Diaz-Balzac C A et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Identification of novel loci interacting with the Kallmann Syndrome gene kal-1"

Diaz-Balzac C A, Buelow H E

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Kallmann syndrome (KS) is characterized by two major physiological deficits, anosmia and hypogonadism. These deficits are the likely result of a neuronal targeting defect of the olfactory axons and a migration failure of GnRH-secreting neurons to the hypothalamus. To date, five genes associated with this syndrome have been identified, namely, KAL1, FGFR1, FGF8, PROKR2, and PROK2; though these only account for approximately 30% of all KS cases. The first gene identified was KAL1, which encodes a secreted cell adhesion protein named anosmin-1 and is responsible for the X-linked form of the KS. Misexpressing the homolog of anosmin-1/KAL1 in Caenorhabditis elegans causes a highly penetrant axonal branching phenotype. In a small pilot modifier screen of the kal-1 gain of function phenotype we have previously isolated a total of seven alleles including mutations in the Heparan sulfate (HS) 6O-sulfotransferase, the HS epimerase and the PAPS (phosphoadenosyl-phosphosulfate) transporter 1. We have expanded this screen to identify more loci that genetically interact with kal-1. To date, we have isolated an additional 19 candidate mutations including 18 suppressor mutants and one enhancer mutation of the branching phenotype. We will report on the progress of characterizing the newly identified mutations. Characterization of the identified genes should afford us to gain a deeper understanding of how kal-1 acts during the development of the nervous system and the role of the extracellular matrix in this process. Additionally, these genes may represent candidate genes to cause Kallmann Syndrome in humans.

Saied-Santiago, K. et al. (2017) International Worm Meeting "Genetic and biochemical analyses between the Heparan sulfate proteoglycans and Wnt signaling proteins in Caenorhabditis elegans."

Bulow, H., Saied-Santiago, K., Townley, R., Diaz-Balzac, C.

[International Worm Meeting, 2017]

Heparan sulfates (HS) are unbranched glycans containing substantial modification patterns in the way of acetylation, sulfation, and epimerization. These extracellular polysaccharides are bound through conserved serine residues to core proteins to form Heparan sulfate proteoglycans (HSPGs). HSPGs are known to regulate cell adhesion, motility, and signaling by modulating protein-protein interactions. Moreover, they have been implicated as co-factors of many morphogens and extracellular proteins including FGF, Wnt, Slit/Robo, among others. Extensive research in Caenorhabditis elegans has established a role of the Wnt family genes in anterior-posterior cell migrations and axon pathfinding. However, the mechanisms regulating Wnts and the frizzled receptors in these processes are largely unknown. To investigate the role of HSPGs with Wnt signaling during cell migration, we performed a genetic analysis between these gene networks in C. elegans. Our findings show that syndecans and glypicans, both membrane-bound proteoglycans, function to promote migrations and determine final cellular positions in Caenorhabditis elegans. Double mutant analyses between HSPG genes demonstrate distinct redundancies among different anterior-posterior neuronal migration events. Moreover, HSPGs genetically interact with Wnt-ligands and frizzled receptors in a context-dependent manner to mediate cell migration and axon guidance. Based on our genetic analyses, we hypothesize that the transmembrane SDN-1/Syndecan forms a biochemical complex with MIG-1/Frizzled and EGL-20/Wnt. Co-immunoprecipitation experiments suggest that the Frizzled receptor MIG-1/Fz physically interacts with the EGL-20/Wnt-ligand and SDN-1/Syndecan in an in vitro heterologous system. Future work will focus on the structure-function analysis of SDN-1, including the involvement of its conserved heparan sulfate attachment sites, and their significance for biochemical interactions with Wnt signaling proteins. We propose that HSPGs act redundantly as co-receptors of Wnt-signaling to regulate defined neuro-developmental cell and axonal migrations.

Rahman, M. et al. (2017) International Worm Meeting "Uncovering interacting factors of Leukoctye Cell-Derived Chemotaxin 2 (lect-2) in dendritogenesis."

Diaz-Balzac, C. A., Bulow, H. E., Rahman, M.

