Ray, Shinjini et al. (2021) International Worm Meeting "Regulation of syncytial germline mechanics by the actin capping protein CAP-1"

Zaidel-Bar, Ronen, Agarwal, Priti, Ray, Shinjini

[International Worm Meeting, 2021]

Actin cytoskeleton remodelling is important for executing dynamic cellular processes during tissue morphogenesis and physiology. Actin filaments in the cytoskeletal network are dynamic structures that polymerise in a regulated manner. The actin filament barbed end, a hotspot of filament assembly, is regulated by competition among actin nucleators, elongation factors and capping protein, which in turn control filament length and network stability and is also capable of generating force by pushing against other structures. Although actin nucleation and elongation factors have been relatively well studied, the role of actin capping proteins in-vivo remains poorly understood. We investigated the role of the actin capping protein CAP-1 in the C. elegans germline, a contractile tissue. Our observations show that endogenously tagged CAP-1 colocalises with the actin cytoskeleton along cell boundaries and rachis bridges in the germline. CAP-1 knockdown in the germline exhibits a disrupted structure characterised by a narrow and meandering rachis, as well as multinucleate germ cells and abnormal oocytes. Further, we observed an increase in levels of F-actin and also in the levels of the actin nucleators CYK-1 and ARX-2. In order to investigate if the structural changes observed in the rachis are accompanied by changes in contractility, we checked the levels of absolute and phosphorylated myosin II and observed their levels to be elevated in CAP-1 depleted background as compared with control, indicating an increase in contractility. The increase in contractility is likely responsible for the constricted and meandering rachis, pulling at the membranes and also resulting in multinucleate cells, in regions where the connecting membrane detached. In conclusion, we show that CAP-1 is essential for maintenance of germline function, it regulates actomyosin structure and levels to maintain the structural integrity of the germline.

Zhang YH et al. (1995) International C. elegans Meeting "A C. elegans Pax-6 HOMOLOG GOVERNS THE IDENTITY OF A PERIPHERAL SENSE ORGAN"

Emmons SW, Zhang YH, Hall DH

[International C. elegans Meeting, 1995]

How do cells find their correct neighbors during the differentiation of precise tissue architecture? This problem arises in the assembly of sense organs in C. elegans. In the male tail, each sensory ray has a distinct morphogenetic identity. Mutations that alter ray identities disrupt the specificity of ray assembly, resulting in fused rays. The mab-18 locus is required for expression of the ray 6 identity. mab-18 consists of the homeodomain portion of a C. elegans Pax-6 gene. cDNAs that rescue the mab-18 mutant phenotype start within a large intron separating Pax-6-like paired and homeodomains. Reporter gene expression shows that a promoter within the intron is activated during ray 6 development and remains active in the adult. It is also transiently activated during ray 8 development. We propose that the mab-18 transcription factor regulates expression of ray 6-specific cell recognition and adhesion molecules. These may be expressed by the ray structural cell. By eliminating the neurons with a laser, we have shown that the morphogenetic properties of rays are determined by the structural cell. In a wild type background the ray 6 structural cell generates a normal ray 6 with thick, tapering morphology. In a mab-18 mutant background it forms a thin, cylindrical ray at the position of ray 4. If ray 4 is present, the ray 6 structural cell fuses with the ray 4 structural cell. When neurons are present, both ray 4 and ray 6 neurons are incorporated into the fused ray.

Scott Everet Baird (2001) International C. elegans Meeting "Ray pattern and HOM-C gene variation in Caenorhabditis briggsae."

Scott Everet Baird

[International C. elegans Meeting, 2001]

