Worm Breeder's Gazette 11(5): 52

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.

lin-11-lacZ Fusion Gene is Expressed in Vulval Cells and Neurons

Gwen Freyd and Bob Horvitz

Mutations in the gene lin-11 cause the 2  vulval precursor cells to 
divide symmetrically instead of asymmetrically.  Mutant animals cannot 
lay eggs and are slightly uncoordinated.  We previously reported that 
lin-11 encodes a protein with both a homeodomain and two copies of a 
cysteine-rich region that potentially binds metal ions, called LIM (
Nature 344, 876,1990).  Two other C.  elegans proteins, mec-3 (Way & 
Chalfie, Cell 54, 5,1988) and ceh-14 (Burglin & Ruvkun, WBG 11:2, 52,
1990) and one mammalian protein, Isl-1 (Karlsson et al., Nature 344, 
879,1990), also contain a homeodomain and two copies of the LIM motif.
We have determined the minimal region of genomic DNA required for 
lin-11 function as assayed by germline transformation.  We coinjected 
the rol-6 plasmid pRF4 (Mello et al., WBG 11:1,18,1989) with either 
the genomic cosmid clone ID6 or subclones of ID6 into lin-11 mutant 
animals and looked for roller progeny that have the ability to lay 
eggs.  We have identified an approximately 10.5 kb region of the 
cosmid ID6 that is sufficient for lin-11 rescue.
To identify cells in which lin-11 is expressed, we constructed a 
translational fusion between lin-11 and lacZ.  The fusion was made 
using the vector pPD22.04 (obtained from Andy Fire), which contains a 
nuclear localization sequence, a synthetic intron, and unc-54 3' 
sequences in addition to lacZ.  The clone used for microinjection 
contained most of the minimal region of genomic DNA required for lin-
11 rescue, but lacked 3 kb of genomic DNA encoding the carboxyl end of 
the lin-11 protein.  The expected fusion product contains two copies 
of the lin-11 LIM domain, but not the homeodomain.
The lin-11-lacZ fusion plasmid was coinjected with pRF4 into wild-
type worms and stably transmitting lines were examined.  Animals 
stained with X-gal (according to the procedure of Jeff Way, personal 
communication) have blue nuclei in the head, ventral nerve cord, vulva,
and tail.  Staining appears in embryos and the pattern changes during 
larval development as described below.
More than 20 cells in the vulval region stain during the L3 and L4 
larval stages, the time period in which the vulva is being formed in 
the hermaphrodite and the 2  cells undergo an asymmetric cell division.
This staining disappears by the time animals reach adulthood.  We 
are in the process of trying to determine the identities of the cells 
in the vulval region.  We suspect that both daughters of the 2  cells 
express the fusion protein.  In addition, 1  cell descendants and/or 
gonadal cells and/or muscle cells might also express the fusion 
At least one nucleus in the pharynx stains and preliminary 
identification suggests it might be the neuron I5.  Eight neurons in 
the lateral ganglion stain and are probably the left-right pairs of 
the neurons RIC, AIZ, ADF, and ADL (identified by Cori Bargmann).  ADF 
and ADL are amphid sensory neurons and AIZ and RIC are interneurons.  
These neurons stain embryonically, and the staining persists 
throughout adulthood.  Six cells in the ventral nerve cord begin to 
stain during larval development and continue to stain through 
adulthood.  We believe that these cells are the VC neurons because of 
their number and positions.  It is possible that lin-11 expression in 
some of these six classes of neurons is required for the animal to 
move properly, accounting for the slightly Unc phenotype of lin-11 
mutants.  We are testing the tentative identifications and functions 
of these cells by crossing the lin-11-lacZ fusion into mutants and by 
laser ablation.