New directions in craniofacial morphogenesis

AbstractThe vertebrate head is an extremely complicated structure: development of the head requires tissue–tissue interactions between derivates of all the germ layers and coordinated morphogenetic movements in three dimensions. In this review, we highlight a number of recent embryological studies, using chicken, frog, zebrafish and mouse, which have identified crucial signaling centers in the embryonic face. These studies demonstrate how small variations in growth factor signaling can lead to a diversity of phenotypic outcomes. We also discuss novel genetic studies, in human, mouse and zebrafish, which describe cell biological mechanisms fundamental to the growth and morphogenesis of the craniofacial skeleton. Together, these findings underscore the complex interactions leading to species-specific morphology. These and future studies will improve our understanding of the genetic and environmental influences underlying human craniofacial anomalies

Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways.

Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors such as Activin, FGF and BMP in the control of early cell fate decisions of human pluripotent stem cells. This analysis resulted in the development and validation of chemically defined culture conditions for achieving specification of human embryonic stem cells into neuroectoderm, mesendoderm and into extra-embryonic tissues. Importantly, these defined culture conditions are devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Importantly, the growth factor roles defined using these culture conditions similarly drove differentiation of mouse epiblast stem cells derived from post implantation embryos, thereby reinforcing the hypothesis that epiblast stem cells share a common embryonic identity with human pluripotent stem cells. Therefore the defined growth factor conditions described here represent an essential step toward the production of mature cell types from pluripotent stem cells in conditions fully compatible with clinical use ant also provide a general approach for modelling the early steps of mammalian embryonic development

Activin/Nodal signalling maintains pluripotency by controlling Nanog expression

The pluripotent status of embryonic stem cells (ESCs) confers upon them the capacity to differentiate into the three primary germ layers, ectoderm, mesoderm and endoderm, from which all the cells of the adult body are derived. An understanding of the mechanisms controlling pluripotency is thus essential for driving the differentiation of human pluripotent cells into cell types useful for clinical applications. The Activin/Nodal signalling pathway is necessary to maintain pluripotency in human ESCs and in mouse epiblast stem cells (EpiSCs), but the molecular mechanisms by which it achieves this effect remain obscure. Here, we demonstrate that Activin/Nodal signalling controls expression of the key pluripotency factor Nanog in human ESCs and in mouse EpiSCs. Nanog in turn prevents neuroectoderm differentiation induced by FGF signalling and limits the transcriptional activity of the Smad2/3 cascade, blocking progression along the endoderm lineage. This negative-feedback loop imposes stasis in neuroectoderm and mesendoderm differentiation, thereby maintaining the pluripotent status of human ESCs and mouse EpiSCs

Contrary to mESCs, EpiSCs are responsive to culture conditions controlling differentiation of hESCs into extra-embryonic tissues, neuroectoderm and mesendoderm.

<p>(A) Expression of pluripotency markers (Oct-4, Nanog) and neuroectoderm (Sox2, Sox1, Six3, Tuj1 and Gbx2), extra-embryonic markers (Cdx2, Hand1, Sox7, GATA6) and mesendoderm markers (Brachyury, Mixl1, Eomes, Sox17) during differentiation of mouse EpiSCs using the culture conditions developed with hESCs. EpiSCs were differentiated following the conditions described above for hESCs (SB+FGF for neuroectoderm, BMP4 for extra-embryonic tissues, three step protocol for mesendoderm). Following the ninth day after plating, RNAs were extracted and expression of the denoted genes was analysed using Q-PCR. Normalized expression is shown as the mean±SD from two informative experiments. (B) Expression of pluripotency markers (Oct-4, Nanog, SSEA-1), extra-embryonic markers (CDX2, Sox7, GATA4), neuroectoderm markers (Sox1, Nestin, βIII tubulin) and mesendoderm markers (Brachyury and Sox17) in EpiSCs differentiated using the conditions developed for hESCs. Expression of the genes denoted was analysed by immuno-fluoresence analyses. Nuclei are shown by Hoechst staining. Scale Bar 100 µM. (C) Expression of pluripotency markers (Oct-4), extra-embryonic markers (CDX2, GATA6), neuroectoderm markers (Sox2, Six3) and mesendoderm markers (Brachyury and Sox17) in mESCs differentiated using the method developed with hESCs. mESCs were differentiated in CDM as described for hESCs and then the expression of the genes denoted was analysed by Real-Time PCR. Normalized expression is shown as the mean±SD from three experiments.</p

Generation of mesendoderm using a combination of Activin, FGF2 and BMP4.