Ray pattern is one of the most variable morphological characters in rhabditid nematodes. Within Caenorhabditis , a modified ray pattern in which ray 3 is posterior of the anus and frequently fused with ray 4 is diagnostic for C. briggsae . This diagnostic character is based on observations of a limited number of C. briggsae strains. Recently, a C. briggsae strain, PB800, was obtained that exhibited an elegans ray pattern at a high frequency. Prompted by this observation, ray pattern variation has been assessed in four other C. briggsae strains, AF16, HK104, HK105, and VT847. AF16 and VT847 exhibited predominately the canonical briggsae pattern whereas HK104, HK105, and PB800 exhibited briggsae and elegans patterns at approximately equal frequencies. PB800 also exhibited a sixless phenotype, in which ray six is morphological transformed and fused with ray 4, at an approximately 1% frequency. In C. elegans this phenotype results at high frequency from mutations mab-18 and mab 21 and at low frequency from haploinsufficiency of egl-5 . Moreover, altered levels egl-5 and mab-5 expression can mimic the canonical C. briggsae ray pattern. Thus, ray pattern variation in C. briggsae may result from allelic variation in C. briggsae HOM-C genes. To test this hypothesis, PCR product length polymorphisms have been identified in the C. briggsae homolog of egl-5 ( Cb_egl-5 ). These Cb_egl-5 polymorphisms will be tested for linkage to variation in ray pattern. If linkage is detected, additional experiments will be conducted to map the C. briggsae ray pattern variants to a single locus.

Siu Wah Choy et al. (2004) East Asia Worm Meeting "Characterization of a transcriptional repressor complex in male sensory ray patterning of C. elegans"

Siu Wah Choy, King L Chow, Yau Ming Wong

[East Asia Worm Meeting, 2004]

SIN-3, a histone deacetylase corepressor associated molecule, has longed been identified as a repression mediator in transcription. Yet, few studies have evaluated its role as a developmental regulator. The isolation of C. elegans SIN-3 through yeast two-hybrid screen as an interacting partner of MAB-21 provides the first clue to this notion. Among the regulatory genes that pattern sensory ray structure, mab-21 is required for the specification of the ray 6 identity. To evaluate the developmental requirement of sin-3 , expression analysis with fluorescent reporter was examined. Result revealed that sin-3 was not turned on ubiquitously but was found only in a subset of cells. It was first detected at the L1 stage in some head neurons. Expression could be seen in the ventral nerve cord, ray structural cells from ray 1 to ray 6 and neurons in the tail and the head region in adults. Most importantly, overlapping expression of mab-21 with sin-3 in the ray 6 structural cell lends strong support to the temporal spatial requirement of these two molecules to interact during ray 6 differentiation. Furthermore, Trichostatin-A treatment blocking the histone deacetylase activity in worms resulted in a plethora of ray phenotypes, indicating that histone deacetylase is critical for ray cells identity determination. Yeast two-hybrid system was further employed to dissect this C. elegans corepressor complex components. Results suggested that there was an important link between BMP signaling pathway and transcriptional repressor in ray patterning. Moreover, novel SIN-3 associated corepressor components in C. elegans were identified through the two-hybrid screen using SIN-3 as a bait. Among several positive clones obtained, some appeared to be required for ray patterning and displayed various ray phenotypes in RNAi knockdown animals. The results suggest that these novel interacting partners are indeed required specifically for ray patterning. Their properties as well as their role in ray cell identity determination will be discussed.

David C.K. Leung et al. (2008) "Identification of MAB-22 targets mediating sensory ray assembly in C. elegans"

David C.K. Leung, Alfred W.H. Chan, King L. Chow

[2008]

"Each male tail sensory ray is composed of one structural cell and two neuronal cells within the hypodermis. Previous studies demonstrated that elimination of the structural cells but not the neuronal cells by laser or genetic ablation could result in a ray missing phenotype, thus highlighting the importance of structural cells for ray formation. The mab-22(bx59) mutant shows an extensive ray-missing phenotype at the non-permissive temperature of 25°C. The phenotype is caused by the assembly failure of the corresponding cellular constituents in a ray. Indeed, mab-22 encoding a T-box containing transcription factor-TBX-2 is also expressed in structural cells. These results suggest that the structural cells and the activity of mab-22 there play an essential role in ray assembly. To gain insight into the molecular events underlying the ray assembly process, molecular and in silico analyses as well as a candidate gene approach were adopted to identify the structural cell-expressed genes regulated by MAB-22. These candidate target genes were validated by subjecting them to a structural cell-specific reporter screen to differentiate their roles in ray cell lineage or assembly process. Finally, we focused on seven of these genes shown to be required for the ray assembly process. Preliminary data revealed that inx-12, encoding a gap junction subunit, is transcriptionally regulated by MAB-22 in structural cells. Interestingly, the RNAi ray missing phenotype for both inx-12 and its paralog inx-13, is similar implicating their common biological roles in ray development. Besides, they co-localize to form punctuate plaques at the interface of the ray structural cells and hypodermis, raising the possibility that these two innexins function together. Future experiments will test how these potential functional pairing of innexins organize the ray cells into a functional organ. Both the phenotypic characterization and genetic analyses of the rest of the putative ray assembly genes will be reported in this presentation. (This study is supported by the Research Grants Council, Hong Kong)."

