL-20. Alternatively, CAM-1 could encode a component of a separate signaling pathway that antagonizes EGL-20. A series of genetic and molecular experiments are proposed to distinguish between these hypotheses. 2. To further characterize the role of Abelson oncogene ABL-1 in cell migration and identify genes that act in the ABL-1 pathway. The Abelson oncogene plays a central role in the growth cone migrations of other organisms. In C. elegans, ABL-1, like CAM-1, antagonizes the activity of EGL-20 in HSN migration. Experiments are proposed to determine which portions of ABL-1 function in migration, to determine where ABL-1 functions, and to identify molecules that act with ABL-1. 3. To define the roles of VAB-8/UNC-51 interactions in cell and growth cone migrations. VAB-8 interacts physically with the conserved serine/threonine kinase UNC-51. Preliminary experiments also suggest that UNC-51 can phosphorylate VAB-8L. The functional significance of the VAB-8/UNC-51 interactions and the role of VAB-8 phosphorylation will be tested.

DESCRIPTION (provided by applicant): A fundamental question in development is how cells are specified. Elucidating mechanisms of cell-type specification is essential to the understanding and eventually treating human diseases, such as cancer, where this process is altered. One mechanism that specifies cell type is Asymmetric Cell Division, where a cell divides to produce two daughter cells that adopt distinct fates. Both intracellular asymmetric molecules and cell signaling appear to polarize neuroblasts, but a connection between the two mechanisms has been lacking. We have found that signaling is essential for certain asymmetric divisions that produce apoptotic cells and neural precursors and have identified molecules that are asymmetrically distributed in these divisions. The overall goal of our research is to understand how both cell intrinsic mechanisms and signaling regulate the fates of the neurons that are generated during these divisions. The proposal has three aims. 1) We propose to determine how the cytohesin GRP-1 regulates asymmetric cell divisions. Our model proposes that GRP-1 functions in V5 to regulate signaling between V5 and asymmetrically dividing neuroblasts, and we will test this idea rigorously for several divisions that require GRP-1 function. We will test the model that PI3K regulates GRP-1 and mediates its effects though ARF-1 and ARF-6. GRP-1's surprising localization to the nucleus raises the interesting possibility that cytohesins may function there, and we will ask whether GRP-1 carries out its functions in the nucleus. Finally, we will pursue RNG-1 and CNT-2, two molecules that may function in the GRP-1 pathway. 2) We propose to determine how Wnts regulate asymmetric divisions of the Q neuroblast, which also require both PIG-1 and GRP-1 function. Our preliminary experiments indicate that three Wnts and two Frizzled receptors are involved in these divisions. We will test the roles of the remaining Wnts and Wnt receptors, characterize the effects of these mutations on the divisions in more detail and test whether Wnt signaling acts in the GRP-1 or PIG-1 pathways. 3) Our analysis of GRP-1 and Wnt signaling indicates that cell signaling regulates asymmetric cell divisions. We will continue our EMS screens and begin RNAi screens to identify molecules that function in these signaling events, as well as molecules that act with HAM-1 and PIG-1.

