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.

Nishiwaki K et al. (1990) Worm Breeder's Gazette "Molecular analysis of the emb-5 locus"

Nishiwaki K, Sano T, Miwa J

[Worm Breeder's Gazette, 1990]

emb-5 is a maternally expressed gene and is required for achieving the correct timing of E cell division at gastruIation. The second E cell division occurs prematurely at the 26-cell temperature-sensitive emb-5 mutants, while in the wiId-type it takes place at the 44-cell stage (1, 2). As previously reported (WBG 11/1:39), we have identified the two Tc1s linked to the emb-5 gene in Bergerac BO. The genomic phage clones isolated by Tc1 flanking probes have been assigned on the physical map by Drs. J. Sulston and A. Coulson and found to be separated by about 400 kb. Based on both genetic and physical map data, we estimated that one of the two Tc1s is about 40 kb or one cosmid length to the right of emb-5.We have succeeded in transforming the ts emb-5 mutant hc61 first by injecting a mixture of three cosmids, C16B3, C38D4, and W05E6 and then by a single C38D4 cosmid (Figure 1). Transformants were usually sick but still produced a few viable embryos at nonpermissive temperature. So far the search for RFLPs in emb-5 mutants and putative intragenic revertants of hc61 with C38D4 as a probe has not been successful. Probably, these strains have small polymorphisms or point mutations. There are at least 8 different mRNAs transcribed from the C38D4 genomic region. However, we observed no differences in size or amount of any of these transcripts between the wild-type and the mutants tested. We are trying to rescue hc61 with DNAs subcloned from C38D4 and are also engaged in research to identify mutation sites. [See Figure 1]

Nishiwaki K et al. (1991) Worm Breeder's Gazette "Molecular cloning of the emb-5 gene"

Miwa J, Nishiwaki K, Sano T

[Worm Breeder's Gazette, 1991]

Temperature-sensitive maternal effect mutations in the emb-5 gene (1) show premature second E cell division and abnormal gastrulation at nonpermissive temperatures. Embryos arrest as balls of several hundred differentiated cells around the lima bean stage (2). We previously reported identification of a Bergerac Tc1 closely linked to emb-5 (WBG 11, 1, 39). We obtained several overlapping cosmids (from J. Sulston and A. Coulson) closely linked to this Tc1 containing region so that some of the cosmids possibly contain the emb-5 gene. We injected 4 overlapping cosmids individually into emb-5(hc61) according to the method of C. Mello and V. Ambros (personal communication). Of these, two overlapping cosmids, C56E12 and C17H4, were found to have transforming activity, indicating that the activity was within a 20kb overlapping region. To limit the region harboring the transforming activity, we examined transformation after cutting C56E12 with various restriction enzymes, as described by S. Kim et al. (3), and found that region within the 10.3kb fragment. Germline transformants grew slowly at nonpermissive temperature and revealed a wide range of E cell phenotypes from mutant type to completely rescued type similar to wild type. Using an internal 1.7kb SalI fragment ( SalI digestion resulted in no rescue) as a probe, we detected a single 5.4kb mRNA in both wild type and hc61. We obtained several cDNA clones from B. Barstead's cDNA library with the same probe. One of the clones had an insert of about 3.6 kb cDNA and the sequence of this cDNA revealed the 1121 aa C-terminal portion of the putative gene product. No significant homology was found when this amino acid sequence was compared with those for other proteins in the SwissProt and the NBRF databases. Preliminary results showed amino acid change from Ala to Glu at 663rd aa from C-terminus in hc61. Currently, we are attempting lacZ fusion experiments using the vectors provided by A. Fire (4) to understand the localization of the gene product.

Nishiwaki K et al. (1987) Worm Breeder's Gazette "isolation and characterization of revertants of emb-1(hc57)."