Douglas S Portman et al. (2001) International C. elegans Meeting "Neuronal fate specification by bHLH proteins in the C. elegans ray sublineage"

Douglas S Portman, Scott W Emmons

[International C. elegans Meeting, 2001]

The use of repeated sublineages is likely to be a common theme in development. In the nervous system, sublineages provide an ideal opportunity to examine the integration of mechanisms that specify general neuronal properties with those that specify the more restricted characteristics of subtype identity. We have been studying the sensory rays of the C. elegans male tail to characterize the regulatory network that controls selection of the ray precursor cells and establishes the four distinct fates of their progeny. During the male L3 stage, nine bilateral pairs of posterior seam cells are specified to become the ray precursor cells, or Rn cells. Through the execution of the ray sublineage, each Rn cell clonally gives rise to the three cell types of each ray (the ray structural cell Rnst and the ray neurons RnA and RnB) as well as a cell that undergoes programmed cell death. Superimposed on the ray sublineage are patterning mechanisms that generate differences between corresponding cells of different rays, including structural cell morphology and ray neuron neurotransmitter identity. The atonal -family bHLH transcription factor LIN-32 is expressed in all of the ray precursor cells, and is required to specify their fates as neuronal precursors. Because strong lin-32 alleles cause nearly complete ray loss, and ectopic expression of lin-32 can specify ectopic ray sublineages, lin-32 can be thought of as a ray proneural gene, acting early to implement commitment to the ray sublineage. However, lin-32 has later functions in ray development as well. By using weak lin-32 alleles along with ray-neuron-specific GFP markers, we have found that loss of lin-32 function can disrupt multiple steps in the ray sublineage, uncoupling cell fate specification in one branch from that of another. lin-32 function is also required for the terminally-differentiated characteristics of ray neurons and ray structural cells. These findings suggest that lin-32 has additional independent functions after the specification of the Rn cell, and are supported by recent results in Drosophila demonstrating that atonal functions in multiple steps of neuronal development as well. To better understand these multiple functions of lin-32 , we screened for suppressors and enhancers of the ray loss phenotype of a weak lin-32 allele and recovered two alleles of the gene hlh-2 . hlh-2 encodes CeE/Da, the worm member of the E/ daughterless family of general bHLH heterodimerization partners. Our hlh-2 mutations enhance the multiple ray defects caused by weak lin-32 alleles, and disrupt the formation of DNA-binding LIN-32:HLH-2 complexes in vitro . Together, these results indicate that lin-32 and hlh-2 are generally required throughout the ray sublineage for multiple fate-specification events, and imply that the LIN-32:HLH-2 complex has multiple, independent targets for different steps in ray development. To identify targets of the LIN-32:HLH-2 complex that implement ray cell fates, we are taking a microarray-based approach in collaboration with Stuart Kim (Stanford University). Using the hermaphrodite-lethal mutation dpy-28(y1ts) , we have compared whole-genome mRNA expression patterns in adult male populations lacking rays ( hlh-2; lin-32 mutants) to those with excess rays ( lin-22 mutants). This approach has thus far identified two new genes expressed in rays, the Onecut-homeodomain transcription factor ceh-39 and the G-protein coupled receptor C03A7.3. We are generating additional data in order to identify a more complete set of ray genes, and are using methods to identify sequence motifs enriched in the promoters of ray-specific genes. We plan to expand this analysis by profiling the gene expression patterns of wild-type animals from mid-L3 to adult, in order to be able to visualize changes in transcription during the ray sublineage.

Ip, KK et al. (2011) International Worm Meeting "The nature of tbx-2 expression pattern in C. elegans male tail."

