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Fig. 1. NKX2.1 activation in foregut endoderm occurs in close proximity to liver
specification. (A) Sagittal view of a mouse embryo at the 6-somite stage,
demonstrating tissue regions isolated for RT-PCR analysis and used for explant
assays. Broken line indicates plane of embryo sections in C-F. (B) RT-PCR
analysis of total RNA from designated tissue regions at different somite
stages. Tissue microdissected from day 10 embryos (E10), with and without
reverse transcriptase (-RT), used as positive and negative controls,
respectively. Actin probe used as control for total RNA integrity; FOXA2 probe
is used to ensure actin levels reflected isolated endoderm. (C-J) Embryo
sections at designated somite stages demonstrating expression of albumin
(green) and NKX2.1 (red) in mouse foregut endoderm by double
immunofluorescence. (C-F) Transverse embryo section at the 10-somite stage
encompassing foregut and region of cardiac mesoderm. (C) Boxed area in DAPI
image (to detect nuclei) is magnified in D-F. Yellow signal in merged image
(F) reflects nonspecific (present in all filters) fluorescent staining of
acellular material frequently seen in the foregut pocket (also seen in
Fig. 2B)
(Kaufman, 1992;
Jung et al., 1999). (D,E) More
cells in the foregut endoderm express albumin than NKX2.1 protein. Markers
(boxed magnification) are colocalized in merged image (F). (G-J) Albumin and
NKX2.1 expression in E9.5 sagittal embryo section. Boxed area in G (DAPI
image) is magnified in H-J and encompasses the cardiac and presumptive lung
(LuR) and liver (LiR) regions. (K-T) Immunostaining of single cells generated
from ventral endoderm isolated at 10- and 14-somite stages. Endoderm cells
were dissected away from adjacent mesoderm, dispersed, spun down onto a slide,
double stained with antibodies for albumin and NKX2.1, and counterstained with
DAPI. Positive cells tended to occur in aggregates. K-N reflect the same cells
at the 10-somite sage; (O-R) same cells at 14-somite stage double-stained with
albumin and NKX2.1. Arrows indicate cells that express both albumin and
NKX2.1. Merged image with DAPI reflects expected cytoplasmic localization of
albumin (O) and nuclear localization of NKX2.1 (R). The granular nature of the
fluorescent signal was also seen in control adult tissues treated identically.
(S) Quantification of albumin-positive, NKX2.1-positive and
albumin/NKX2.1-positive ventral endoderm cells relative to total cells on
cytospin slides at 8-, 10- and 14-somite stages.
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