Parker B Antin

PMID: 15294868;Abstract:

During embryonic development, the first blood vessels are formed through the aggregation and subsequent assembly of angioblasts (endothelial precursors) into a network of endothelial tubes, a process known as vasculogenesis. These first vessels generally form in mesoderm that is adjacent to endodermal tissue. Although specification of the angioblast lineage is independent of endoderm interactions, a signal from the endoderm is necessary for angioblasts to assemble into a vascular network and to undergo vascular tube formation. In this study, we show that endodermally derived sonic hedgehog is both necessary and sufficient for vascular tube formation in avian embryos. We also show that Hedgehog signaling is required for vascular tube formation in mouse embryos, and for vascular cord formation in cultured mouse endothelial cells. These results demonstrate a previously uncharacterized role for Hedgehog signaling in vascular development, and identify Hedgehog signaling as an important component of the molecular pathway leading to vascular tube formation.

Neural crest stem cells can be isolated from differentiated cultures of human pluripotent stem cells, but the process is inefficient and requires cell sorting to obtain a highly enriched population. No specific method for directed differentiation of human pluripotent cells toward neural crest stem cells has yet been reported. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering. In this report, we use small-molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for coculture on feeder layers or cell sorting to obtain a highly enriched population. Critical to this approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 12-14 d, pluripotent cells are efficiently specified along the neuroectoderm lineage toward p75(+) Hnk1(+) Ap2(+) neural crest-like cells with little or no contamination by Pax6(+) neural progenitors. This cell population can be clonally amplified and maintained for >25 passages (>100 d) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells, and mesenchymal precursor cells. Neural crest-like stem cell-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes, and adipocytes. In sum, we have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.

PMID: 23264563;PMCID: PMC3634647;Abstract:

Endothelia in the atrioventricular (AV) canal of the developing heart undergo a prototypical epithelial mesenchymal transition (EMT) to begin heart valve formation. Using an in vitro invasion assay, an extracellular matrix protein, Olfactomedin-1 (OLFM1), was found to increase mesenchymal cell numbers in AV canals from embryonic chick hearts. Treatment with both anti-OLFM1 antibody and siRNA targeting OLFM1 inhibits mesenchymal cell formation. OLFM1 does not alter cell proliferation, migration or apoptosis. Dispersion, but lack of invasion in the presence of inhibiting antibody, identifies a specific role for OLFM1 in cell invasion during EMT. This role is conserved in other epithelia, as OLFM1 similarly enhances invasion by MDCK epithelial cells in a transwell assay. Synergy is observed when TGFβ2 and OLFM1 are added to MDCK cell cultures, indicating that OLFM-1 activity is cooperative with TGFβ. Inhibition of both OLFM1 and TGFβ in heart invasion assays shows a similar cooperative role during development. To explore OLFM1 activity during EMT, representative EMT markers were examined. Effects of OLFM1 protein and anti-OLFM1 on transcripts of cell-cell adhesion molecules and the transcription factors Snail-1, Snail-2, Twist1 and Sox-9 argue that OLFM1 does not initiate EMT. Rather, regulation of transcripts of Zeb1 and Zeb2, secreted proteases and mesenchymal cell markers by both OLFM1 and anti-OLFM1 is consistent with regulation of the cell invasion step of EMT. We conclude that OLFM1 is present and necessary during EMT in the embryonic chick heart. Its role in cell invasion and mesenchymal cell gene regulation suggests an invasion checkpoint in EMT where OLFM1 acts to promote cell invasion into the three-dimensional matrix. © 2013. Published by The Company of Biologists Ltd.

PMID: 9882479;Abstract:

Previous studies have identified two signaling interactions regulating cardiac myogenesis in avians, a hypoblast-derived signal acting on epiblast and mediated by activin or a related molecule and an endoderm-derived signal acting on mesoderm and involving BMP-2. In this study, experiments were designed to investigate the temporal relationship between these signaling events and the potential role of other TGFβ superfamily members in regulating early steps of heart muscle development. We find that while activin or TGFβ can potently induce cardiac myogenesis in pregastrula epiblast, they show no capacity to convert noncardiogenic mesoderm toward a myocardial phenotype. Conversely, BMP-2 or BMP-4, in combination with FGF-4, can readily induce cardiac myocyte formation in posterior mesoderm, but shows no capacity to induce cardiac myogenesis in epiblast cells. Activin/TGFβ and BMP-2/BMP-4 therefore have distinct and reciprocal cardiac-inducing capacities that mimic the tissues in which they are expressed, the pregastrula hypoblast and anterior lateral endoderm, respectively. Experiments with noggin and follistatin provide additional evidence indicating that BMP signaling lies downstream of an activin/TGFβ signal in the cardiac myogenesis pathway. In contrast to the cardiogenic-inducing capacities of BMP-2/BMP-4 in mesoderm, however, we find that BMP-2 or BMP-4 inhibits cardiac myogenesis prior to stage 3, demonstrating multiple roles for BMPs in mesoderm induction. These and other published studies suggest a signaling cascade in which a hypoblast-derived activin/TGFβ signal is required prior to and during early stages of gastrulation, regulated both spatially and temporally by an interplay between BMPs and their antagonists. Later cardiogenic signals arising from endoderm, and perhaps transiently from ectoderm, and mediated in part by BMPs, act on emerging mesoderm within cardiogenic regions to activate or enhance expression of cardiogenic genes such as GATA and cNkx family members, leading to cardiac myocyte differentiation.