DESCRIPTION (provided by applicant): Neuronal cell body and growth cone migrations shape the overall pattern and connectivity of nervous systems. The objective of the proposed research is to investigate the mechanisms that control neuronal migrations. Understanding these basic mechanisms could lead to insights into how damaged nervous systems might be repaired. The proposal has three specific aims. 1) To determine how VAB-8 and the Trio homolog UNC-73 regulate guidance receptors. A large variety of axon trajectories are guided by a few conserved guidance molecules. Many guidance receptors are broadly expressed posing the question of how neurons select among specific guidance cues to initiate and terminate directed growth. We have found that the kinesin-related molecule VAB-8 regulates the sensitivity to guidance cues by controling the levels of their receptors at the cell surface, VAB-8 acts though the conserved Rac GEF UNC-73/Trio. The focus of this aim is to show that the physical interactions between UNC-73, VAB-8 and the guidance receptors SAX-3/Robo, UNC-5 and UNC-40 mediate the effects of VAB-8 on these receptors, to show that the effects of VAB-8 are mediated by Rac signaling and to define the mechanism that promotes the accumulation of these receptors at.the cell surface. 2) To determine how Wnt signaling, VAB-8 and UNC-73 interact. Wnts are conserved glycoproteins that control the migrations of growth cones along the A/P axis of C. elegans and mammals. In C. elegans Wnts also regulate neuronal polarity alng this axis. Our results indicate that VAB-8 can regulate Wnt signaling through the MIG-1 Frizzled receptors and that MIG-1 and the second Frizzled receptor LIN-17 antagonize one to control the polarity of the PLM mechanosensory neuron. We propose experiments that will define how these molecules act together to regulate neuronal polarity and screens to define additional molecules that act in the Wnt signaling pathways involved in axon guidance and neuronal polarity. 3) To determine whether ABL-1, CRML-1 and UNC-53 inhibit the function of VAB-8L and UNC-73. Our genetic experiments indicate that these conserved signaling molecules regulate axon guidance by inhibiting VAB-8 and UNC-73 signaling. We propose to test this hypothesis and define the mechanisms that they employ to regulate VAB-8 and UNC-73.

DESCRIPTION (provided by applicant): The enclosed proposal represents a competitive renewal of an established graduate training program in Genetics at UC Berkeley. We request funds to support 17 graduate trainees in the areas of Developmental Genetics and Genomics, Cell &Systems Genetics, and Population Genetics and Evolution. Students enter this inter-disciplinary training program after gaining admission into one of four Departments on campus. Two of the Departments, Molecular and Cell Biology (MCB) and Integrative Biology (IB), are located within the College of Letters and Sciences. A third Department, Plant and Microbial Biology (PMB), is located in the College of Natural Resources. Students can also enter the training program through the School of Public Health (SPH). Students are selected into the program based on an interest in one of the broadly defined areas of genetics that are sponsored by this training grant. They are also selected based on merit and future promise as independent investigators. Despite their diverse backgrounds, the training program includes a number of mechanisms to ensure that all of the graduate students obtain rigorous training in genetics. Once they enter the program, students are free to select any one of 41 different faculty mentors located in the four aforementioned performance sites. These faculty employ a variety of genetic and genomic methods to study a broad spectrum of problems in cell, developmental, and evolutionary biology, including metazoan and plant patterning, embryogenesis, sex determination, cell determination and differentiation, morphogenesis, and organogenesis. The study of cellular processes in the areas of signal transduction, DNA replication, transcription, cell trafficking and the cytoskeleton, transcription, and DNA replication are the focus of many faculty. Other faculty use genetic approaches to study evolutionary processes such as the evolution of chordates and patterning in invertebrates. In the first year of the program, trainees complete advanced lecture, laboratory, and seminar courses. Students in PMB and MCB are also required to complete three 10-week laboratory rotations with potential dissertation mentors. All students commence their dissertation research by the beginning of the second year. They are also required to complete an oral qualifying exam that is administered by four faculty from at least two different departments within the training program. All students are required to complete at least two semesters of teaching as graduate instructors during the second and third years of the training program. In addition to formal course offerings in the different areas of genetics, students are expected to participate in a variety of seminar programs, joint lab meetings, journal clubs, and retreats that are sponsored by the Genetics Training Program.