Miwa J, Nishiwaki K

[Worm Breeder's Gazette, 1987]

Temperature-sensitive maternal mutations in the emb-1 gene cause defects in the migration and fusion of pronuclei and in the pseudocleavage of a fertilized egg. The gene function is required within a short period around fertilization. We are isolating and characterizing revertants of emb-1 mutations to investigate emb-1 gene function and to analyze events around fertilization. With EMS mutagenesis we have independently isolated 4 revertants from emb-1( hc57) III, which can grow at the nonpermissive temperature of 25 C. Two of them were shown to be dominant mutations and mapped to linkage group I and II. Two others were semidominant and mapped to linkage group III. The former two revertants were further characterized to show that maternal expression of the mutations was necessary and sufficient for the suppression of emb-1(hc57). Cytological observation has revealed that these revertants in the hc57 background exhibited weak pseudocleavage and lacked male pronuclear migration at 25 C. This suggests that the restoration of the emb-1 gene function is incomplete and that normal pseudocleavage and male pronuclear migration are not absolutely necessary for the development of C. elegans. DAPI staining of the hc57 mutant detected more stained materials in the female pronucleus than in the male pronucleus and no polar bodies in fertilized eggs. With rhodamine-phalloidin staining, we found that the hc57 mutant exhibited rapid aggregation of microfilaments after fertilization at 25 C we are looking into a possible defect in meiosis after fertilization.

Nishiwaki K et al. (1989) Worm Breeder's Gazette "identification of Tc1s near emb-5 gene."

Nishiwaki K, Miwa J

[Worm Breeder's Gazette, 1989]

Temperature-sensitive maternal effect mutations in the emb-5 gene (1) show premature second E cell division and abnormal gastrulation at nonpermissive temperature. Embryos arrest as balls of cells without normal morphogenesis (2). Understanding of the emb-5 gene function at the molecular level should provide a good insight into the mechanism of morphogenesis in animal development. We are trying to clone the emb-5 gene by chromosome walking from Tc1s linked to emb-5 and transformation experiments. We have genetically mapped the emb-5 gene and located it between unc-79 and dpy-17 on linkage group III. To identify Tc1s near the emb-5 gene in Bergerac BO or TR679, we have outcrossed these strains ten times to Bristol N2-derived emb-5 mutant strains doubly marked with unc-79 and dpy-17 or singly with unc-79 and constructed strains with Bristol background except for the region around emb-5. Southern analysis of Tc1-containing restriction fragments for the constructed strains and their parental strains has revealed two Bergerac-associated Tc1s. Three factor mapping has assigned one fragment about 0.28 mu to the left of the emb-5 gene and the other about 0.03 mu to the right. We also found one TR679- associated Tc1-containing fragment closely linked to emb-5, although its map position has not been accurately determined. Using the flanking DNAs of the two Bergerac-associated Tc1s as probes, we have isolated EMBL3 N2 genomic clones. Phage clones corresponding to these Tc1 flanking sequences have been sent to Drs. John Sulston and Alan Coulson, Cambridge, England, for physical mapping.

Nishiwaki K et al. (1990) Worm Breeder's Gazette "On cell division sequence of C. elegans early embryogenesis"

Miwa J, Nishiwaki K

[Worm Breeder's Gazette, 1990]

The early embryogenesis of C. elegans appears to follow an invariably rigid cell division sequence. However, we do not really know how rigid this sequence is for normal embryogenesis. We report here our observation that the cell division sequence of C. elegans early embryogenesis can vary without affecting the hatching efficiency or the fertility of hatchees. We observed embryos with the same preparation method as described by Sulston et al.(1), where we used Difco purified agar (PA) for supporting embryos instead of Difco Bacto- agar. Cover glasses were sealed with vaseline. Experiments were carried out at 25 C. When we used 5% PA, we consistantly observed that AB(16) occurred 0.5-3 minutes before P(3)(2) (division of P3 to P4+D) and that MS(8) occurred 2-4.5 minutes before D(2) (5/5) in the N2 development. These cell division sequences were reversed when 2% PA was used; that is, P3(2) occurred 0.5-1 minute before AB(16) and D occurred 2-4 minutes before MS(8) (3/4). In one of the four cases, MS(8) occurred before D2 as in the 5% PA, although P3(2) occurred before AB(16). Almost all of some 50 embryos so far examined in each condition hatched normally. We tested the fertility of the worms that showed the embryonic cell division sequence typically expected for the % agar used. All of these worms grew fertile with essentially normal brood sizes. The results show that the cell division sequence of C. elegans early embryogenesis is not absolutely rigid and allows some range of variations without affecting the normal development and the fertility. We think that the observed variations in the cell division sequence resulted from difference in the pressure applied on the embryos. The embryos on the 5% PA looked flatter than those on the 2% PA under Nomarski optics. The 5% PA must have exerted more pressure on the embryos than did the 2% PA. Then, we may expect the division sequence in the 2% PA to be closer to that under no pressure Shierenberg et al observed the embryos prepared in water under a cover glass sealed with petrolatum and plastic-foam cushions (condition without pressure) to exhibit the division sequence of P3(2) -> AB(16) and D(2)-> MS8(2), which is indeed identical to that observed with the 2% PA. ---In addition to variations in the cell division sequence, we also observed variations in the cell division axes of E cells under the two different agar conditions. Both Ea and Ep divided in parallel and dorsal-ventral in the 2% PA, whereas the division axis of Ep inclined posteriorly in the 5% PA. Neither of the division axis variations resulted in any abnormal development or infertility.

