Worm Breeder's Gazette 12(4): 72 (October 1, 1992)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Mup-4 Mutations Cause Embryonic Defects in Muscle and Hypodermal Organization

Beth Gatewood, Elizabeth Bucher

Dept. of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, 19104-6058

We are investigating a previously uncharacterized essential zygotic gene which we have called mup-4 (III) (muscle position defect). mup-4 was identified in a genetic mosaic screen designed to isolate essential lineage specific zygotic genes(1) and is defined by 5 alleles ( ar60 , mg23 , mg26 , mg30 , mg36 :mg alleles kindly provided to us by Bruce Wightman). mup-4 was of interest since, in addition to having AB lineage specific requirements, we initially observed that mup-4 mutants arrest as three-fold embryos and show a body wall muscle position defects reminiscent of mup-1 mutant phenotype(2). Nomarski observation shows that muscle misposition is not the result of generalized cell death or necrosis that might alter correct muscle spatial organization. Mosaic analysis shows that mup-4 is not a muscle specific protein (such as deb-1 or myo-3 that when mutant cause muscle position defects), but is essential in AB lineage (only one body wall muscle descendant). These phenotypic and mosaic analyses suggest that mup-4 is required for the spatial organization of muscle by functioning in either hypodermis or neurons.

To identify the cellular basis and the time of onset of the defect, we are using Nomarski observation of developing embryos in combination with immunofluorescent staining of mutants with antibodies directed at hypodermal and muscle specific antigens kindly provided to us by Barstead, Waterson, Kagawa and Bogaert from the MRC collection). These studies reveal that mup-4 ( ar60 )shows variable expressivity (we have not yet quantitated the arrested populations). mup-4 ( ar60 )mutants arrest at: bean stage (don't elongate); two-fold (perhaps similar to paralyzed-at-two-fold); and at three-fold, although some have been observed to hatch (Mup). Muscle cell misposition is evident at the earliest detectable stage of muscle differentiation (about bean stage) in mutants stained for the muscle protein paramyosin (R224). These mutants likely correspond to the earliest arrest population. We analyzed mup-4 mutants for components of muscle and hypodermal attachment structures, including a muscle attachment antigen (MH25) and hypodermal intermediate filament protein overlying muscle (MH4). These antigens are expressed and their position, like the muscle, is aberrant. Furthermore, muscle (R224) and hypodermal intermediate filament antigen (MH4) still co-localize in three-fold arrested embryos. Therefore, muscle and hypodermis are able to interact even in aberrant positions (see also mup-12 ).We have also observed muscle twitching consistent with the stable attachment of muscles to the hypodermis.

Our current working hypothesis is that mup-4 functions in hypodermis and mutations result in hypodermal defects, thus affecting muscle position. This hypothesis is supported by MH27 antibody staining of the hypodermal desmosomes that reveals bean stage arrested embryos having patches of hypodermal staining on one side of the embryo (perhaps similar to J. Rothman's zen mutants). In three-fold Mups, the hypodermis encloses the embryo, but the cells are irregularly shaped and twisted around the worm. Time lapse video microscopy, in combination with hypodermal cell specific markers will examine hypodermal cell number and positions to determine possible transformation of cell fate or differentiation and morphogenesis defects. We are undertaking cloning of mup-4 to determine its specific role in embryonic development. To this end, we have mapped mup-4 to the right of sma-3 and approximately 0.02 cM left of ncl-1 . We are now mapping mup-4 relative to lin-39 ( ceh-15 ).

Literature Cited:

1. E. Bucher and I. Greenwald. Genetics 128:281-292 (1991).

2. P.Y. Goh and T. Bogeart. Development 111:667-681 (1991).