DESCRIPTION (provided by applicant): Understanding how axons find their synaptic targets is a fundamental problem in neurodevelopment. While great progress has been made in elucidating the mechanisms of axon guidance, less is known about how axons fasciculate within specific regions of a nerve bundle. We propose to study this problem in C. elegans with a focus on the HSN axon. The HSN axon extends along the ventral nerve cord after the entire nervous system has been established. The HSN growth cone fasciculates along the PVQ and PVP axons, and the short-term goal of this project is to identify the cues that define the HSN's ability to grow along its specific path in the nerve bundle. While little is known about how axons fasciculate within mammalian nerves, recent findings suggest that sorting of olfactory axons within the nerve are important in their targeting at the olfactory bulb. The HSN may provide a model for how such nerves are organized in mammals. The proposal has three specific aims. In the first aim, the interface between the different axons will be labeled using GRASP. In the second aim, RNAi approaches to identify genes involved in HSN axon fasciculation will be developed. These approaches should bypass the lethal effects caused by RNAi of essential genes and indirect effects caused by earlier defects in the fasciculation of other axons. In the third aim, the approaches will be tested to elucidate the role of the neurotransmitter acetylcholine in axon fasciculation.. PUBLIC HEALTH RELEVANCE: Understanding how these axons find their targets is a fundamental problem in neurodevelopment. While great progress has been made in elucidating the mechanisms of axon guidance, less is known about how axons grow along specific regions of a nerve bundle. We propose to study this problem in C. elegans, a model organism that has been instrumental in defined conserved molecules that function axon guidance.

DESCRIPTION (provided by applicant): Understanding how neuroblasts and neurons polarize is a fundamental problem in neurodevelopment. We propose to study this problem in C. elegans with a focus on how polarity is established along the C. elegans anterior/posterior (A/P) axis. C. elegans has been an important model for the nervous system development. In both C. elegans and the spinal cord, the same molecules guide migrating axons along the dorsal/ventral and A/P axes. Wnt glycoproteins, for example, guide axons toward the anterior in both organisms. In C. elegans, Wnts are the main regulators not only of axon guidance, but also of neuroblast and neuronal polarity. We propose to define the common mechanisms that Wnts use to regulate these two processes. In addition, disregulation of Wnt-pathway components contributes to many forms of cancer. Understanding the mechanisms of Wnt regulation and signaling has potential implications for spinal cord repair and cancer. PUBLIC HEALTH RELEVANCE: All cells exhibit polarity. Polarity is particularly important during nervous system development, where the proper establishment of polarity is essential for neuroblasts to divide to produce neurons, for neurons to migrate and for axons to find their synaptic targets. We propose to study how neuronal polarity is regulated in model organism C. elegans by the family of secreted Wnt glycoproteins.

The asymmetric divisions of neural progenitors are an important regulator of neural fate. Studies over the past 20 years have identified mechanisms that distribute developmental potential and orient the spindle in these divisions. Much less, however, is known about a subset of these divisions that produce daughter cells of different sizes, what I refer to here as Daughter Cell Size Asymmetry (DCSA). DCSA has been observed in cell divisions that range from the C. elegans zygote to mouse cortical progenitors. We propose to study DCSA in the Q.a and Q.p neural progenitors, where it contributes to the apoptotic fate. These cells provide an excellent model for the study of DCSA because they use two different mechanisms for DCSA: Q.a divides asymmetrically by a myosin-dependent, spindle-independent mechanism, and Q.p by a spindle-dependent mechanism. These studies have important implications for human health: dysregulation of cortical progenitor divisions can result in lissencephaly and microcephaly, and Wnt signaling, which is dysregulated in several cancers, regulates Q division asymmetry. The overall goal of our studies is to understand how these types of asymmetric divisions regulated. Our work has identified both Wnt signaling and the conserved molecules PIG-1, HAM-1 and TOE-2 as playing crucial roles in DCSA. Our working model proposes that the localization of PIG-1 controls membrane extension through GTPases of the Rho family and spindle movement through trimeric G proteins. It also proposes that Wnt signaling regulates both the myosin-dependent, spindle-independent and the spindle-dependent mechanisms through the PIG-1 and that HAM-1, TOE-2 and MAP kinase signaling switch Wnt signaling from a default spindle-dependent mechanism to a spindle-independent mechanism. A combination of genetic, molecular and imaging approaches will be used to test these hypotheses.