Corrigendum: Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

This corrects the article DOI: 10.1038/nature18614.

Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.

Hhex regulates the specification and growth of the hepatopancreatic ductal system.

Significant efforts have advanced our understanding of foregut-derived organ development; however, little is known about the molecular mechanisms that underlie the formation of the hepatopancreatic ductal (HPD) system. Here, we report a role for the homeodomain transcription factor Hhex in directing HPD progenitor specification in zebrafish. Loss of Hhex function results in impaired HPD system formation. We found that Hhex specifies a distinct population of HPD progenitors that gives rise to the cystic duct, common bile duct, and extra-pancreatic duct. Since hhex is not uniquely expressed in the HPD region but is also expressed in endothelial cells and the yolk syncytial layer (YSL), we tested the role of blood vessels as well as the YSL in HPD formation. We found that blood vessels are required for HPD patterning, but not for HPD progenitor specification. In addition, we found that Hhex is required in both the endoderm and the YSL for HPD development. Our results shed light on the mechanisms directing endodermal progenitors towards the HPD fate and emphasize the tissue specific requirement of Hhex during development.

On the development of the hepatopancreatic ductal system.

The hepatopancreatic ductal system is the collection of ducts that connect the liver and pancreas to the digestive tract. The formation of this system is necessary for the transport of exocrine secretions, for the correct assembly of the pancreatobiliary ductal system, and for the overall function of the digestive system. Studies on endoderm organ formation have significantly advanced our understanding of the molecular mechanisms that govern organ induction, organ specification and morphogenesis of the major foregut-derived organs. However, little is known about the mechanisms that control the development of the hepatopancreatic ductal system. Here, we provide a description of the different components of the system, summarize its development from the endoderm to a complex system of tubes, list the pathologies produced by anomalies in its development, as well as the molecules and signaling pathways that are known to be involved in its formation. Finally, we discuss its proposed potential as a multipotent cell reservoir and the unresolved questions in the field.

Vascular development in the vertebrate pancreas.

The vertebrate pancreas is comprised of a highly branched tubular epithelium, which is intimately associated with an extensive and specialized vasculature. While we know a great deal about basic vascular anatomy of the adult pancreas, as well as islet capillaries, surprisingly little is known about the ontogeny of its blood vessels. Here, we analyze development of the pancreatic vasculature in the mouse embryo. We show that pancreatic epithelial branches intercalate with the fine capillary plexus of the surrounding pancreatic mesenchyme. Endothelial cells (ECs) within this mesenchyme are heterogeneous from the onset of organogenesis. Pancreatic arteries take shape before veins, in a manner analogous to early embryonic vessels. The main central artery forms during mid-gestation, as a result of vessel coalescence and remodeling of a vascular plexus. In addition, we show that vessels in the forming pancreas display a predictable architecture that is dependent on VEGF signaling. Over-expression of VEGF disrupts vascular patterning and arteriovenous differentiation within the developing pancreas. This study constitutes a first-time in-depth cellular and molecular characterization of pancreatic blood vessels, as they coordinately grow along with the pancreatic epithelium.

Wnt4 is essential to normal mammalian lung development.

Wnt signaling is essential to many events during organogenesis, including the development of the mammalian lung. The Wnt family member Wnt4 has been shown to be required for the development of kidney, gonads, thymus, mammary and pituitary glands. Here, we show that Wnt4 is critical for proper morphogenesis and growth of the respiratory system. Using in situ hybridization in mouse embryos, we identify a previously uncharacterized site of Wnt4 expression in the anterior trunk mesoderm. This expression domain initiates as early as E8.25 in the mesoderm abutting the tracheoesophageal endoderm, between the fusing dorsal aortae and the heart. Analysis of Wnt4(-/-) embryos reveals severe lung hypoplasia and tracheal abnormalities; however, aortic fusion and esophageal development are unaffected. We find decreased cell proliferation in Wnt4(-/-) lung buds, particularly in tip domains. In addition, we observe reduction of the important lung growth factors Fgf9, Fgf10, Sox9 and Wnt2 in the lung bud during early stages of organogenesis, as well as decreased tracheal expression of the progenitor factor Sox9. Together, these data reveal a previously unknown role for the secreted protein Wnt4 in respiratory system development.

Crosstalk between the developing pancreas and its blood vessels: an evolving dialog.

Growth and development of embryonic organs goes hand in hand with development of the vascular system. Blood vessels have been known for centuries to supply nutrients and oxygen to all cell types in an organism, however, they have more recently been shown to provide specific cues required for the formation and functionality of a number of tissues. Here, we review the role of blood vessels during pancreas formation, from early specification of the initial pancreatic bud, to its growth and maturation. The overarching theme that emerges from the many studies carried out in the past decade is that the vasculature likely plays diverse and changing roles during pancreas organogenesis. Blood vessels are required for endocrine specification at the onset of pancreatic budding, while only a few days later, blood vessels suppress pancreatic branching and exocrine differentiation. In this review, we summarize our understanding to date about the crosstalk between the pancreas and its vasculature, and we provide a perspective on the promises and challenges of the field.

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.

EphB3 marks delaminating endocrine progenitor cells in the developing pancreas.

