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Fig. 5. Lhx5 regulates Sfrp1a and Sfrp5 expression. (A-I) Lhx5
regulates Sfrp1a expression. (A-C) Animal pole views, dorsal to the right.
(D-F) Dorsal views, bud stage, sfrp1a expression. (G-I) Flat mounts
in glycerol; dorsal views, rostral to the top. 2 ng (H) or 4 ng (I) of
lhx5 morpholino was injected. (J-M) Lhx5 cell autonomously
regulates Sfrp1a expression. Transplanted cells are labeled in red.
sfrp1a transcripts are highlighted by white arrowheads and are
apparent as blue dots in the narrow cytoplasm around the nuclei of
transplanted cells in controls (J, ctl) and lhx5 mRNA-injected cells
transplanted into lhx5 morpholino injected hosts (M). sfrp1a
transcripts are not detected in lhx5-en injected cells transplanted
into control hosts (K). In transplanted lhx5 morpholino-injected
cells, sfrp1a expression is reduced (L). (J-L) 90% epiboly stage; (M)
one-somite stage. (N) Mapping of sfrp1a upstream enhancers by
transient GFP reporter expression in the forebrain. Fragments are numbered
using the first nucleotide of the Sfrp1a start codon as the reference. GFP
expression in the forebrain was scored at the 3 somite stage and the Prim-5
stage. The number of + signs indicate relative GFP expression levels.
(O) Lhx5 binds to the sfrp1a promoter. Input represents 0.2%
(2 µl, 2x) or 0.1% (1 µl, 1x) of starting material for each
immunoprecipitation reaction. 2 µl (2x) or 1 µl (1x) of purified DNA
product from each sample was used in the PCR. ctl., uninjected embryos treated
in parallel; inj., lhx5-GFP mRNA-injected embryos. Mock
immunoprecipitation with no antibody fails to amplify from either uninjected
or injected groups (data not shown). (P-U) Lhx5 regulates Sfrp5
expression. Flat mounts in glycerol, dorsal views, rostral to the top. (R,T,U)
2 ng (R,T) or 4 ng (U) of lhx5 morpholinos was injected. Scale bar in
U: 250 µm for A-F; 100 µm for G-I; 8 µm for J-M; 100 µm for
P-U.
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