Chow, KL, Ip, KK

[International Worm Meeting, 2011]

Development of C. elegans male tail sensory rays involves multiple processes such as ray lineage, ray assembly, patterning and morphogenesis. Previously we identified tbx-2 as a gene responsible for ray assembly during male tail development. Ray assembly defect can be found in all rays in tbx-2 mutants, but the expression pattern of tbx-2 in adult male rays was restricted to the structural cells of rays 1, 5, 7 and occasionally in R4B neurons, as revealed by GFP transcriptional reporter. tbx-2 is under its own negative regulation in all rays via direct binding of the protein to a negative regulatory element on its locus region; disruption of which results in up-regulation of tbx-2 expression in all ray structural cells in male tail. We are interested in how tbx-2 expression pattern is only subjected to tight negative feedback in rays other than ray 1, 5, 7 and what its significance is. Several candidates of modifiers of tbx-2 expression, including ray patterning genes such as the SMADS, and ceh-43, a homeobox gene, were evaluated. tbx-2 reporter activity was altered in SMADS mutants. tbx-2 expression is reduced in ray 5 in general. Site specific mutation of a ceh-43 putative binding site - CAATTA - on promoter of tbx-2 transcriptional GFP reporter results in loss of GFP signal in rays, hinting a direct regulation of tbx-2 by ceh-43. Protein-DNA binding assays will be carried out to confirm this direct association. Meanwhile, additional upstream components, e.g., lin-32, and its interaction with ray assembly genes has been examined. Our final goal is to delineate the developmental program leading to ray assembly and patterning. The expression pattern of the tbx-2 also hinted ray-specific function of tbx-2 on ray identity control. We have investigated this feature using neurotransmitter (NT) production as a ray identity marker in tbx-2 mutants, where changes in ray specific NT marker expression was noted. For example, flp-5, originally expressed in neuron B of rays 1, 5, 7 is also expressed in ray 4 of tbx-2 mutant males. Subsequent temperature shift experiment showed that tbx-2 is required for the determination step, but not for the maintenance step. Whether such changes are a result of ray identity alteration executed by tbx-2 expression remains to be resolved. (This study is supported by Research Grants Council, Hong Kong).

Portman DS et al. (2000) East Coast Worm Meeting "The bHLH transcription factors LIN-32 and HLH-2 specify multiple aspects of ray development in the C. elegans male tail"

Emmons SW, Portman DS

[East Coast Worm Meeting, 2000]

During the male L3 larval stage, nine pairs of posterior hypodermal cells begin to execute the ray sublineage, generating the two neurons and one structural cell that comprise each ray. We are interested in understanding how the fates of ray neuroblasts and differentiated ray cells are determined, and how the asymmetric cell divisions of the ray sublineage allocate these fates. We have found that two genes encoding bHLH transcription factors, lin-32 (belonging to the atonal family of proneural genes) and hlh-2 (the worm ortholog of the E/daughterless family of heterodimerization partners), are required for, and expressed during, ray development. In both lin-32 and hlh-2; lin-32 mutants, a spectrum of ray development defects can be observed. In strong loss-of-function backgrounds, the ray precursor cell usually gives rise only to hypodermal cells, suggesting an early role for these genes in neuronal commitment. In weaker alleles, a variety of partially-successful sublineages occurs. Execution of the anterior and posterior branches of the sublineage can become uncoupled from each other; that is, A-type ray neurons can be generated in the absence of their cognate ray structural cells, and vice versa. This implies that the two branches of the sublineage independently require lin-32 and hlh-2 function. Finally, when ray cells are generated, they often have abnormal or absent processes. We conclude that lin-32 and hlh-2 are required for several cell-fate determination steps, from early commitment to ray fate, to intermediate fates in the ray sublineage and the establishment of differentiated characteristics. To understand these functions at a biochemical level, we are studying the DNA-binding and heterodimerization properties of these proteins, and have found that LIN-32 and HLH-2 can heterodimerize and bind to E-box-containing sequences in vitro. We are currently determining the effects of mutations on these activities and investigating the sequence-specificity of DNA binding. In collaboration with Stuart Kim, we have also begun to use microarrays to identify potential LIN-32:HLH-2 target genes expressed during the ray sublineage and in mature rays.

