Hedgehog signaling is required for the differentiation of ES cells into neurectoderm

Abstract

Mouse embryonic stem cells can differentiate in vitro into cells of the nervous system, neurons and glia. This differentiation mimics stages observed in vivo, including the generation of primitive ectoderm and neurectoderm in embryoid body culture. We demonstrate here that embryonic stem cell lines mutant for components of the Hedgehog signaling cascade are deficient at generating neurectoderm-containing embryoid bodies. The embryoid bodies derived from mutant cells are also unable to respond to retinoic acid treatment by producing nestin-positive neural stem cells, a response observed in cultures of heterozygous cells, and contain cores apparently arrested at the primitive ectoderm stage. The mutant cultures are also deficient in their capacity to differentiate into mature neurons and glia. These data are consistent with a role for Hedgehog signaling in generating neurectoderm capable of producing the appropriate neuronal and glial progenitors in ES cell culture.

Directing the differentiation of embryonic stem cells to neural stem cells

“…Studies also indicate that Sonic Hedgehog promotes both the survival and proliferation of NSCs and neuroblasts during adult neurogenesis (Machold et al, 2003;Ahn and Joyner, 2005;Palma et al, 2005). Our initial studies, using ESCs carrying mutations in Hedgehog signaling components, suggested that this signal is critical for the differentiation of primitive ectoderm to neurectoderm (Maye et al, 2004). Our more recent analysis, using Hedgehog antagonists and the Sox1-GFP reporter system, has established two roles for Hedgehog signaling in ESC neurogenesis ( Fig.…”

“…ESC-derived embryoid bodies are similar to embryoid bodies derived from teratocarcinoma stem cells, first described by Stevens and Pierce (Pierce and Dixon, 1959;Stevens, 1959) and subsequently demonstrated to undergo a similar pattern of differentiation to isolated inner cell masses cultured in vitro (Martin et al, 1977). When mouse ESCs are removed from their feeder layer, and placed in suspension culture in the absence of the growth factor LIF, which promotes maintenance of the pluripotential state (Smith et al, 1988), they form aggregates, which within 2-4 days consist of an outer layer of hypoblastlike cells (extraembryonic visceral endoderm) surrounding an epiblastlike core (primitive ectoderm; Martin et al, 1977;Rathjen et al, 2002;Maye et al, 2004). At this stage, the embryoid body resembles the anterior prestreak stage embryo, with the epiblast-like core able to generate derivatives of all three primary germ layers; definitive endoderm, mesoderm, and ectoderm (Keller, 2005).…”

“…At this stage, the embryoid body resembles the anterior prestreak stage embryo, with the epiblast-like core able to generate derivatives of all three primary germ layers; definitive endoderm, mesoderm, and ectoderm (Keller, 2005). The embryoid body core continues to express the ESC marker Oct4 and begins expressing the primitive ectoderm marker FGF5 (Hebert et al, 1991;Rathjen et al, 2002;Maye et al, 2004). Between days 6 and 8, the core undergoes cavitation and forms an inner epithelial layer (Coucouvanis and Martin, 1995).…”

“…Cells within this layer can be committed to definitive ectoderm, characterized by Sox2 and Otx2 expression (Rathjen et al, 2002;Maye et al, 2004). Subsequently, a morphological transformation to columnar epithelium, resembling the neural tube, is accompanied by the expression of neurectoderm-specific markers such as Sox1 and Six3 (Rathjen et al, 2002;Maye et al, 2004).…”

“…Embryoid bodies derived from ESCs carrying loss of function mutations for components of the Hedgehog signaling pathway are able to generate primitive ectoderm, but not neurectoderm or neurons (Maye et al, 2004). The Hedgehog signaling cascade is best known for its role in ventral patterning (Briscoe et al, 2000;Wichterle et al, 2002); however, several recent studies support an additional role in NSC survival (Britto et al, 2000;Machold et al, 2003;Cayuso et al, 2006) and proliferation (Machold et al, 2003;Ahn and Joyner, 2005;Palma et al, 2005) in the embryonic and adult mouse brain.…”