BACKGROUND: Understanding the process by which pancreatic beta-cells acquire their "fate" is critical to the development of in vitro directed differentiation protocols for cell replacement therapies for diabetics. To date, these efforts are hampered by a paucity of markers that distinguish pancreatic endocrine cells at different stages of differentiation. RESULTS: Here, we identify EphB3 as a novel pro-endocrine marker and use its expression to track delaminating islet lineages. First, we provide a detailed developmental expression profile for EphB3 and other EphB family members in the embryonic pancreas. We demonstrate that EphB3 transiently marks endocrine cells as they delaminate from the pancreatic epithelium, prior to their differentiation. Using a Tet-inducible EphB3(rtTA-lacZ) reporter line, we show that short-term pulse-labeled EphB3(+) cells co-express Pdx1, Nkx6.1, Ngn3, and Synaptophysin, but not insulin, glucagon, or other endocrine hormones. Prolonged labeling tracks EphB3(+) cells from their exit from the epithelium to their differentiation. CONCLUSIONS: These studies demonstrate that pro-endocrine cell differentiation during late gestation, from delamination to maturation, takes approximately 2 days. Together, these data introduce EphB3 as a new biomarker to identify beta-cells at a critical step during their step-wise differentiation and define the timeframe of endocrine differentiation.

Insulin-producing ß-cells regenerate ectopically from a mesodermal origin under the perturbation of hemato-endothelial specification.

To investigate the role of the vasculature in pancreatic ß-cell regeneration, we crossed a zebrafish ß-cell ablation model into the avascular npas4l mutant (i.e. cloche). Surprisingly, ß-cell regeneration increased markedly in npas4l mutants owing to the ectopic differentiation of ß-cells in the mesenchyme, a phenotype not previously reported in any models. The ectopic ß-cells expressed endocrine markers of pancreatic ß-cells, and also responded to glucose with increased calcium influx. Through lineage tracing, we determined that the vast majority of these ectopic ß-cells has a mesodermal origin. Notably, ectopic ß-cells were found in npas4l mutants as well as following knockdown of the endothelial/myeloid determinant Etsrp. Together, these data indicate that under the perturbation of endothelial/myeloid specification, mesodermal cells possess a remarkable plasticity enabling them to form ß-cells, which are normally endodermal in origin. Understanding the restriction of this differentiation plasticity will help exploit an alternative source for ß-cell regeneration.

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.

Membrane association and remodeling by intraflagellar transport protein IFT172.

The cilium is an organelle used for motility and cellular signaling. Intraflagellar transport (IFT) is a process to move ciliary building blocks and signaling components into the cilium. How IFT controls the movement of ciliary components is currently poorly understood. IFT172 is the largest IFT subunit essential for ciliogenesis. Due to its large size, the characterization of IFT172 has been challenging. Using giant unilamellar vesicles (GUVs), we show that IFT172 is a membrane-interacting protein with the ability to remodel large membranes into small vesicles. Purified IFT172 has an architecture of two globular domains with a long rod-like protrusion, resembling the domain organization of coatomer proteins such as COPI-II or clathrin. IFT172 adopts two different conformations that can be manipulated by lipids or detergents: 1) an extended elongated conformation and 2) a globular closed architecture. Interestingly, the association of IFT172 with membranes is mutually exclusive with IFT57, implicating multiple functions for IFT172 within IFT.

HHEX is a transcriptional regulator of the VEGFC/FLT4/PROX1 signaling axis during vascular development.

Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.

Lipid metabolism in mucous-dwelling amitochondriate protozoa.

Entamoeba, Giardia, and trichomonads are the prominent members of a group known as 'mucosal parasites'. While Entamoeba and Giardia trophozoites colonise the small intestine, trichomonads inhabit the genitourinary tracts of humans and animals. These protozoa lack mitochondria, well-developed Golgi complexes, and other organelles typical of higher eukaryotes. Nonetheless, they have developed unique metabolic pathways that allow them to survive and multiply in the small intestine and reproductive tracts by scavenging nutrients from the host. Various investigators have shown that these protozoa are unable to synthesise the majority of their own lipids and cholesterol de novo; rather, they depend mostly on supplies from outside sources. Therefore, questions of how they transport and utilise exogenous lipids for metabolic purposes are extremely important. There is evidence suggesting that these parasites can take up the lipids and cholesterol they need from lipoprotein particles present in the host and/or in the growth medium. Studies also support the idea that individual lipid and fatty acid molecules can be transported without the help of lipoproteins. Exogenous phospholipids have been shown to undergo fatty acid remodelling (by deacylation/reacylation reactions), which allows these protozoa to alter lipids, bypassing the synthesis of entirely new phospholipid molecules. In addition, many of these amitochondriates are, however, capable of elongating/desaturating long-chain fatty acids, and assembling novel glycophospholipid molecules. In this review, progress in various aspects of lipid research on these organisms is discussed. Attempts are also made to identify steps of lipid metabolic pathways that can be used to develop chemotherapeutic agents against these and other mucosal parasites.

Rgs16 and Rgs8 in embryonic endocrine pancreas and mouse models of diabetes.

Diabetes is characterized by the loss, or gradual dysfunction, of insulin-producing pancreatic beta-cells. Although beta-cells can replicate in younger adults, the available diabetes therapies do not specifically target beta-cell regeneration. Novel approaches are needed to discover new therapeutics and to understand the contributions of endocrine progenitors and beta-cell regeneration during islet expansion. Here, we show that the regulators of G protein signaling Rgs16 and Rgs8 are expressed in pancreatic progenitor and endocrine cells during development, then extinguished in adults, but reactivated in models of both type 1 and type 2 diabetes. Exendin-4, a glucagon-like peptide 1 (Glp-1)/incretin mimetic that stimulates beta-cell expansion, insulin secretion and normalization of blood glucose levels in diabetics, also promoted re-expression of Rgs16::GFP within a few days in pancreatic ductal-associated cells and islet beta-cells. These findings show that Rgs16::GFP and Rgs8::GFP are novel and early reporters of G protein-coupled receptor (GPCR)-stimulated beta-cell expansion after therapeutic treatment and in diabetes models. Rgs16 and Rgs8 are likely to control aspects of islet progenitor cell activation, differentiation and beta-cell expansion in embryos and metabolically stressed adults.

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.