PMC:4502371 / 14315-18861 JSONTXT

{"project":"2_test","denotations":[{"id":"25917920-11470828-43299632","span":{"begin":1234,"end":1238},"obj":"11470828"},{"id":"25917920-11470828-43299633","span":{"begin":2172,"end":2176},"obj":"11470828"},{"id":"25917920-2071016-43299634","span":{"begin":2615,"end":2619},"obj":"2071016"}],"text":"Characterization of a mua-3(uy19) mutation\nWe acquired RB1547 from the Caenorhabditis Genetics Center (CGC), which contains a deletion in sta-2. We observed a high rate of lethality during the L4 molt at 25° (Figure 1A). After outcrossing against N2 two times, we found the lethality was not linked to the deletion in sta-2. After a series of SNP mappings, we located the lethal mutation on chromosome III close to unc-119. Subsequent whole genome sequencing revealed a 488-bp (131 amino acids) in-frame deletion in the muscle attachment abnormal-3, mua-3 gene (see Materials and Methods) (Figure 1, B and C).\nFigure 1 Characterization of a newly isolated mua-3 allele, uy19. (A) Temperature-dependent lethality of the newly isolated mua-3 mutant. The numbers of dead animals and total animals were counted to calculate percent survival. Animals were synchronized and plated as L1s and the percentage of survival was measured after they passed the L4 molt; 61 hr for 15°, 40 hr for 20°, and 30 hr for 25°. The numbers are average percent survival ± SEM. (B) A schematic drawing of the exons and introns of mua-3 gene and the location of the deletion. (C) A schematic drawing of the domain structure of the mua-3 gene (Bercher et al. 2001) and the location of the deletion. (D) Time course of death of the mua-3(uy19) mutants at 25°. Very few deaths were observed until 30 hr from L1. Then, within 3–4 hr, more than 90% were dead. The time window of death was somewhat wider than a worm’s molting period (approximately 2 hr) because the mutants are not perfectly synchronized, and thus the time to reach the molt varies among the animals. The numbers are average percent survival ± SEM. (E) A phylogenetic tree to show MUA-3 is a homolog of human fibrillins. Compared fibrillin homologs are C. elegans MUA-3, C. elegans MUP-4, C. elegans FBN-1, Drosophila melanogaster DP, Homo sapiens FBN1, FBN2, and FBN3. H. sapiens EYS sequence was included as an outgroup member to infer the root of this unrooted tree. The mutants showed a lethal phenotype similar to the previously reported mua-3 mutants, confirming that the lethality is due to a mutation in mua-3 (Bercher et al. 2001). From transmission electron microscopy (TEM) observations of detachment zones of their mua-3 mutants, Bercher and others suggested that MUA-3 acts at the apical hypodermal surface to maintain the attachment of the hypodermis to the basal cuticle and that the primary defect in mua-3 mutants is the failure of these attachments. But unlike the old allele mua-3(ar62) that die during various stages as early as at L1 (Bucher and Greenwald 1991), the new mutants grow normally until the L4 molt without any visible defects in development, and within 2–3 hr of molting, most die (Figure 1D) (30 hr to 33 hr from L1s is when most of the death occurs). At the L1, L2, and L3 stages, mutants rarely died. The occasional escapers after the L4 molt looked pale and sick immediately after molting, but within 2 hr they appeared normal and reproduced normally (data not shown).\nWhen we examined the cause of sudden death during L4 molt, we found that internal organs such as the pharynx and gonad were detached and misplaced (Figure 2, A, D, and E). The gonad develops normally before molting at nonpermissive temperatures (Figure 2, B and C). This shows that MUA-3 is required for the organ attachment, consistent with the previous finding by Bercher et al. 2001. The sudden death during the L4 molt could suggest that mechanical strain during the L4 molt is sufficiently stressful to cause all the tearing in our mutants.\nFigure 2 The phenotypes of mua-3. (A) Representative DIC microscopy image of mua-3(uy19) mutants dying during L4 molt. White arrow shows a normal-looking gonad arm with germ cell nuclei. The red arrow shows misplaced and detached gonad. Scale bar = 30 µm. A, anterior; P, posterior; D, dorsal; and V, ventral. (B and C) The gonad structures of mua-3(uy19) at early L4 stage before molt show normal development of gonads at 25°. Scale bar = 10 µm. (D and E) The tips of the pharynxes of two dying mua-3(uy19) were detached from the tips of the mouths. Scale bar = 20 µm The sequence of MUA-3 shares high homology with that of the human FBN1, mutations in which are the cause of Marfan syndrome (Figure 1E). Our results may suggest that the detachment of internal organs during the L4 molt could mimic certain aspects of Marfan pathology such as aneurysm or aortic dissection in the heart caused by weak connective tissue integrity under mechanical stress."}