<p>(A) Colony morphologies formed in response to the three-step protocol to differentiate hESCs into mesendoderm progeny. H9 cells were grown for 2 days in CDM/PVA+Activin 10 ng/ml+FGF2 12 ng/ml, then for 72 hours in CDM/PVA+SU5402 10 µM+Activin 5 ng/ml. The next 4 days, cells were grown in CDM/PVA+Activin 30 ng/ml+FGF2 20 ng/ml+BMP4 10 ng/ml. Images of the same colonies were captured every day for 9 days. Scale Bar 200 µM. (B) Effect of different combinations and doses of Activin, FGF2 and BMP4 on the differentiation of hESCs grown in CDM/PVA. Following the third day in CDM/PVA+SU5402 10 µM, H9 cells were induced to differentiated in CDM/PVA supplemented with different combination of growth factors. RNAs were extracted after 3 days and expression of the denoted genes was analysed using Q-PCR. Normalized expression is shown as the mean±SD from two informative experiments. hESCs grown in CDM+Activin+FGF or differentiated in CDM+SB431542+FGF2 were used as negative controls. (C) Expression of specific markers for mesendoderm in hESCs differentiated in CDM/PVA supplemented with Activin, FGF2 and BMP4 in the three step protocol. Nuclei are shown by Hoechst staining. Scale Bar 100 µM. (D) Microarray gene expression heat map to compare human embryonic stem cells (ESC) grown in CDM supplemented with Activin and FGF and mesendoderm cells generated in CDM/PVA supplemented with Activin, FGF and BMP4 (LE). Up-regulation is coloured in shades of red and down-regulation in shades of blue according to the log z scale shown at the bottom of the heat map. Gene names marked with an asterisk denote genes that pass a significant differential regulation threshold. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006082#s4" target="_blank">Material and Methods</a>). (E) Fraction of cells expressing the definitive endoderm marker CXCR4 and the mesendoderm/mesoderm marker PDGFαR after induction of differentiation in CDM PVA in the presence of increasing doses of Activin. H9 cells were differentiated following the three step protocol described above in the presence of Activin (30 or 100 ng/ml). Fraction of cells expressing CXCR4 or PDGFαR was determined using FACS 8 days after plating. (F) RT-PCR analyses for the expression of liver markers (Albumin, HNF4, αFP), gut marker (CDX2) and cardiac markers (ANF, α Actinin, α-1 Channel) in endoderm progenitors grown in media containing serum. Endoderm progenitors generated using the three step protocol were differentiated in media containing three different FBS lots. RNAs were extracted after 5 and 10 days of differentiation and the expression of genes expressed was analysed using RT-PCR.</p

Identification of culture conditions for inducing neuroectoderm and mesendoderm differentiation in CDM-PVA.

<p>H9 hESCs were grown for different periods in CDM-PVA supplemented with different growth factors (Activin, FGF2 or BMP4) or chemical inhibitors SU5402 (SU) for FGF. Then, immunostaining analyses were used to determine the fraction of cells expressing the pluripotency marker Oct-4, the neuroectoderm marker Sox2, the mesoderm marker Brachyury, and the endoderm marker Sox17. The levels of marker-expressing cells were divided into five arbitrary categories: 0 for absence of expression, Very Low (VL) for <1% expressing cells, Low (L) for <5% expressing cells, Moderate (M) for >10% expressing cells, High (H) for >50% expressing cells. Indicated in blue are conditions allowing the generation of neuroectoderm (expressing Sox2) and in red those inducing mesendoderm (cells expressing Brachyury or Sox17).</p

Identification of culture conditions for inducing neuroectoderm and mesendoderm differentiation in CDM-BSA.

<p>H9 hESCs were grown for different periods in CDM-BSA supplemented with different growth factors (Activin, FGF2 or BMP4) or chemical inhibitors [SB431542 (SB) for Activin/Nodal, SU5402 (SU) for FGF or BIO for GSK3β/Wnt]. Then, immunostaining analyses were used to determine the fraction of cells expressing the pluripotency marker Oct-4, the neuroectoderm marker Sox2, the mesoderm marker Brachyury, and the endoderm marker Sox17. The levels of marker-expressing cells were divided into five arbitrary categories: 0 for absence of expression, Very Low (VL) for <1% expressing cells, Low (L) for <5% expressing cells, Moderate (M) for >10% expressing cells, High (H) for >50% expressing cells. Indicated in blue are conditions allowing the generation of neuroectoderm (expressing Sox2) and in red those inducing mesendoderm (cells expressing Brachyury or Sox17).</p