Nishiwaki K et al. (1994) Worm Breeder's Gazette "Tissue Distribution of the EMB-5 Protein During Development."

Sano T, Nishiwaki K, Tabuse Y, Miwa J

[Worm Breeder's Gazette, 1994]

Tissue Distlibution of the EMB-S protein during development Kiyoji Nishiwaki, Tohru Sano, Yo Tabuse, and Johji Miwa Fundamental Research Laboratories, NEC Corporation, Miyukigaoka, Tsukuba 305, Japan The emb-5 gene is required for the correct timing of gut precursor cell division during gastrulation' 2 and for the proliferation of germ cells during postembryonic development3. emb 5 encodes the protein EMB-5 homologous to the yeast nuclear protein SPT6, which has been shown to affect the transcription of a variety of genes and suggested to play a role in chromatin assembly or modification4. To understand the tissue distribution patten of EMB-5 during development, we have conducted the epitope-tagging analysis using the antigenic epitope of hemagglutinin of influenza virus (HA I ). The emb-5 gene epitope-tagged at the 5' end was active and rescued the emb-5 mutant phenotype although more weakly than did the wild type gene. Using a strain with chromosomally integrated DNA(epitope tagged emb-5 + rol-6d), we found that the anti-epitope antibodies stained interphase nuclei of various cell types, although they did not stain early embryonic cells before the 5 l-cell stage and germ cells in all stages including mature oocytes and sperm. (We used the polyclonal HA. 11 antibody from BAbCO because the monoclonal antibody 12CA5 from Boehringer gave substantial background staining of worm nuclei.) On the other hand, the antibodies against an EMB-5 C-terminal peptide stained all the interphase nuclei except for the germ cell nuclei before the adult stage in the same strain, although the mature sperm were not stained as in the case of the anti-epitope antibodies. In embryos, the germline precursor cells P0 to P3 appeared to be stained but not the germline cells P4, Z2, and Z3. These results suggest that EMB-5 may have modifications on its N-terminus in early embryonic cells and adult germ cells, and that it may have modifications on both of its N- and C-termini or not be present in germ cells before the adult stage and mature sperm. It is possible that EMB-5 is not required for the mature sperm lacking chromatin proteins. It is interesting that the early embryonic cells and germ cells, where the temperature-sensitive emb-5 mutations show remarkable effects, seem to have modified EMB-5 proteins. However, because we are analyzing the artificially introduced epitope-tagged gene, we have to be careful in interpreting its expression pattem. We are currently preparing antibodies against an N-terminal peptide and several internal peptides of EMB-5. 1. Miwa et al. (1980). Dev. Biol. 76, 160-174. 2. Schierenberg et al.(l980). Dev. Biol.76, 141-159. 3. Nishiwaki, Miwa, and Hedgecock, unpublished. 4. Nishiwaki eJ al. (1993). Mol. Gen. Genet. 239, 313-322.

Nishiwaki K et al. (1988) Worm Breeder's Gazette "the emb-1 and emb-3 gene functions are required for the meiosis after fertilization."

