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.
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(+).
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.