Reprogramming Postnatal Human Epidermal Keratinocytes Toward Functional Neural Crest Fates.

During development, neural crest (NC) cells are induced by signaling events at the neural plate border of all vertebrate embryos. Initially arising within the central nervous system, NC cells subsequently undergo an epithelial to mesenchymal transition to migrate into the periphery, where they differentiate into diverse cell types. Here we provide evidence that postnatal human epidermal keratinocytes (KC), in response to fibroblast growth factor 2 and insulin like growth factor 1 signals, can be reprogrammed toward a NC fate. Genome-wide transcriptome analyses show that keratinocyte-derived NC cells are similar to those derived from human embryonic stem cells. Moreover, they give rise in vitro and in vivo to NC derivatives such as peripheral neurons, melanocytes, Schwann cells and mesenchymal cells (osteocytes, chondrocytes, adipocytes, and smooth muscle cells). By demonstrating that human keratin-14+ KC can form NC cells, even from clones of single cells, our results have important implications in stem cell biology and regenerative medicine. Stem Cells 2017;35:1402-1415.

Genome-wide identification of Wnt/ß-catenin transcriptional targets during Xenopus gastrulation.

The canonical Wnt/ß-catenin signaling pathway plays multiple roles during Xenopus gastrulation, including posteriorization of the neural plate, patterning of the mesoderm, and induction of the neural crest. Wnt signaling stabilizes ß-catenin, which then activates target genes. However, few targets of this signaling pathway that mediate early developmental processes are known. Here we sought to identify transcriptional targets of the Wnt/ß-catenin signaling pathway using a genome-wide approach. We selected putative targets using the criteria of reduced expression upon zygotic Wnt knockdown, ß-catenin binding within 50kb of the gene, and expression in tissues that receive Wnt signaling. Using these criteria, we found 21 novel direct transcriptional targets of Wnt/ß-catenin signaling during gastrulation and in addition have identified putative regulatory elements for further characterization in future studies.

A comparative morphologic and morphometric study about geniculate ganglion development in man and in rat.

CONCLUSIONS: Human-rat geniculate ganglion (GG) have multiple origins: (1) An initial proximity (20 µm) to the endocranial foramen of the IAM, suggests neural crest induction; and (2) The influence of epibranchial placodes: the tensor tympani muscle (TTM) and the otic apical coil. OBJECTIVES: This study was undertaken to determine the comparative development of human-rat GG. MATERIALS AND METHODS: A light microscopic study of the GG in human material obtained from spontaneous abortions at 9, 13, 14, 17, 18, and 30 weeks, and one neonate was done. This study examined Webster rat embryos and a post-natal series. Specimens were fixed in Bouin fluid, embedded in paraffin, cut, and stained with H&E. The histomorphometric data were obtained with image analysis software. RESULTS: The human fetus of 9 weeks presents two neuronal groups in the VII nerve: one near (20 µm) the IAM endocranial foramen, foraminal, and the other, tympanic. Neonate GG is located between the TTM and the cochlear apex (inwards). In the 16 day old rat embryo GG is placed within a canal containing the stapedial artery. In the adult rat the GG and the stapedial artery are placed within the IAM.

The use of human pluripotent stem cells for the in vitro derivation of cranial placodes and neural crest cells.

Due to their intrinsic differentiation potential, human pluripotent stem cells (hPSCs) hold remarkable promise for their use in cell-based therapies as well as an in vitro model for early human embryogenesis and for modeling disease. During the development of the human embryo, transient structures such as the neural crest (NC) and the cranial placodes (CPs) are specified in the first 3-4 weeks of gestation. Because of this early occurrence and a scarce availability of embryos for research purposes, these transient structures remain largely unexplored in humans. Hence, investigators are now exploiting in vitro differentiation of hPSC to unveil these early events and to generate NC and CP cells in vitro. Derivatives of the NC and CPs will contribute to the formation of very important organs, including most of the peripheral nervous system (NC) and the sensory organs of the head (CP). There are many diseases and conditions that affect NC and CP derivatives, thus a better knowledge of how these structures specialize, and the derivation of functional NC and CP cells for therapeutic applications will have an impact on the understanding and treatment of these disorders. Here, we discuss the current state of the art in directing hPSCs into NC or CP cells, which in spite of their importance is still in its infancy.

Role of Sp5 as an essential early regulator of neural crest specification in xenopus.

BACKGROUND: The neural crest (NC) is a multipotent embryonic cell population, which is induced by an integration of secreted signals including BMP, Wnt, and FGF and, subsequently, NC cell fates are specified by a regulatory network of specific transcription factors. This study was undertaken to identify a role of Sp5 transcription factor in vertebrates. RESULTS: Xenopus Sp5 is expressed in the prospective neural crest regions from gastrulation through the tadpole stages in early development. Knockdown of Sp5 caused severe defects in craniofacial cartilage, pigmentation, and dorsal fin. Gain- and loss-of-function of Sp5 led to up- and down-regulation of the expression of NC markers in the neural fold, respectively. In contrast, Sp5 had no effect on neural induction and patterning. Sp5 regulated the expression of neural plate border (NPB) specifiers, Msx1 and Pax3, and these regulatory factors recovered the expression of NC marker in the Sp5-deficient embryos. Depletion of Sp5 impaired NC induction by Wnt/ß-catenin or FGF signal, whereas its co-expression rescued NC markers in embryos in which either signal was blocked. CONCLUSIONS: These results suggest that Sp5 functions as a critical early factor in the genetic cascade to regulate NC induction downstream of Wnt and FGF pathways.

sizzled function and secreted factor network dynamics.

Studies on the role of the E-box binding transcription factor Snail2 (Slug) in the induction of neural crest by mesoderm (Shi et al., 2011) revealed an unexpected increase in the level of sizzled RNA in the dorsolateral mesodermal zone (DMLZ) of morphant Xenopus embryos. sizzled encodes a secreted protein with both Wnt and BMP inhibitor activities. Morpholino-mediated down-regulation of sizzled expression in one cell of two cell embryos or the C2/C3 blastomeres of 32-cell embryos, which give rise to the DLMZ, revealed decreased expression of the mesodermal marker brachyury and subsequent defects in neural crest induction, pronephros formation, and muscle patterning. Loss of sizzled expression led to decreases in RNAs encoding the secreted Wnt inhibitor SFRP2 and the secreted BMP inhibitor Noggin; the sizzled morphant phenotype could be rescued by co-injection of RNAs encoding Noggin and either SFRP2 or Dickkopf (a mechanistically distinct Wnt inhibitor). Together, these observations reveal that sizzled, in addition to its established role in dorsal-ventral patterning, is also part of a dynamic BMP and Wnt signaling network involved in both mesodermal patterning and neural crest induction.