Worm Breeder's Gazette 10(2): 111

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.

Genetics and Molecular Biology of lin-10, a Gene that Specifies Pn.p Cell Fates

Stuart Kim and Bob Horvitz

Figure 1

Each of the six Pn.p cells in the vulval equivalence group (P3.p to 
P8.p) has the same developmental potential.  These cells can undergo 
any one of three cell lineages depending primarily on the proximity of 
a regulatory cell, the anchor cell.  When the anchor cell is near, Pn.
p cells undergo vulval cell lineages (termed the 1  and 2  cell 
lineages), and when the anchor cell is distant, they undergo a non-
vulval hypodermal cell lineage (termed the 3  cell lineage).  lin-10 
is involved in specifying Pn.p cell lineages; mutations in this gene 
cause all Pn.p cells to express the 3  cell lineage, resulting in a 
Vulvaless (Vul) phenotype.  lin-10 probably acts in Pn.p cells and 
thus may be involved in signal reception, signal transduction or in 
the control of gene expression.
Mutations in most genes involved in Pn.p cell determination have 
pleiotropic effects, and in at least some cases, this is because these 
genes control other cell lineages.  To see if lin-10 is also involved 
in specifying the fates of non-vulval cells, we wanted to determine 
its null phenotype.  Our results suggest that lin-10 function is 
required only in the Pn.p cells, since a complete lack of lin-10 gene 
activity results in a Vul phenotype.  First, ten lin-10 alleles have 
been isolated by screening for Vul mutants to date.  Thus, Vul 
mutations in lin-10 arise about as frequently as mutations in any gene 
involved in Pn.p cell determination and are isolated more frequently 
than alleles of genes such as lin-3 and let-23, which have a lethal 
null phenotype.  Second, the Vul phenotype is not enhanced when lin-10(
e1439) is placed in trans to a deficiency.  Third, we isolated eight 
lin-10 alleles by screening for mutations that failed to complement 
lin-10(e1439).  One allele, n1638, appears to be amber-suppressible.  
All of these alleles result in a Vul phenotype when homozygous, with 
no obvious pleiotropic effects.  Thus, lin-10 appears to control 
specifically the fates of only three cells: P5.p, P6.p and P7.p.
Using the approach of transposon tagging, we have cloned a gene that 
is probably lin-10 (see figure below and CSH abstracts, 1987).  In two 
mut-2-induced  a Tc1 element has inserted 
into a 10 kb intron of a candidate lin-10 transcription unit.  This 
gene produces a 1.6 kb mRNA, and its expression is reduced both in the 
transposon insertion alleles and in an EMS-induced lin-10 point mutant,
n299.  These results suggest that this transcription unit corresponds 
to the lin-10 gene.  Based upon preliminary microinjection experiments,
a cosmid containing this gene appears able to rescue the Vul 
phenotype of a lin-10 strain.
[See Figure 1]
We have isolated three full-length lin-10 cDNA clones.  Sequence 
analysis of these and other cDNA clones shows that this gene encodes a 
protein (407 amino acids) that is not homologous to sequences in any 
of the protein databases (NBRF/PIR Protein Database including the 
prerelease sequences and the Pasteur Institute Protein Database) nor 
is it striking in its primary structure.
To determine when and where lin-10 is expressed, we have isolated 
anti-lin-10 antisera.  When these antisera are used to probe Western 
blots, one major band (45 kd) is observed in samples derived from N2, 
and this band is reduced in intensity in samples derived from either 
of the transposon-insertion alleles or from a point mutant, n299.  
This result suggests that these antisera are specific for lin-10.  The 
anti-lin-10 antisera were then used to stain worms in situ.  Embryos 
stain the strongest, from the single cell stage to just before 
hatching.  This staining is reduced in a strain carrying lin-10(n1299),
a transposon insertion allele.  The embryonic expression of this gene 
is considerably earlier than the time of vulva formation, which is in 
the third larval stage.  One possibility is that the low level of 
expression during the third larval stage is sufficient for vulval 
determination.  Another possibility is that lin-10 acts early in 
development, endowing cells with the potential to undergo vulval 
induction at some later time.  These possibilities might be 
distinguished by determining the temperature-sensitive period of lin-
10(n1509ts).  The anti-lin-10 antisera detect an antigen localized to 
the cytoplasm, suggesting that lin-10 does not bind DNA nor does it 
function as an extracellular receptor.
Staining with anti-lin-10 antisera is observed in most or all cells 
in the embryo.  There are a variety of genes that, like lin-10, 
display a specific mutant phenotype but are expressed more generally (
e.g.  the human retinoblastoma oncogene).  Such a pattern of 
expression can be rationalized in two general ways.  First, at the 
cellular level, lin-10 is similar to genes with wild-type null 
phenotypes (e.g.  unc-93 and act-3), since in cells in which lin-10 is 
expressed the loss of lin-10 activity has no effect.  Perhaps in these 
cells, but not in the Pn.p cells, there is an activity that is 
redundant with lin-10.  Second, although lin-10 is expressed in many 
embryonic cells, it might function only in Pn.p cells.  For example, 
lin-10 might be regulated at the posttranslational level so that 
active gene product appears in only P5.p, P6.p and P7.p.  
Alternatively, lin-10 may play a role in signal transduction and 
function only in those cells that actually receive a signal.  Finally, 
since Pn.p cell determination is controlled by a genetic pathway 
containing at least seventeen genes, the expression of only some of 
the genes in this pathway may be specific to Pn.p cells.  As a result, 
lin-10 may have functional consequences only in Pn.p cells because 
only in these cells are all of the genes of the lin-10 pathway 

Figure 1