Gallegos, Maria E. (2009) International Worm Meeting "A discovery-based molecular techniques course aimed to develop reagents for rab gene function studies."

Gallegos, Maria E.

[International Worm Meeting, 2009]

I have developed a discovery-based lab course at California State University, East Bay (CSUEB) entitled Advanced Molecular Techniques. This lab course has three aims: 1) to promote the National Science Education Standards mandate to replace "cookbook" lab courses with inquiry-based learning; 2) to teach a variety of molecular biology techniques applicable in both academia and industry settings and 3) to develop molecular tools useful to the C. elegans community for gene function studies. During the quarter, students learn a variety of standard molecular techniques in addition to QuikChange Site-Directed Mutagenesis (Stratagene) and Gateway Recombinational Cloning (Invitrogen). To prepare for and execute each step of the project, students must also master software tools such as NCBI BLAST (NLM), CLUSTAL W (EMBL-EBI), Primer Generator (Lawler and Turchin), A Plasmid Editor (Wayne Davis) and Image J (NIH), all of which are available free online. Students use these software tools and molecular techniques to modify Orfeome entry clones and create expression vectors containing dominant negative and constitutive active forms of the rab monomeric GTPases (entry clones kindly provided by Dr. Kang Shen). To date we have focused on the rab family of GTPases for several reasons: The rab family is manageable in size with 22 members. The average coding sequence is short, about the length of a traditional sequencing run. Dominant negative and constitutive active forms are well characterized. And finally, rab GTPases play important and diverse roles in cell and developmental biology. Each pair of students is assigned a single gene. One will create the dominant-negative while the other will create the constitutive active form. Thus each student owns a unique project with unique solutions while still having access to backup reagents (when necessary) and advice from peers. To provide each student a unique project, I needed to reduce costs. The QuikChange Site-Directed Mutagenesis kit by Stratagene is the most expensive component of this course thus each year we have also experimented with its protocol. Importantly, our results indicate that you do not need to use PAGE purified mutagenic primer pairs (as recommended), there is no reduction in mutagenesis efficiency if half the reaction volume is used and you can still get a sufficient number of transformants with half the volume of competent cells. Our progress will be reported. Any tools that have been created are freely available to the worm community.

Gallegos, Maria et al. (2015) International Worm Meeting "Cellular and Molecular Mechanisms Underlying Neurite Termination and Refinement in C. elegans."

Avina, Monika, Gallegos, Maria, Azevedo, Joshua, Das, Pranti, Ndungu-Case, Catherine, Serra, Lorrayne, Tara, Boshika, Flores, Alejandro

[International Worm Meeting, 2015]

In the posterior half of the worm, gentle touch is first detected by the mechanosensory neuron PLM. As expected for a spatially restricted function, the PLM dendrite occupies the posterior half of the animal. How is the termination point of the PLM dendrite established? Exuberant outgrowth followed by stereotyped pruning is a common feature in the development of neural circuits in vertebrates. In C. elegans, PLM also follows this pattern of growth (Gallegos and Bargmann 2004). Initially, the PLM dendrite extends rapidly, bypassing its target site then undergoes regulated shortening to terminate just posterior to the anterior mechanosensory neuron, ALM. My lab is interested in both molecular and cellular factors that contribute to this process. To date we have identified worm orthologs of the yeast RAM pathway that function during the regulated shortening step of PLM development: SAX-1 (an NDR kinase), F09A5.4 (an NDR kinase cofactor) and SAX-2 (a scaffold protein). More recently, genetic evidence suggests that vesicle trafficking may regulate PLM length both within and in parallel to RAM pathway signaling. For example, rab-10, a GTPase involved in polarized secretion leads to PLM overextension, a defect not enhanced in the absence of sax-2. We are also interested in identifying the cell or tissue that might initiate PLM dendrite termination. One candidate, BDU, is an interneuron that makes both gap junction and synaptic connections with mechanosensory circuit neurons. Interestingly, BDU extends a posterior neurite that makes contact with the PLM neurite tip at the right time and place to promote PLM termination (Gallegos unpublished data and Zhang et al 2013). To test the role that BDU might play in PLM termination, we created unc-86 mosaic animals that allow PLM development in the absence of ALM and BDU. Our preliminary data indicates that the PLM dendrite extends significantly longer on average in mosaic animals that lack ALM and BDU in comparison to non-mosaic controls suggesting that BDU is required to confine the PLM dendrite to the posterior half of the animal in a contact-dependent mechanism. In our initial experiments, however, BDU was not visible. We are redoing these experiments using GFP markers that light up both BDU and PLM to ensure that their development is normal in nonmosaic controls. We are also attempting to use the free Duplication qDp3 to confirm our findings.

