Ondine Cleaver

Endothelial signaling during development.

Blood vessels perfuse all tissues in the body and mediate vital metabolic exchange between tissues and blood. Increasing evidence, however, points to a direct role for paracrine signaling between blood vessel cells and surrounding target organ cells, during embryonic development and cell differentiation. Understanding the nature of this signaling and its heterogeneity, both in the embryo and in adult tissues, may not only provide insights into mechanisms for normal developmental cell fate decisions, but could also lead to novel targeted therapeutic approaches for a variety of diseases such as heart disease, diabetes or cancer.

Role of endothelial cells in early pancreas and liver development

Liver and pancreas initially develop by budding from the embryonic endoderm. The formation of these organs coincides with the appearance of endothelial cells (ECs) adjacent to the endoderm. ECs either develop in situ in organs, or are recruited by organs and are induced to form blood vessels. Recent reports on liver and pancreas have now shown that ECs also induce essential steps in organ formation such as morphogenesis and cell differentiation. This review summarizes reports on EC signaling during organogenesis and cell differentiation.

Dependence of mouse embryonic stem cells on threonine catabolism.

Threonine Required Embryonic stem (ES) cells divide rapidly, raising the possibility that they might exist in a metabolic state that facilitates rapid growth. By monitoring the abundance of common metabolites in mouse ES cells, Wang et al. (p. 435; published online 9 July) found altered levels of metabolites involved in carbon metabolism. Measurement of messenger RNA levels revealed unusually high expression of the gene encoding threonine dehydrogenase. In addition, in growth experiments, mouse ES cells were critically dependent on the amino acid threonine. Mouse embryonic stem cells exist in a high-flux metabolic state comparable to that of rapidly dividing bacteria. Measurements of the abundance of common metabolites in cultured embryonic stem (ES) cells revealed an unusual state with respect to one-carbon metabolism. These findings led to the discovery of copious expression of the gene encoding threonine dehydrogenase (TDH) in ES cells. TDH-mediated catabolism of threonine takes place in mitochondria to generate glycine and acetyl–coenzyme A (CoA), with glycine facilitating one-carbon metabolism via the glycine cleavage system and acetyl-CoA feeding the tricarboxylic acid cycle. Culture media individually deprived of each of the 20 amino acids were applied to ES cells, leading to the discovery that ES cells are critically dependent on one amino acid—threonine. These observations show that ES cells exist in a high-flux backbone metabolic state comparable to that of rapidly growing bacterial cells.

Epithelial dynamics of pancreatic branching morphogenesis

The mammalian pancreas is a highly branched gland, essential for both digestion and glucose homeostasis. Pancreatic branching, however, is poorly understood, both at the ultrastructural and cellular levels. In this article, we characterize the morphogenesis of pancreatic branches, from gross anatomy to the dynamics of their epithelial organization. We identify trends in pancreatic branch morphology and introduce a novel mechanism for branch formation, which involves transient epithelial stratification and partial loss of cell polarity, changes in cell shape and cell rearrangements, de novo tubulogenesis and epithelial tubule remodeling. In contrast to the classical epithelial budding and tube extension observed in other organs, a pancreatic branch takes shape as a multi-lumen tubular plexus coordinately extends and remodels into a ramifying, single-lumen ductal system. Moreover, our studies identify a role for EphB signaling in epithelial remodeling during pancreatic branching. Overall, these results illustrate distinct, step-wise cellular mechanisms by which pancreatic epithelium shapes itself to create a functional branching organ.

Prospective isolation of skeletal muscle stem cells with a Pax7 reporter.

Muscle regeneration occurs through activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to make new myofibers. We used a transgenic Pax7‐ZsGreen reporter mouse to prospectively isolate stem cells of skeletal muscle by flow cytometry. We show that Pax7‐expressing cells (satellite cells) in the limb, head, and diaphragm muscles are homogeneous in size and granularity and uniformly labeled by certain cell surface markers, including CD34 and CD29. The frequency of the satellite cells varies between muscle types and with age. Clonal analysis demonstrated that all colonies arising from single cells within the Pax7‐sorted fraction have myogenic potential. In response to injury, Pax7+ cells reduce CD34, CD29, and CXCR4 expression, increase in size, and acquire Sca‐1. When directly isolated and cultured in vitro, Pax7+ cells display the hallmarks of activation and proliferate, initially as suspension aggregates and later distributed between suspension and adherence. During in vitro expansion, Pax7 (ZsGreen) and CD34 expression decline, whereas expression of PSA‐NCAM is acquired. The nonmyogenic, Pax7neg cells expand as Sca1+ PDGRα+ PSA‐NCAMneg cells. Satellite cells expanded exclusively in suspension can engraft and produce dystrophin+ fibers in mdx−/− mice. These results establish a novel animal model for the study of muscle stem cell physiology and a culture system for expansion of engraftable muscle progenitors.

