The
nob-1 (IIIR) gene is defined by two loss of function alleles,
ct223 and
ct351, which result in the Nob (no back end) L1 lethal phenotype, and one reduced function allele,
ct230, which results in viable animals with lumpy, misshapen tails, similar to those resulting from mutations in
vab-7, the C. elegans even-skipped homologue. To understand the basis for the gross posterior defects of Nob-1 embryos, we have analyzed tissue differentiation and posterior embryonic lineages in animals homozygous for
ct223 using a "4D" microscope. Despite the severity of the Nob-1 phenotype, we found no alterations in lineages that give rise to posterior gut, hypodermis, muscle and neurons. To determine if these tissues differentiate properly, we examined the expression of a variety of tissue-specific markers. With two exceptions, all gut, hypodermal and muscle markers assayed are expressed in the proper spatial and temporal patterns. The two exceptions have drawn our attention, however, to patterning and morphogenetic defects in posterior gut and hypodermis. First, staining with MH27 antibody to zonula adherens junctions reveals defects in the posterior ventral hypodermis: P5/6 through P11/12 fail to migrate to the ventral midline at the proper time and in a reproducible spatial order. In addition, the posterior lateral seam cells fail to elongate along the anterior-posterior axis. The animals eventually manage to enclose, but we believe these defects in hypodermal cell movement and morphogenesis account for the overall Nob morphology. Currently, we are using a C. elegans
pax-3/7::GFP reporter (kindly provided by Andrew Chisholm), which is expressed in ventral hypodermal cells during morphogenesis, to describe enclosure in Nob-1 embryos in greater detail and to determine if Nob-1 mutations affect the expression of this marker. Second, two observations suggest that
ct223 results in a posterior-to-anterior transformation in the gut. In wild-type embryos, a lacZ reporter construct of
pal-1, the C. elegans caudal homologue, is expressed in
int5L/R, two cells that switch their places along the anterior-posterior axis during gut morphogenesis. In Nob-1 embryos, two other gut cells, tentatively identified as
int7L/R, express the reporter and also appear to undergo movements that switch their positions, such that they reside ultimately anterior to
int6L/R. Currently, we are using laser ablation to confirm the identity of the extra pal::lacZ expressing cells and determine if elimination of these cells can rescue any aspects of the Nob phenotype. In other experiments, analysis of a Nob-1 germ line mosaic indicated that embryonic expression of
nob-1 is sufficient for normal embryogenesis. These experiments taken together suggest that
nob-1 may regulate later events in A/P patterning and morphogenesis through specification of the posterior boundary of
pal-1 expression. Finally, since
nob-1 still resides in a gap on the physical map, we are continuing screens for Tc1 alleles in various mutator backgrounds, in preparation for molecular cloning.