Worm Breeder's Gazette 13(5): 75 (February 1, 1995)

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.

Scoping Out Mutants in EXCruciating Detail

Matthew Buechner [1], David Hall [2], and Ed Hedgecock [1]

[1] Johns Hopkins University, Baltimore, MD, 21218
[2] Albert Einstein College of Medicine, Bronx, NY, 10461

      We have nearly completed an electron-microscopic survey of exc 
mutants, in which the long hollow processes (canals) of the excretory 
canal cell form large cysts.  Together with results obtained by staining 
the canal cell with fluorescent wheat-germ agglutinin, these micrographs 
support our hypothesis that the defects in these mutants are focused at 
the apical surface of this polarized epithelial cell.
      Electron micrographs of wild-type canals show a long, narrow 
central lumen throughout the canal, surrounded by an electron-dense 
material apparently required to maintain the lumenal structure, and by a 
series of myriad connected coated vesicles forming canaliculi that open 
into the central lumen.  This central structure excludes the other 
organelles, such as mitochondria, Golgi, and microtubules to the edges of 
the canal cell processes.  The canal is extensively gap-junctioned to the 
hypoderm on one side, and forms a basement membrane on the other.   The
mutants preserve many of these elements.  Gap junctions appear  normal, as
do the canalicular vesicles and microtubules.  The most  striking changes
in some of the mutants appear in the electron-dense  lumenal coating.  In
DIC views of exc-6 (rh103) (located very close to par-2 on IIIL) animals,
the lumen repeatedly splits up into multiple wide  channels that meander
and coalesce.  This is reflected in serial-section  electron micrographs
where the lumen splits into two lumena, each  surrounded by an electron-
dense coat, then rejoin to make a single lumen.   This gene may encode a
controlling element that determines how much  lumen or lumenal coat is to
be made, and where it is to be placed.  Similar  defects may be seen in
exc-8 (rh210) (X) mutants.
      The exc-7 (rh252) mutants show, in addition to moderate-sized  cysts
primarily at the canal termini, a series of large non-canalicular 
vesicles at the canal tips that may reflect an inability to degrade
lumenal  material properly.  The canal lumen is known to form a
carbohydrate  material that is presumably the basis for staining by wheat-
germ  agglutinin.  Intriguingly, a chitinase has been found by the genome 
sequencing project in the area of IIC where exc-7 maps.
      Most strikingly, in mutants such as exc-2 (XL) and exc-5 (IVC) that 
form very large round cysts, the lumenal coat no longer is completely 
attached to the lumen.  In a few micrographs, this density is seen
attached  to the lumen at a few points, but otherwise floating freely in
the canal.   Around many large cysts, the density has bunched up to one
side of the  cyst.  The purpose of the density may be to regulate the
spacing of  canaliculi as well as to maintain the shape of the lumen;
where the  density has bunched up, the canaliculi are often excluded.
      These defects are all consistent with an inability to maintain 
properly an apical "basement membrane" required for cell shape.  This is 
confirmed by the recent sequencing results of Steven Jones in Dave 
Baillie's laboratory, that the let-653 gene (defects of which cause 
formation of lethal excretory canal cysts in early larval stages) encodes
a  mucin.  Mucins are secreted and membrane-bound highly hydrophilic O-
linked glycoproteins found at the apical surfaces of most epithelia.  We 
are now preparing to sequence the other exc genes to determine if they are 
related to this mucin.