Biphasic Ngn3 expression in the developing pancreas

Ngn3 is a bHLH transcription factor critical for the specification of endocrine cells in the pancreatic Islets of Langerhans. Previous studies in mouse embryos have reported transient expression of Ngn3 in scattered cells within the developing pancreatic epithelium during midgestation (Schwitzgebel et al. [ 2000 ] Development 127:3533–3542). Specifically, these Ngn3‐expressing cells have been shown to be progenitor cells fated to give rise to islet endocrine cells (Gradwohl et al. [ 2000 ] Proc Natl Acad Sci USA 97:1607–1611). Here, we characterize the expression of Ngn3 transcripts and protein throughout pancreatic development. Interestingly, we identify and define a dramatic and previously unnoticed gap in developmental Ngn3 expression. We show that both Ngn3 transcript and protein expression occur in two distinct temporal waves, the first occurring early from approximately E8.5 to E11.0, and the second initiating at approximately E12.0. Strikingly, this observed biphasic expression correlates with the “first” and “second” transitions, which encompass two distinct waves of embryonic endocrine differentiation. In addition, our studies demonstrate that Ngn3 transcripts are markedly more widespread in the pancreatic epithelium than NGN3 protein, indicating that post‐transcriptional regulation is likely to play a critical role during endocrine differentiation. Developmental Dynamics 237:3270–3279, 2008.

Blood vessels restrain pancreas branching, differentiation and growth

How organ size and form are controlled during development is a major question in biology. Blood vessels have been shown to be essential for early development of the liver and pancreas, and are fundamental to normal and pathological tissue growth. Here, we report that, surprisingly, non-nutritional signals from blood vessels act to restrain pancreas growth. Elimination of endothelial cells increases the size of embryonic pancreatic buds. Conversely, VEGF-induced hypervascularization decreases pancreas size. The growth phenotype results from vascular restriction of pancreatic tip cell formation, lateral branching and differentiation of the pancreatic epithelium into endocrine and acinar cells. The effects are seen both in vivo and ex vivo, indicating a perfusion-independent mechanism. Thus, the vasculature controls pancreas morphogenesis and growth by reducing branching and differentiation of primitive epithelial cells.

BMP and BMP receptor expression during murine organogenesis.

Cell-cell communication is critical for regulating embryonic organ growth and differentiation. The Bone Morphogenetic Protein (BMP) family of transforming growth factor beta (TGFbeta) molecules represents one class of such cell-cell signaling molecules that regulate the morphogenesis of several organs. Due to high redundancy between the myriad BMP ligands and receptors in certain tissues, it has been challenging to address the role of BMP signaling using targeting of single Bmp genes in mouse models. Here, we present a detailed study of the developmental expression profiles of three BMP ligands (Bmp2, Bmp4, Bmp7) and three BMP receptors (Bmpr1a, Bmpr1b, and BmprII), as well as their molecular antagonist (noggin), in the early embryo during the initial steps of murine organogenesis. In particular, we focus on the expression of Bmp family members in the first organs and tissues that take shape during embryogenesis, such as the heart, vascular system, lungs, liver, stomach, nervous system, somites and limbs. Using in situ hybridization, we identify domains where ligand(s) and receptor(s) are either singly or co-expressed in specific tissues. In addition, we identify a previously unnoticed asymmetric expression of Bmp4 in the gut mesogastrium, which initiates just prior to gut turning and the establishment of organ asymmetry in the gastrointestinal tract. Our studies will aid in the future design and/or interpretation of targeted deletion of individual Bmp or Bmpr genes, since this study identifies organs and tissues where redundant BMP signaling pathways are likely to occur.

HoxA3 is an apical regulator of haemogenic endothelium

During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial–haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells.

Autophagy is essential for cardiac morphogenesis during vertebrate development

Genetic analyses indicate that autophagy, an evolutionarily conserved lysosomal degradation pathway, is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryogenesis. To address this question, we examined the role of autophagy in the early development of zebrafish, a model organism for studying vertebrate tissue and organ morphogenesis. Using zebrafish that transgenically express the fluorescent autophagy reporter protein, GFP-LC3, we found that autophagy is active in multiple tissues, including the heart, during the embryonic period. Inhibition of autophagy by morpholino knockdown of essential autophagy genes (including atg5, atg7, and becn1) resulted in defects in morphogenesis, increased numbers of dead cells, abnormal heart structure, and reduced organismal survival. Further analyses of cardiac development in autophagy-deficient zebrafish revealed defects in cardiac looping, abnormal chamber morphology, aberrant valve development, and ectopic expression of critical transcription factors including foxn4, tbx5, and tbx2. Consistent with these results, Atg5-deficient mice displayed abnormal Tbx2 expression and defects in valve development and chamber septation. Thus, autophagy plays an essential, conserved role in cardiac morphogenesis during vertebrate development.