Gallegos, Maria E. et al. (2009) International Worm Meeting "A candidate gene approach to identify additional genes that function in the sax-1/sax-2 pathway to regulate mechanosensory neurite termination."

Das, Pranti, Karamchedu, Padma, Chandramouli, Priya, Gallegos, Maria E.

[International Worm Meeting, 2009]

In C. elegans, sax-1 and sax-2 regulate neurite termination and the maintenance of neuron morphology. Specifically, in sax-1 and sax-2 loss-of-function mutants, the neurite of the posterior mechanosensory neuron (PLM) extends beyond its normal site of termination anterior to the ALM cell body. In addition, ectopic neurites extend from the cell soma of most if not all neurons, a mutant phenotype that worsens as the animal ages. sax-1 and sax-2 are likely orthologs of budding yeast RAM pathway members, CBK1 and TAO3, respectively. Additional members of this pathway include KIC1, SOG2/HYM1, and MOB2. We are taking a reverse genetic approach to determine the role that putative RAM pathway members and candidate downstream effectors play in PLM neurite termination and maintenance of neuron morphology in C. elegans. Specifically, we have used single and combinatorial RNAi to knockdown gene activity of putative MOB2 and KIC1 orthologs. Genes examined in this RNAi study include gck-1, gck-4, cst-1/cst-2, T12B3.4 and F38H4.10. We are also using deletion allels to test the role that candidate downstream effectors, rab-8 and rab-10, play in PLM neurite termination. rab-8 and rab-10 are most closely related to yeast SEC4, a RAB GTPase involved in polarized secretion. Interestingly, yeast Cbk1p binds Sec2p, the guanyl-nucleotide exchange factor for Sec4p. Results will be presented.

Maria E Gallegos et al. (2005) International Worm Meeting "Mechanosensory neurite termination and tiling are directed by SAX-2 and the SAX-1 kinase."

Cori Bargmann, Maria E Gallegos

[International Worm Meeting, 2005]

Animals possess mechanosensory neurons that extend sensory dendrites just beneath the body surface to detect and transmit tactile information. To identify the precise location of an external stimulus, mechanosensory neurons restrict dendrite growth to non-overlapping regions, a pattern called tiling. How this regulated pattern of growth is established and maintained is largely a mystery. In particular, molecules that regulate neurite termination and the transition from outgrowth to morphological stability are poorly understood. In C. elegans, two pairs of mechanosensory neuron, ALM (L/R) and PLM (L/R), mediate escape from mechanical stimuli applied to the anterior and posterior half of the animal, respectively. To respond appropriately, the anteriorly-directed sensory processes of each neuron pair tile the body surface along the antero-posterior axis. sax-1 and sax-2, members of a conserved molecular pathway, regulate mechanosensory tiling in C. elegans by controlling termination of neurite growth. Specifically, the PLM process fails to terminate properly, leading to overlap of anterior and posterior mechanosensory neurons. Furthermore, over-expression of sax-2 in PLM causes premature termination of the PLM neurite, suggesting that SAX-2 may function cell autonomously to deliver an instructive signal to inhibit neurite growth. How does this conserved molecular pathway control neurite length? sax-2 encodes a large conserved protein with HEAT/Armadillo repeats that functions with sax-1, an S/T kinase. To better understand how sax-2 functions, I am currently using GFP fusions and immunocytochemistry to characterize the subcellular localization of SAX-2 in whole animals and primary neuron culture. My preliminary results show that sax-2 is expressed in a variety of cells and its subcellular localization is punctate. Experiments to further characterize the SAX-2 puncta are underway.

Maria E Gallegos et al. (2004) West Coast Worm Meeting "Mechanosensory neurite termination and tiling are directed by sax-2 and the sax-1 kinase."

