Cell fates and fusion in the C. elegans vulval primordium are regulated by the EGL-18 and ELT-6 GATA factors — apparent direct targets of the LIN-39 Hox protein
Kyunghee Koh1,*,
Sara M. Peyrot2,
Cricket G. Wood1,
Javier A. Wagmaister2,
Morris F. Maduro1,
David M. Eisenmann2 and
Joel H. Rothman1,
1 Department of Molecular, Cellular and Developmental Biology, University of
California, Santa Barbara, CA 93106, USA
2 Department of Biological Sciences, University of Maryland Baltimore County,
Baltimore, MD 21250, USA
* Present address: Department of Genetics, University of Pennsylvania,
Philadelphia, PA 19104, USA
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Fig. 1. Phenotypes of egl-18 chromosomal mutants. (A) Surface view of a
wild-type L1 larva. Alae are clearly visible as two unbroken lines along the
length of the body. (B) Surface view of an egl-18(n475) L1 larva,
showing breaks in alae (arrows) and a twisted body (the basis for the Rol
phenotype). (C) Wild-type vulval opening at the `Christmas tree' (L4) stage
larva. (D) Defective vulval opening of an egl-18(n475) larva at the
L4 stage. (E,F) Adult hermaphrodites, showing the wild-type vulva (E) and the
protruding vulva (Pv1) phenotype (F). In these and subsequent photos, anterior
is towards the left and dorsal is towards the top.
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Fig. 2. Mutations in the egl-18 gene. Boxes represent exons and lines
represent introns. The nature of each mutation is described in the lower part
of the figure. The numbers in parentheses indicate the position of the changed
bases in the genomic sequence relative to the egl-18 ATG.
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Fig. 3. Vulval phenotypes of egl-18(RNAi) animals rescued for lethality by
wEx1070. (A) Nomarski image of a wild-type vulval opening at the
`Christmas tree' (L4) stage. (B) Nomarski image of an egl-18(RNAi);
wEx1070 animal, in which all of the six P3.p-P8.p cells (arrows) did not
divide and appear to have fused. (C) ajm-1::GFP (adherens junction
marker) expression in a wildtype animal at the early L3 stage. All six VPCs
(arrowheads) are clearly demarcated by ajm-1::GFP. One of the VPCs is
partially out of focus in this image. (D) ajm-1::GFP expression in an
egl-18(RNAi); wEx1070 animal at a similar stage to the animal shown
in C. All but one VPC are in the process of fusion as indicated by fragmented
ajm-1::GFP expression (arrows). One VPC remains unfused as shown by a
complete ring (arrowhead).
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Fig. 5. (A) Hox/PBC-binding sites in the egl-18 and elt-6 genomic
region and GFP reporter constructs. Open reading frames of egl-18 and
elt-6 are indicated by black and gray boxes, respectively. Introns
and 5' and 3' UTRs are indicated by lines. Triangles indicate
consensus Hox/PBC-binding sites, two of which in the second intron of
egl-18 are labeled sites 1 and 2. (B) Alignment of C.
elegans site 1 and the corresponding C. briggsae sequence. The
region corresponding to C. elegans site 2 is not conserved in C.
briggsae.
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Fig. 6. LIN-39 and CEH-20 bind cooperatively to two consensus Hox/PBC binding sites
in vitro. The bottom arrow indicates bands corresponding to probes bound to
LIN-39 alone and the top arrow indicates bands corresponding to probes bound
to LIN-39/CEH-20 heterodimers. In the first four lanes, an oligonucleotide
(Antp/Exd) containing a binding site for a Drosophila Hox protein,
Antennapedia, and its co-factor, Extradenticle, is used as a positive control.
LIN-39 and CEH-20 bind the wild-type site 1 (S1) and site 2 (S2) efficiently,
but not the mutated sites (S1M and S2M). Site 2 shows greater binding affinity
for LIN-39/CEH-20 heterodimers than does site 1, and LIN-39 alone can bind
site 2 but not site 1.
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© The Company of Biologists Ltd 2002
800 bp enhancer and a basal promoter. (A) L2 larva, in
which all VPCs (P3.p-P8.p) show approximately equal levels of GFP expression
(arrows). All six VC neurons show GFP expression, although not all are visible
in this focal plane. Arrowhead indicate a VC neuron. (B) L3 larva shortly
before VPC divisions, showing stronger GFP expression in P5.p-P7.p cells than
in P4.p and P8.p. (C) L3 larva after the first VPC cell divisions. Daughters
of P6.p show higher GFP expression than daughters of P5.p and P7.p. GFP
expression is not detectable in P4.px and P8.px cells. (D) Early-L3 larva
showing expression of an elt-6 transcriptional fusion pKK41 in
P5.p-P7.p cells. pKK41 contains 