[International Worm Meeting, 2017]

Dendrite development is essential for the transmission and processing of sensory stimuli. The mechanisms of dendritogenesis are not fully understood, but abnormalities in dendrite morphology have been found in several neurological disorders. We use the multi-dendritic C. elegans PVD neurons, which have complex menorah-like dendritic arbors, as a model to study the genes involved in dendrite development. Studies from our lab and others have shown that a conserved cell-adhesion complex, comprised of MNR-1/Menorin and SAX-7/L1CAM, acts from the skin to regulate PVD dendrite branching through the transmembrane receptor, DMA-1/LRR-TM, deemed the 'menorin' complex. Recently, we determined that the conserved gene, Leukocyte Cell-Derived Chemotaxin 2, or lect-2/Chondromodulin II, also functions to pattern PVD dendrites. Though implicated in immune response and a range of pathologies, the developmental functions of lect-2/ChM-II have not fully been characterized. Acting from an entirely different tissue- the body-wall muscle- LECT-2/ChM-II is a diffusible factor that is a key player in the 'menorin' complex. Its localization is fully dependent on SAX-7/L1CAM (but not MNR-1/Menorin or DMA-1/LRR-TM) and double mutant analyses show that lect-2 acts in the same genetic pathway as mnr-1/Menorin, sax-7/L1CAM, and dma-1/LRR-TM. We propose that LECT-2/ChM-II functions as not only a co-factor to the 'menorin' complex, but also in concert with other genes from potentially different tissues. In order to elucidate other binding factors of LECT-2/ChM-II, we are performing two forward genetic screens. In the first, we aim to isolate modifiers of a lect-2/ChM-II hypomorphic allele. In the second, we aim to isolate genes required for correct localization of LECT-2/ChM-II with the help of a FACS based Worm Sorter. In preliminary experiments, we have isolated novel alleles of not only sax-7/L1CAM, but of potentially novel interactors, which we aim to characterize in future experiments. Overall, we aim to gain a deeper and fuller understanding of lect-2/ChM-II and the interplay of different tissues and factors involved in dendrite development-eventually providing insight into developmental mechanisms and potential diagnostic approaches for neurodevelopmental disorders.

Salazar, C. J. et al. (2019) International Worm Meeting "A novel Rab GTPase restricts dendrite branching."

Bulow, H. E., Grant, B. D., Diaz-Balzac, C. A., Salazar, C. J.

[International Worm Meeting, 2019]

Dendrite development depends on numerous extracellular and intracellular cues to ensure proper structure and function. However, the regulatory mechanisms of dendrite development remain incompletely understood. To better understand dendrite development, we are utilizing the PVD somatosensory neuron with its highly stereotyped 'menorah'-like dendrites. The Menorin genetic pathway consists of several factors that function from different tissues to promote PVD dendrite development. While progress has been made in understanding factors that promote the formation of dendritic branches, much less is known about factors that restrict branching. During a genetic screen, we have identified a locus that encodes for a putative, uncharacterized Rab GTPase, which we name rab-X. Time course analyses determined that the number of branching points was significantly increased in adult animals. This suggests rab-X suppresses dendritic branching into adulthood. A transcriptional reporter shows expression to be in the epidermis from early embryonic through adult stages and transgenic expression of a rab-X cDNA in the epidermis is sufficient to rescue the mutant phenotype. Genetic analyses show that the Menorin pathway is largely epistatic to rab-X, indicating it may function in the same genetic pathway. In addition, we found that a rescuing N-terminal translational RAB-X fusion displays intracellular, perinuclear localization. We will present our progress to (1) determine whether mutations in rab-X affect localization of components of the Menorin pathway, to (2) determine the subcellular localization of rab-X, and to (3) determine if GTPase activity of rab-X is necessary for restricting dendritic growth.

Tang, L.T.-H. et al. (2019) International Worm Meeting "The TIAM-1 guanine nucleotide exchange factor shapes somatosensory dendrites independently of Rac1 guanine nucleotide exchange activity by controlling F-actin localization."

Ramierz-Suarez, N., Tang, L.T.-H., Diaz-Balzac, C., Salzberg, Y., Rahman, M., Lazaro-Pena, M., Bulow, H.