Maria E Gallegos, Cori I Bargmann

[West Coast Worm Meeting, 2004]

Neurons possess an astonishing diversity of morphologies. While great strides have been made in understanding mechanisms of axon and dendrite guidance, less is known about cues that regulate neurite termination and the transition from outgrowth to stability. Neurite termination is particularly important in vision and somatosensation. In these sensory systems, an array of neurons restrict their dendrites to non-overlapping regions to provide accurate information about stimulus location, a pattern called tiling. How this pattern of dendrite termination develops is poorly understood. In C. elegans , two pairs of mechanosensory neurons (left and right) tile the surface of the animal along the anteror-posterior axis. The cell body and sensory process of ALM (L/R) and PLM (L/R) respond to gentle touch in the anterior and posterior half of the animal, respectively. During wild-type development, we found that the growing PLM sensory process did not terminate precisely at its final position. Instead, the PLM neurite overlapped transiently with ALM, and then resolved to a non-overlapping receptive field along the body surface. Resolution of overlap occurred during a discrete period of paused or slowed PLM process growth, between an early period of rapid outgrowth and a later period of maintenance growth where PLM growth matched animal growth to maintain a constant PLM length to animal length ratio. sax-1 and sax-2 , members of a conserved molecular pathway, regulate mechanosensory tiling in C. elegans by controlling dendrite termination. The sensory process of PLM fails to slow between the active growth and maintenance growth phases in sax-2 mutants, leading to sustained overlap of anterior and posterior mechanosensory processes. sax-2 encodes a large conserved protein with HEAT/Armadillo repeats that functions with sax-1 , an NDR S/T kinase in PLM termination and in other developmental pathways. Over-expression of sax-2 caused premature termination of the PLM mechanosensory neurite, suggesting that SAX-2 can deliver an instructive signal to inhibit neurite growth.

Maria E Gallegos et al. (2001) International C. elegans Meeting "sax-1 and sax-2 act in parallel with unc-34 to inhibit neurite outgrowth in the adult worm."

Maria E Gallegos, Cori Bargmann, Jennifer A Zallen

[International C. elegans Meeting, 2001]

Neurite outgrowth is a precisely regulated process. Furthermore, evidence suggests that the growth potential of neurons decreases with age. Both cell extrinsic and cell intrinsic factors have been implicated in this decline. We are interested in factors that prevent aberrant neurite outgrowth in the adult. These negative-acting factors could play a role in the potential of axons to regenerate following injury in humans. The initial outgrowth and guidance of axons are normal in sax-1 and sax-2 mutants, but at later larval stage and in the adult, sax-1 and sax-2 mutants have neurons with enlarged, irregular shaped cell bodies and ectopic neurites. Similar phenotypes have been seen in mutants that disrupt neuronal activity and function, but neuronal function is apparently normal in sax-1 and sax-2 mutants. This observation suggests that sax-1 and sax-2 may act more directly with the actin cytoskeleton to regulate cell shape and inhibit ectopic neurite outgrowth. To ask if repulsive axon guidance factors also play a role in regulating neurite outgrowth in the adult, we have begun to make double mutants of sax-1 and sax-2 with various axon guidance mutants including unc-34 , the worm homolog of Enabled. Evidence suggests that Enabled mediates repulsive guidance through the sax-3 / robo guidance receptor. Indeed, unc-34; sax-1 and unc-34; sax-2 double mutants have a strongly enhanced ectopic neurite outgrowth defect in late larval stages and the adult. This result suggests that sax-1 and sax-2 may act in redundant parallel pathways with unc-34 to inhibit ectopic neurite outgrowth at late larval stages. Alternatively, sax-1 and sax-2 may create a permissive environment for axon guidance pathways to respond to cues present in the adult animal. sax-1 encodes a serine/threonine kinase related to the Ndr protein kinase family in humans (62% id.) and flies (60 % id.). The function of Ndr kinase in neurons is unknown, but the Dm-NDR kinase and other closely related kinases have been shown to affect cell shape and polarity in nonneuronal cells. Since double mutant analysis places sax-1 and sax-2 in the same genetic pathway, I am in the process of cloning sax-2 in order to identify other components of this kinase pathway. I now have rescue with a small pool of cosmids. Progress in cloning and additional phenotypic characterization will be presented.

Maria E Gallegos et al. (2002) West Coast Worm Meeting "sax-1 and sax-2 maintain neuron morphology in larval and adult stages in C. elegans."

Jennifer A Zallen, Cori Bargmann, Maria E Gallegos

[West Coast Worm Meeting, 2002]

To generate a neural circuit, neurons send a characteristic number of axons and dendrites along stereotyped paths towards their final targets. Once these targets are reached, mature neurons increase in size with the animal, but stop sending out new processes and maintain a stable morphology. Evidence suggests that the maintenance of neuronal morphology is actively regulated throughout adulthood. For example, mature neurons from the peripheral nervous system (PNS) of vertebrates can switch from a state of maintenance to a state of re-growth following axotomy. What are the molecular mechanisms that initiate and maintain the transition from neurite growth to stability?

