The excretory canal cell of C.elegans provides a simple single-cell model for the study of tubular formation in epithelial cells. EXC proteins regulate apical (lumenal) diameter of the excretory canals.
exc-9 mutants exhibit penetrant wide, meandering canals. In addition, hermaphrodites show non-penetrant tailspike defects, and males show non-penetrant ray defects and lowered mating efficiency.
We previously identified
exc-9 as F20D12.5.
exc-9 encodes a small single LIM domain protein, which likely mediates protein-protein interactions. EXC-9 shows high homology to CRIP (Cysteine-Rich Intestinal Protein), first identified as a protein from the mammalian intestine and heart, but also reported in invertebrate neurons.
I integrated the
exc-9 promoter region into a GFP vector. This construct is expressed in several tissues including the canals, tailspike, DTCs, several neurons, and UTSE. Overexpression of this construct in N2 animals sometimes causes arrest at the 2-fold stage, a phenotype similar to that caused by overexpression of the canal-specific homeobox gene
ceh-6.
Low levels of expression of a translational GFP construct of
exc-9 partially rescued the cystic canal phenotype of
exc-9, but high levels of expression caused “unextended canals” - in these animals, canals have a normal lumenal diameter but fail to grow from the cell body. This phenotype can also be caused by overexpression of
exc-5 (guanine exchange factor), or by mosaic loss of laminin function. We used this phenotype to look for genetic interactions between
exc-9 and other genes. Worms with unextended canals resulted when
exc-9 was overexpressed in N2 or in
exc-2,
exc-4,
exc-9, or
sma-1 mutants, but no effect on the canals was observed when
exc-9 was overexpressed in
exc-5 mutants; i.e. loss of EXC-5 function rescued the
exc-9 overexpression phenotype. Conversely, worms with unextended canals were created when
exc-5 was overexpressed in an
exc-9 background, i.e. loss of EXC-9 function did not prevent the effects of
exc-5 overexpression.
Similarly, expression of our
exc-9 construct partially prevented cyst formation in
exc-9 mutants, as well as in
exc-2 (not cloned yet) and
exc-4 (CLC channel) mutants.
We interpret these results to suggest that EXC-9 functions upstream to signal EXC-5 to regulate cytoskeletal dynamics (likely via CDC-42) to regulate tubule diameter. EXC-9 may in turn depend upon EXC-2 and EXC-4 function to perform this regulation.