Douglas S. Portman et al. (2007) International Worm Meeting "Wnt-regulated asymmetric expression of the Atonal/MATH-family factor LIN-32 patterns the progeny of the ray neuroblast."

Douglas S. Portman, Renee M. Miller

[International Worm Meeting, 2007]

Each sensory ray in the C. elegans male tale is made of three cells that descend from a single precursor, the ray neuroblast. Through two rounds of asymmetric division, the ray neuroblast gives rise to two sensory neurons (RnA and RnB), the ray structural cell (Rnst) and a programmed cell death (Rn.aap). This provides an excellent model in which to explore the genetic mechanisms that determine sex-specific neural and glial cell fates. The development of all three ray cell types requires the function of the atonal-class bHLH factor LIN-32. In lin-32 null mutants, very few rays develop; those that do form require the activity of HLH-2, the E/Daughterless-family factor that heterodimerizes with LIN-32. Consistent with this proneural-like role, lin-32 expression in the ray sublineage is first detectable in the common precursor of all three ray cells, the ray neuroblast Rn.a. Interestingly, however, once the Rn.a cell divides, lin-32 expression becomes asymmetric, persisting only in Rn.aa. This pattern repeats itself when Rn.aa divides, such that lin-32 remains on in Rn.aaa (which becomes the RnA neuron) but not its sister Rn.aap (the dying cell). This unexpected finding suggests that asymmetric lin-32 expression could be important for the patterning cell fates among the four progeny of each Rn.a cell. Recent results suggest that asymmetric lin-32 expression does indeed have an important function in ray development. In lin-17/Fz mutants, we find that both asymmetric lin-32 expression and ray cell fate is disrupted. These animals exhibit ectopic expression of lin-32 during the ray sublineage, indicating that Wnt signaling is necessary to restrict lin-32 to the anterior branch of the ray sublineage. Moreover, the RnA marker dat-1 is expressed in many additional cells in the lin-17 male tail, suggesting that ectopic lin-32 expression may promote RnA-like fate in the RnB neuron. To test this, we have expressed lin-32 throughout the late ray sublineage with a heat-shock promoter. In these animals, we observe loss of the RnB marker cwp-4 and some ectopic expression of dat-1. Together, these findings indicate that lin-32 acts not only as a proneural gene in the ray neuroblast but also to specify neural subtype among its descendants.

Stephen S H Ho et al. (2001) International C. elegans Meeting "Delineation of enhancer elements and trans-acting factors controlling mab-21 activity in sensory ray differentiation."

Stephen S H Ho, King L Chow

[International C. elegans Meeting, 2001]

In C. elegans male tail, the major peripheral sensory structures comprise of nine pairs of ray sensilla embedded in a cuticular fan. Each ray has its own specific morphological and functional identity as defined by a group of regulatory genes. Among them, mab-21 regulates ray 6 identity. To establish the requirement of mab-21 in normal development, we dissect the regulatory elements in the mab-21 locus using both rescue and reporter assay approaches. A minimal genomic fragment that can efficiently rescue the mab-21 mutant sensory ray phenotype was defined. Using green fluorescent protein (GFP) as the reporter system, we demonstrate that mab-21 locus contains multiple transcriptional regulatory elements including hypodermal, neuronal and structural cell enhancer. The hypodermal enhancer lies in the 5' flanking sequence of the gene, which direct expression of the reporter in body and tail hypodermal tissue. A neuronal enhancer is located in intron 4. This element generates fluorescent signal in the neurons of pharyneal bulb, ventral nerve cord and the sensory ray. The 0.7kb enhancer residing in the 3' flanking sequence of the locus directs the expression of the reporter in the sex muscle, a subset of sensory ray neurons and structural cells of ray 2 and 6. We present data showing that not all these enhancer elements are required for ray differentiation. Both hypodermal and neuronal enhancer are dispensable for specification of the ray 6 identity, as demonstrated by rescue experiment with mab-21 mutants. The only cis -element essential for ray 6 morphology determination is the 3' enhancer. Delimitation of this enhancer element, identification of its associated trans -acting factors are in progress. The outcome of such experiments would provide insight about the regulation of mab-21 gene and indication of its tissue specificity with reference to sensory ray 6 differentiation .