[International Worm Meeting, 2019]

Development of dendritic arbors is crucial for nervous system assembly, but the intracellular mechanisms that govern these processes remain incompletely understood. The somatosensory PVD neurons in C. elegans is an excellent model to study the genetic and molecular determinants of dendritic branching and growth due to its stereotypical structure. Previously it has been shown that the DMA-1 leucine rich transmembrane (LRR-TM) receptor is expressed in PVD and forms a complex with the cell adhesion molecules MNR-1/Menorin, SAX-7/L1CAM from the epidermis and cytokine LECT-2/Chondromodulin II secreted from muscle, thereby providing spatial cues to developing PVD dendrites. However, the molecular machinery downstream of DMA-1 remained unclear. Through forward genetic screens, we uncovered three more genes necessary for PVD dendrite morphogenesis that interact with DMA-1 in PVD: hpo-30, tiam-1, and act-4. We further demonstrated through double mutant analysis and rescue experiments that the guanine nucleotide exchange factor TIAM-1 and ACT-4/Actin function with DMA-1 in PVD to pattern higher order branches by localizing F-actin to the distal ends of developing dendrites, while secondary and tertiary branch growth is modulated by TIAM-1 and DMA-1 in parallel pathways. Biochemical experiments show that DMA-1 is part of a biochemical complex with TIAM-1 through PDZ ligand-domain interaction, which localizes TIAM-1 to quaternary branches. Surprisingly, we found that TIAM-1 appears to function independently of its canonical Rac1 guanine nucleotide exchange factor activity, and may directly modulate actin dynamics. We also found through colocalization and time-lapse imaging of tagged DMA-1 and HPO-30 that HPO-30 regulates the internalization of DMA-1, which in turn influences PVD dendrite development. Collectively, our experiments suggest that the DMA-1/LRR-TM receptor on PVD dendrites may control aspects of dendrite patterning by directly modulating F-actin dynamics.

Desse, V. et al. (2019) International Worm Meeting "SAX-7/L1CAM in the maintenance of neuronal architecture."

Perrat, P., Blanchette, C., Desse, V., Benard, C.

[International Worm Meeting, 2019]

Whereas remarkable advances have been made in understanding neuronal development, the mechanisms that maintain the nervous system architecture lifelong are poorly understood. How does the nervous system established during embryogenesis subsequently maintain its integrity throughout life, despite the animal's growth, the addition of new neurons, maturation processes, body movements, and aging? The evolutionarily conserved gene sax-7, homologous to mammalian L1CAM, is one of the factors required for maintaining the structural integrity of ganglia and fascicles in C. elegans1. Thus, sax-7 not only contributes to nervous system development (roles in guidance and fasciculation of a subset of developing neurites)2, but is also required for maintaining the organization of neural architecture after its initial development. Indeed, some neural structures that initially develop normally become disorganized in animals lacking the function of sax-7. With heat shock assays, we show that expression of sax-7S(+) during larval stages is sufficient to rescue the maintenance defects of sax-7 mutants that were devoid of functional sax-7 during nervous system development. We are characterizing a true null allele (generated using CRISPR/Cas9), as well as the expression pattern of a functional SAX-7S::sfGFP (insertion in the endogenous locus using CRISPR/Cas9), and we find that SAX-7S is present in neurons, at the plasma membrane. We are exploring the structure-function relationship of SAX-7S, a transmembrane protein harboring extracellular Ig and FnIII domains, in the context of maintenance of the nervous system architecture. A conditional knockout of L1CAM in the adult mouse brain results in behavioral and learning defects, indicating that L1CAM plays a post-developmental role in mammals as well. Our studies are expected to help understand the role of SAX-7/L1CAM in conserved molecular mechanisms ensuring neuronal maintenance, which may go awry in neurodegenerative conditions. 1 Pocock, Benard et al 2008; Sasakura et al 2005; Wang, Chen et al 2005; Zallen et al 1999; Ramirez-Suarez et al 2019 2 Diaz-Balzac et al 2015; Salzberg et al 2013; Yip and Heiman 2018, Dong et al 2013; Zhu et al 2017

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.