Nishiwaki K (1995) Worm Breeder's Gazette "Mutations With Extra Turning Phenotype of DTCs."

Nishiwaki K

[Worm Breeder's Gazette, 1995]

Mutations with extra turning phenotype of DTCs Kiyoji Nishiwaki Fundamental Research Laboratories, NEC Corporation, Miyukigaoka, Tsukuba 305, Japan During hermaphrodite development, the two distal tip cells (DTCs) located on the anterior and the posterior ends of the gonad primodium migrate and make the two U-shaped arms of the gonad in rotational symmetry; the anterior arm to the right and the posterior arm to the left of the intestine. Although the migration of DTCs is just more complicated than the other long range migrations, analysis of the DTC migrations is advantageous because the trajectories of the DTCs can be deduced from the shape of the gonad arms (1). I have isolated four recessive mutations in four different genes affecting the turning frequency of DTCs. DTCs normally turn twice when they change their migrating directions from along the ventral muscles to dorsalward and from dorsalward to along the dorsal muscles. However, DTCs in these mutations often make extra turns once or more after the apparently normal two turns. The extra turned arms usually run to the opposite direction along the unoccupied surface of the dorsal muscles. In addition to DTC, some embryonic (HSNs, CANs, ALMs, coelomocytes) and postembryonic migrations (QRpa and QLpa descendants, excretory canals) were observed in these mutants and summarized below. mig(kl O9) X: Both arms usually made extra turns more than twice; 98percent for anterior and 98percent for posterior. No penetrant abnormality was observed for other migrations. migfkl21) ?: Anterior arms often made one extra turn; 42percent for anterior and 15percent for posterior. No penetrant abnormality was observed for other migrations. mig(kl23) IV: Posterior arms often made one extra turn like kl21; 15percent for anterior and 32percent for posterior. Left embryonic coelomocytes were often positioned too anteriorly. mig(kl24) 11: Posterior arms often made one extra turn like kl21; 3percent for anterior and 35percent for posterior. QL migration was reversed and QRpa descendants (SDQ, AVM) were posteriorly misplaced. HSNs were often too posterior. The mutant often had a protruding vulva or none and was slightly uncoordinated. All of the mutations are likely to affect the turning programs of DTCs. It is interesting that the frequencies of extra turns for anterior (right) and posterior (left) arms are not always the same in spite of the symmetry of the arms. Similar observations are reported for dorsal migrations of DTCs in unc-5 and unc-62. There are three phenotypic classes, I) both arms are affected similarly (k109); 2) anterior or right arms are affected more frequently than posterior or left arms (k121); 3) posterior or left arms are affected more frequently than anterior or right arms (k123, kl24). Because no other migration defect was detected in kl09 and kl21, these genes may function specifically for the DTC migration. On the other hand, kl23 and kl24 showed some other migration defects. kl24 was very pleiotropic. So, these genes seem to define more general functions for cell migration and/or cell type specification. I am presently isolating more mutants with an extra turning phenotype too identify a group of genes responsible for this phenotype. 1. Hedgecock et al. (1987). Development 100, 365-382. (2). Hedgecock; et al. (1990). Neuron 2. 61-85.

Hobert O et al. (1996) Worm Breeder's Gazette "LIM-homeobox genes in the worm"

Hobert O, Liu Yanxia, Ruvkun GB

[Worm Breeder's Gazette, 1996]

LIM-homeobox (LHX) genes, which encode proteins with two Zn-finger-like LIM domains and a DNA-binding homeodomain, have been shown to play roles in nervous system development of both invertebrate and vertebrate organisms. Originally, three LIM-homeobox genes were characterised in C.elegans: lin-11 and mec-3 (founding members of the LIM-homeobox family) were identified genetically (Freyd et al., 1990, Way et al., 1988), while the ceh-14 LHX gene was identified in a sequence based screen for homeobox genes (B!rglin et al., 1989). By doing lots of GENEFINDER and BLAST searches, we found that the C.elegans genome project has sequenced 4 more LHX genes, located on cosmids ZC64, C40H5, K03E6 and C04F1, which we termed CeLIM-4,-5,-6 and -7, respectively. We are interested in characterising the function of these genes by expression pattern analysis, and phenotypic analysis of animals bearing mutations in these genes. CeLIM-4 is located on the X chromosome right next to ceh-18. We constructed a CeLIM-4-GFP translational fusion and found CeLIM-4 to be expressed exclusively in several pairs of head neurons of which at least one is an amphid sensory neuron. We are in the process of identifying these cells. CeLIM-4 maps close to the let-43 gene; we are trying to rescue this mutant with CeLIM-4 but think it is unlikely that a mutant head neuron-specific transcription factor would cause lethality. In collaboration with R.Plasterk we have started to search for Tc1-insertions. CeLIM-5 is the C.elegans ortholog of the Drosophila apterous and vertebrate LH2/LHX2-genes. We found CeLIM-5 to be encoded by the ttx-3 gene (see accompanying abstract by Hobert et al.). In adults, CeLIM-5/ttx-3 is exclusively expressed in the AIY interneuron. CeLIM-6 is located on the X chromosome (next to unc-1) and is most closely related to the Lmx-subclass of LHX genes. Like CeLIM-4 and CeLIM-5, CeLIM-6 also shows a neuronal specific expression pattern: A promoter fusion to GFP demonstrates CeLIM-6 to be expressed most strongly in two pairs of head sensory neurons. which we preliminarily identified as the ASG and AWA sensory neurons. In collaboration with R.Plasterk we have started to search for Tc1-insertion for this gene as well. CeLIM-7 maps on chromsome I close to several let-mutations (which we are trying to rescue) and is the ortholog of the islet-class of vertebrate LHX genes. We are in the process of subcloning its promoter to determine its expression pattern. The function of the vertebrate islet-1 gene in motorneuron generation (Pfaff et al., 1996) suggests the possibility of a neuronal expression pattern for this gene as well. We constructed a new lin-11-GFP fusion containing 10 kB of lin-11 promoter sequence and preliminary expression analysis reveals a similar expression pattern similar to a lin-11-lacZ expression pattern reported by Freyd & Horvitz (several pairs of head neurons, including one pair of sensory neurons, VC neurons, vulval cells). We will use this construct to monitor axon morphology of these neurons in the lin-11 mutant background. Preliminary analysis with a different, smaller lin-11-GFP construct generously provided by Sawa & Horvitz revealed VC process morphology defects. So far, for every known subclass of vertebrate LHX genes a C.elegans homolog has been identified. Given this observation and the number of genes that have been estimated to be sequenced already by the GSC (~80%), we don't expect (m)any more LHX genes to exist in the worm. The overlapping expression patterns of several vertebrate LHX genes in the nervous system has led to the proposal of a "LIM-code", in which distinct combinationsof LHX gene expression specify different motor neuron fates (Tsuchida et al., 1994). Although our expression pattern analysis is not complete yet, we note that so far there is no significant overlap of C.elegans LHX gene expression. If this holds true, it would suggest that C. elegans LHX genes may specify the simpler nervous system of the worm in manner that is distinct from the postulated combinatorial LHX interactions that may specify the vertebrate nervous system.