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Fig. 2. Deficiency of definitive endoderm cells in the ventral foregut of
Hex-/- embryos. (A-E) in situ hybridization for Hex
(A), Foxa2 (B,D) and Mrg1 (C,E). Reduced
Foxa2-positive domain in the ventral endoderm (D) corresponds to the
Hex-positive domain of the wild-type embryo (in A); Foxa2 expression
in the notochordal plate (blue arrowheads) was not affected. The Mrg1
expression domain, a marker of the septum transversum mesenchyme, was not
affected in Hex-/- embryos. (F-H) Sagittal sections of a
5-6S embryos; red arrows indicate ventral endoderm (v.e.) and black arrows,
visceral endoderm (visc.) (F) In situ hybridization for Hex showing
the expression domain of Hex in definitive endoderm cells of the
ventral foregut (v.e.) adjacent to the visceral endoderm, contiguous to red
arrow. (G,H) Sections immunostained for Foxa2 (blue nuclei) to
distinguish definitive (Foxa2 positive) and visceral endoderm (Foxa2 negative)
cell domains. (I,J) Diminished phospho-H3 positive cells (black nuclei) are
found in the ventral endoderm of Hex-/- embryos. Red boxes
denote the Hex-positive domain (indicated in F) used in the
quantitation analysis in M. (K,L) Embryos were exposed in vivo to BrdU for 3.5
hours, harvested, fixed, sectioned and stained for BrdU incorporation (purple
nuclei). The colored boxes in K indicate the endoderm domains used for in the
quantitation analysis in N. (M) Percentage of phospho-H3-positive cells in the
ventral lateral endoderm (red boxes in I and J). A total of 80 sections from
wild-type (n=3) and Hex-/- (n=4) embryos
were counted. A statistically significant decrease of phospho-H3-positive
cells is found in the ventral endoderm of Hex-/- embryos.
(N) Percentage of BrdU-positive cells in different endodermal domains:
dorsal-rostral endoderm (blue box), ventral-rostral endoderm (orange box) and
ventral-lateral endoderm (red box). A total of 70 sections from wild-type
(n=3) and Hex-/- (n=4) embryos were
counted. P value was determined by the homoscedastic t-test (one tail
distribution). A statistically significant decreased BrdU incorporation is
found in the ventral-lateral endoderm of Hex-/- embryos;
the lower proliferation rate is not found in other domains of
Hex-/- embryos. (O) In situ hybridization for
Hex. Sagittal section showing the expression domain of Hex
in the ventral endoderm at 8-9S. (P-R) Immunostaining of sagittal sections
from 8-9S embryos comparing the anterior visceral/definitive endoderm border
in the wild-type and Hex-/- embryos. In the
Hex-/- embryo, visceral endoderm cells (strongly Gata4
positive) occupy the position where the definitive endoderm (Foxa2 positive)
normally occurs in the ventral foregut. In all sections, ventral is to the
left, dorsal is to the right.
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-fetoprotein on isolated ventral endoderm
from 2-6S embryos cultured for 48 hours with cardiac mesoderm. Beating cardiac
domains are outlined in red and sometimes lie above or below endoderm domains;
all purple staining is positive, with a subset denoted by arrows. (C-E)
Embryos were exposed in vivo to BrdU for 2 hours, harvested, fixed, sectioned
and stained for BrdU incorporation (purple nuclei). Blue boxes denote lateral
gut endoderm; red boxes denote hepatic endoderm. (F,G) Quantitation of
BrdU-positive endoderm cells relative to total cells in the hepatic (red bars)
and lateral (blue bars) endodermal domains. A total of 24 sections from
wild-type (n=3) and Hex-/-(n=3) embryos
were evaluated; P value was determined by the homoscedastic one tailed t-test.
A lower proliferation rate was detected in the hepatic bud of
Hex-/- embryos. (H,I) TUNEL assay in the hepatic bud of
18S embryos. The regions with a white dotted outline correspond to the blue
and red boxed regions in panels D,E. No evidence for enhanced apoptosis was
detected in Hex-/- embryos; note the positive staining in
the neural tube and amnion in both embryos.
