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EB3/5 cells
This ES cell line developed by Dr. Hitoshi Niwa has been a valuable tool for mouse genetic engineering in our laboratory. EB3/5 cells contain an Oct3-blasticidin resistance gene knockin allele, which enables the selection of pluripotent cells by simple drug administration. These cells can be maintained on feeder-free gelatin-coated dishes.

X-gal staining of a mouse embryo (E11.5) expressing tauLacZ under the Gfr1 promoter. GFR1 is a GPI-anchored cell surface receptor that binds to GDNF with high affinity. Signals are detected in the brain, enteric nervous system, kidneys and peripheral sensory ganglia.

X-gal staining of a mouse embryo (E12.5) expressing tauLacZ under the Ret promoter. RET is a receptor tyrosine kinase that is activated by GDNF family ligands bound to GFR receptors. Signals are detected in the brain, peripheral autonomic and sensory ganglia, and kidneys.

Top view of the head of a Ret tauLacZ/+ embryo (E12.5) subjected to X-gal staining. Signals are detected in the cranial sensory (trigeminal) and parasympathetic (sphenopalatine and otic) ganglia and in the brain (midbrain dopaminergic neurons etc.).

Transmission electron microscopy of enteric neural crest-derived cells in a mouse model of Hirschsprung disease (intestinal aganglionosis). In this mouse model, ENCCs are lost due to caspase-independent non-apoptotic cell death, which leads to the absence of enteric ganglia in the distal colon. These data reveal the crucial role of cell death in the etiology of Hirschsprung disease.

Immunohistochemical detection of GFP (green) and E-cadherin (red) in a developing kidney of GFR1-GFP knockin mice. GFR1 is expressed in the ureteric bud and its surrounding metanephric blastema. E-cadherin is detected in cell-cell junctions of the ureteric bud.

Genetic cell tracing using the Cre-loxP system enables visualization of the morphology of single neurons and their processes (green) against the whole neuronal population (PGP9.5 staining: red) in the developing enteric nervous system.

Neural progenitors in the peripheral ganglia can be selectively expanded in vitro by a neurosphere culture method. These progenitors can differentiate into neurons and glia. This photograph shows the robust neuronal differentiation of neurosphere cells isolated from the parasympathetic ganglia of RET-EGFP knockin embryos. RET-expressing cells (green) co-express PGP9.5, a pan-neuronal marker (red).

Immunostaining of differentiated neurosphere cells generated from mouse sympathetic ganglia. Neurons are recognized by anti-class III tubulin antibody (green; cell bodies and neurites) and anti-Phox2b antibody (blue; nuclei). The nuclei of glial cells are visualized by anti-Sox10 antibody (red).

Immunofluorescent detection of Phox2b (magenta) and PGP9.5 (green) in a whole-mount preparation of mouse embryonic gut (E13.5). Phox2b is expressed in both undifferentiated neural progenitors and neurons. Note that, at this developmental stage, only a subpopulation of Phox2b-positive cells express PGP9.5, a pan-neuronal marker.

Visualization of the RET tyrosine kinase in living enteric neurons. These neurons were isolated from mice engineered to express a RET-EGFP fusion protein under the endogenous Ret promoter. Unlike Ret-deficient mice, which die at birth due to intestinal aganglionosis and kidney agenesis, mice homozygous for the RET-EGFP allele display no abnormalities in RET-dependent organogenesis. RET-EGFP mice serve as a valuable platform for examining the localization and trafficking of RET in living cells and tissues.

Phase contrast view of the same movie as above. Note the highly dynamic morphological changes of the growth cone after its contact with a mesenchymal cell of the gut.

Time-lapse imaging analysis of enteric neural crest-derived cells (ENCCs) in the hindgut of RET-EGFP homozygous mice (E12.5). This movie was taken by examining a gut organ culture using a laser confocal microscope. In migrating ENCCs, RET-EGFP signals were more prominent in the frontal region in the direction of their migration, suggesting that polarized localization of RET is crucial for cell migration.