Worm Breeder's Gazette 13(5): 60 (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.

daf-12 is a Heterochronic Gene Acting During L2 and L3 Larval Stages

Adam Antebi and Ed Hedgecock

Johns Hopkins University, Department of Biology, 3400 North Charles 
Street, Baltimore, MD  21218.

daf-12 is a nuclear hormone receptor (D. Riddle, P. Larsen, W.H. Yeh,
p.c.)  required for both continuous and dauer development. daf-12 alleles
can be  grouped into six classes on the basis of  their hypodermal (e.g.,
Lin),  gonadal (e.g., Mig) and dauer-related phenotypes (e.g., Daf-d, Daf-
c).     Recently, we have focused on two questions:  (1) When is daf-12
activity  required? (2) What is its relation to known heterochronic genes? 

Time of Daf-12 Action:  At least three phenotypes  are expressed by the L2 
molt. At the L2 molt, the male linker cell fails to reflex dorsally (Mig-
lc  phenotype).  Similarly, the hypodermal seam cells repeat their
previous  division pattern at the L2 molt (Lin-L2 phenotype). Finally,
dauer  morphogenesis fails at the L2 molt in some alleles (Daf-d
phenotype). This  suggests that daf-12(+) transcription is required
earlier, either before or  during the L2 intermolt,  to regulate target
genes by the L2 molt. 
      Preliminary experiments with a heat-sensitive allele (rh193hs) are 
consistent with a requirement for daf-12 by the L2 molt. This allele, 
which is non-Daf at all temperatures, has heat-sensitive Lin and Mig 
phenotypes. Temperature shift experiments show that the TSP for the Lin 
phenotype is during L2. Moreover, late larval defects in the hypodermis 
(e.g., failure of L/A switch) appear to be indirect, since daf-12(+)
activity  before the L2 molt is sufficient to suppress these Lin-L4
phenotypes. The  daf-12(+) activity is need during the L3 stage for
migration of the distal  tip cells of hermaphrodites (Mig-dtc phenotype).
      Our current hypothesis is that daf-12(+)  is required by the L2 
intermolt to advance L3 programs of gonadogenesis and hypodermal fates 
during continous development.  Under dauer-inducing conditions, daf-12(+) 
promotes dauer formation in diverse tissues.  
Interaction with Heterochronic Mutants:  To elucidate the role of daf-12 
in temporal control, we are examining genetic interactions with known 
heterochronic genes. We have adapted the model from Ambros and Moss 
(1994), to include daf-12  (see Figure 1) based on epistasis experiments 
summarized below.  The crux of this model is that  LIN-14 and perhaps 
LIN-28  repress daf-12(+) activity.
      We have looked at double mutants of daf-12(rh61 lof) and loss of 
function alleles of  lin-14  and lin-28,  which cause precocious 
development,  and also with lin-4 and lin-29, which cause delayed 
development (Ambros and Horvitz, (1984) Science 226, 409; Chalfie et al. 
(1981) Cell 24, 59).   In the gonad, the Mig phenotype of daf-12  is 
epistatic to these heterochronic mutants in all cases. Thus daf-12 
controls temporal identity in both gonadal and non-gonadal tissues, 
whereas the other heterochronic genes affect primarily non-gonadal fates. 
      In the hypodermis, daf-12  appears to act downstream of lin-14 , but 
upstream of lin-28 and lin-29.   In daf-12(rh61) lin-14(ma135) double 
mutants, daf-12(rh61)  suppresses lin-14(ma135)- induced precocious 
expression of L3 fates and precocious larval-to-adult (L/A) switch.  
Evidently, lin-14(+)  acts through daf-12(+)  to determine the time of L3 
and later fates.  Also lin-4(e912), whose wildtype activity downregulates 
lin-14, is epistatic to daf-12.  lin-4(e912) retarded phenotypes (e.g. 
complete failure of the L/A switch and vulval morphogenesis)  are
epistatic to daf-12(rh61)  (incomplete L/A switch), daf-12(rh62)   (non-
Lin, Daf-c allele), and strains harboring an extrachromosomal array  of
       daf-12(rh61) lin-28(n719) animals express lin-28  precocious 
phenotypes, i.e., vulval morphogenesis during L2 and L/A switch during L3. 
This argues that lin-28(+)  acts either downstream or functions 
independently of daf-12(+). Finally, in daf-12 lin-29(n333) double 
mutants, seam cells completely fail to undergo the L/A switch (i.e. lin-29 
phenotype). Thus lin-29, the ultimate regulator of the L/A switch (Ambros 
(1989) Cell 57, 49), is epistatic to daf-12.

Figure 1. Heterochronic Model (adapted from Ambros and Moss, (1994) TIGS 
10: 123, and references therein)  LIN-14 activity consists of two 
components defined by genetically separable phenotypes.  LIN-14a  activity
promotes L1 fates, and perhaps inhibits L2 programs.  LIN-14b  together
with LIN-28 permit L2 programs, and prevent precocious  expression of L3
fates.  LIN-28 may independently repress precocious  expression of LIN-29.
The LIN-4 antisense RNA down-regulates LIN-14,  allowing expression of L3
fates.  DAF-12 specifies L3 fates.   DAF-12 could directly or indirectly
regulate LIN-29, or  down-regulate  LIN-28 at later times to allow LIN-29
expression.    An unidentified gene  may determine L4 fates.  LIN-29, a Zn
finger protein (A. Rougvie, V.  Ambros, p.c.),  executes the L/A switch. 


              L1      L2           L3         L4          Adult

lin4 -----/lin-14a
    -------------/ lin-14b
                          }----/  daf-12  ---- ?  -----> 
                   lin-28  ----------------------------/  lin-29         
This diagram should be viewed with a non-proportional font.