Miwa J, Nishiwaki K

[Worm Breeder's Gazette, 1988]

In C. elegans, oocytes at the diakinesis stage, which is a final stage of the meiosis-I prophase, quickly enter the metaphase after fertilization. Two polar bodies are successively formed during the meiosis after fertilization and the pronuclei are reconstructed after the completion of meiosis-II. Miwa et al.(1980) and Schierenberg et al.(1980) identified 9 genes required for C. elegans embryogenesis. The gene function of two of these, emb-1 and emb-3, is necessary very early in the embryogenesis. Reconstruction of the pronuclei is defective in emb-1(hc57). Early divisions are variably abnormal in emb-3(hc59). We have studied the possibility that these early abnormalities are caused by defective meiotic events in the mutants. We stained the meiotic spindle and the chromosomes of the fertilized egg with an anti-tubulin antibody and DAPI. The meiotic spindle of N2 was spherical or barrel-like in both meiosis-I and -II, as was observed by Albertson(1984). The 6 bivalent chromosomes were arranged pentagonally with one of them in the center. In the emb-1(hc57) mutant, although the meiosis-I spindle was formed and the homologous chromosomes were separated from each other, no polar body was produced. Both oocyte and sperm chromosomes gradually degraded after the homologs separated and no cell division followed . As for the emb-3( hc59) mutant, about 40% of the embryos examined exhibited the disordered meiosis-I spindle structure and the abnormal arrangement of the paired homologs. Although the homologs were separated, they were all extruded together as a single large polar body. The male pronucleus was, however, reconstructed and the development, which did not appear to be completely abnormal, continued in the haploid state. We observed haploid embryos with several hundred cells. These results indicate that both emb-1 and emb-3 gene functions are required for the meiosis after fertilization, but that each gene plays an apparently different role. The construction of the meiosis-I spindle seems to need the emb-3 gene function, but not the emb-1 gene function. Also, the polar body formation appears to require both the emb-1 gene and possibly the emb-3 gene to function. The emb-3 gene probably acts before the emb-1 gene in meiosis-I. Schierenberg et al. (1985) observed strong cytoplasmic streaming in the wild-type embryos of the mechanically denucleated female pronucleus, which arrested before hatching. We observed the similar abnormality with the emb-3 embryos without the female pronucleus.

Uekusa T et al. (1994) Worm Breeder's Gazette "Molecular Analysis of unc-103"

Nishiwaki K, Miwa J, Uchida MK, Uekusa T

[Worm Breeder's Gazette, 1994]

Molecular analysis of unc-103 Tomoyuki Uekusa (uekusa@exp.cl.nec.cojp)l 2, Kiyoji Nishiwakil, Masaatsu K. Uchida2, and Johji Miwa1 1Fundamental Research Laboratories, NEC Corporation, Miyukigaoka, Tsukllba 305, Japan 2Department of Molecular Pharmacology, Meiji College of Pharmacy; 1-35-23, Setagaya- ku, Tokyo 154, Japan.

Tabuse Y et al. (1987) Worm Breeder's Gazette "progress in tpa-1 gene cloning."

Tabuse Y, Nishiwaki K, Miwa J

[Worm Breeder's Gazette, 1987]

We isolated, from the mutator strains NJ82 and NJ204, five independent spontaneous TPA resistant mutants assigned to the tpa-1 gene. Three of the five mutants contained the 2.4kb fragment that was size-altered due to Tc1 insertion. In the last meeting we proposed that the 2.4kb HindIII fragment is at least a portion of the tpa-1 gene. Now we have found another spontaneous tpa-1 mutant with the Tc1- inserted 2.4kb fragment, and this mutant was isolated from yet another mutator strain RW7097 (a gift from Dr. Waterston/Dr. Mori ) . This result further supports the idea that the 2.4kb fragment constitutes a part of the tpa-1 gene. We have sequenced about 90% of the 2.4kb fragment, so far. It is a fragment with high AT content and several A and T clusters. To understand the relationship between Tc1 insertion and acquisition of resistance to TPA, we have also sequenced the Tc1 flanking region of two of the three mutants (original isolates for MJ562 and MJ563) isolated from NJ82. We found that Tc1 was inserted in the same position in both mutants. Analysis of insertion sites for other mutants are underway. We have also screened a cDNA library, prepared from mRNA of mixed age (kindly provided by Dr. Kimble/Dr. Ahringer). We obtained two cDNA clones using a 1.5kb fragment as a probe, which is a Tc1 flanking region of the 2.4kb fragment derived from MJ562. Characterization of the cDNA is in progress.