Friedman LC et al. (1996) Midwest Worm Meeting "mog-3 ETC."

Friedman LC, Kimble JE

[Midwest Worm Meeting, 1996]

The fem-3 gene is regulated post-transcriptionally to effect the switch from spermatogenesis to oogenesis (Barton et al., 1987; Ahringer and Kimble, 1991). The mog genes are excellent candidates for encoding trans-acting regulators of fem-3 to achieve this switch (Graham and Kimble, 1993; Graham et al., 1993; Gallegos and Kimble, unpublished). We will describe our initial efforts to clone mog-3 and to characterize it genetically.

Babadi, Nasrin et al. (2009) International Worm Meeting "Genetic control of axon guidance and branching: what have we learned from png-1?"

Colavita, Antonio, Gallegos, Maria, Arauz, Claudia, Babadi, Nasrin

[International Worm Meeting, 2009]

Understanding the cellular and molecular mechanisms governing axon guidance and branching is a central issue in developmental neurobiology. PNGases are highly conserved genes that are involved in the proteasomal degradation of misfolded glycoproteins during ER-associated degradation. We have previously reported the role of C. elegans PNGase, png-1 in regulation of axon branching at the vulva. png-1 mutants displayed excessive ectopic branching defects in the VC4 and VC5 motorneurons at the vulva. Additionally, png-1 mutants displayed overextension and branching defect in the DVB and AVL motorneurons. To further understand the role of png-1 in the regulation of axon guidance and branching, we conducted several genetic screens looking for enhancers/suppressors of png-1 associated branching defects. We identified sax-2 and sax-1mutants that enhanced png-1 branching in VC4 and VC5. Similarly, these mutants enhanced png-1 overextension and branching defects at the DVB and AVL. These findings suggest that png-1 and sax-1/sax-2 act in parallel genetic pathways to regulate axon guidance and branching. Furthermore, we analyzed homologues of genes involved in the ER-associated degradation in C.elegans for defects similar to png-1. We identified rad-23 to display overextension and branching defects in the DVB and AVL motorneurons only. Currently we are completing the characterization of these genes and we believe our finding will shed new light on mechanisms involved in axon guidance and branching.

Ablaza, Adriel-John et al. (2013) International Worm Meeting "The Visual Detection of odr-1 22G RNAs via a MosSCI Sensor System."

Juang, Bi-Tzen, L'Etoile, Noelle, Gallegos, Maria, Ablaza, Adriel-John

[International Worm Meeting, 2013]

Caenorhabditis elegans forages for food by distinguishing between various odorants in a dynamic environment. Their sensory neurons have the ability to adapt to persistent odors when the animal is starved (Bargmann, 2006). Adaptation to specific odors takes place in the AWC, a paired olfactory sensory neuron. In the AWC, a transmembrane guanylyl cyclase, odr-1, is required for chemotaxis towards all odorants. Prolonged odor exposure results in decreased chemotaxis, which correlates with a decrease in odr-1 mRNA. Odor adaptation is initiated by the translocation of a protein kinase, EGL-4, into the nucleus of the AWC (L'Etoile et al., 2002; Lee et al., 2010). Nuclear EGL-4 promotes a 22G directed repression of the odr-1 gene thereby initiating long-term odor adaptation (Juang et al., submitted). ChIP indicated that the heterochromatin state at the odr-1 locus increases during AWC odor adaptation and this increase requires 22G RNA synthesis. However, qRT-PCR and ChIP analysis does not offer a dynamic or cell-specific readout of odr-1 22G RNA function. Here we describe our plans to create a fluorescent reporter that is capable of tracking changes in odr-1 22G RNA function in live worms as they adapt to odor. The sensors that detect odr-1 22G RNAs are inserted via Mos Single Copy Insertion (MosSCI). These sensors will have the capability to detect specific odr-1 22G RNA function in the AWC olfactory sensory neuron as well as cells throughout the worm. The creation of a single copy insertion of an odr-1 small RNA sensor will allow us to test our hypothesis that a small RNA-directed pathway is dynamically activated during olfactory adaptation. In addition, this tool will allow us to understand whether odr-1 22G RNA represses transcription of our reporter in other cells in the worm.