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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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