Lung endoderm morphogenesis: gasping for form and function.

The respiratory endoderm develops from a small cluster of cells located on the ventral anterior foregut. This population of progenitors generates the myriad epithelial lineages required for proper lung function in adults through a complex and delicately balanced series of developmental events controlled by many critical signaling and transcription factor pathways. In the past decade, understanding of this process has grown enormously, helped in part by cell lineage fate analysis and deep sequencing of the transcriptomes of various progenitors and differentiated cell types. This review explores how these new techniques, coupled with more traditional approaches, have provided a detailed picture of development of the epithelial lineages in the lung and insight into how aberrant development can lead to lung disease.

Capillarity and active cell movement at mesendoderm translocation in the Xenopus gastrula.

During Xenopus gastrulation, leading edge mesendoderm (LEM) advances animally as a wedge-shaped cell mass over the vegetally moving blastocoel roof (BCR). We show that close contact across the BCR-LEM interface correlates with attenuated net advance of the LEM, which is pulled forward by tip cells while the remaining LEM frequently separates from the BCR. Nevertheless, lamellipodia persist on the detached LEM surface. They attach to adjacent LEM cells and depend on PDGF-A, cell-surface fibronectin and cadherin. We argue that active cell motility on the LEM surface prevents adverse capillary effects in the liquid LEM tissue as it moves by being pulled. It counters tissue surface-tension effects with oriented cell movement and bulges the LEM surface out to keep it close to the curved BCR without attaching to it. Proximity to the BCR is necessary, in turn, for the maintenance and orientation of lamellipodia that permit mass cell movement with minimal substratum contact. Together with a similar process in epithelial invagination, vertical telescoping, the cell movement at the LEM surface defines a novel type of cell rearrangement: vertical shearing.

pmar1/phb homeobox genes and the evolution of the double-negative gate for endomesoderm specification in echinoderms.

In several model animals, the earliest phases of embryogenesis are regulated by lineage-specific genes, such as Drosophila bicoid Sea urchin (echinoid) embryogenesis is initiated by zygotic expression of pmar1, a paired-class homeobox gene that has been considered to be present only in the lineage of modern urchins (euechinoids). In euechinoids, Pmar1 promotes endomesoderm specification by repressing the hairy and enhancer of split C (hesC) gene. Here, we have identified the basal echinoid (cidaroid) pmar1 gene, which also promotes endomesoderm specification but not by repressing hesC A further search for related genes demonstrated that other echinoderms have pmar1-related genes named phb Functional analyses of starfish Phb proteins indicated that, similar to cidaroid Pmar1, they promote activation of endomesoderm regulatory gene orthologs via an unknown repressor that is not HesC. Based on these results, we propose that Pmar1 may have recapitulated the regulatory function of Phb during the early diversification of echinoids and that the additional repressor HesC was placed under the control of Pmar1 in the euechinoid lineage. This case provides an exceptional model for understanding how early developmental processes diverge.

Vertebrate endoderm development and organ formation.

The endoderm germ layer contributes to the respiratory and gastrointestinal tracts and to all of their associated organs. Over the past decade, studies in vertebrate model organisms, including frog, fish, chick, and mouse, have greatly enhanced our understanding of the molecular basis of endoderm organ development. We review this progress with a focus on early stages of endoderm organogenesis including endoderm formation, gut tube morphogenesis and patterning, and organ specification. Lastly, we discuss how developmental mechanisms that regulate endoderm organogenesis are used to direct differentiation of embryonic stem cells into specific adult cell types, which function to alleviate disease symptoms in animal models.

Trim-Away mediated knock down uncovers a new function for Lbh during gastrulation of Xenopus laevis.

We previously identified the protein Lbh as necessary for cranial neural crest (CNC) cell migration in Xenopus through the use of morpholinos. However, Lbh is a maternally deposited protein and morpholinos achieve knockdowns through prevention of translation. In order to investigate the role of Lbh in earlier embryonic events, we employed the new technique "Trim-Away" to degrade this maternally deposited protein. Trim-Away utilizes the E3 ubiquitin ligase trim21 to degrade proteins targeted with an antibody and was developed in mammalian systems. Our results show that Xenopus is amenable to the Trim-Away technique. We also show that early knockdown of Lbh in Xenopus results in defects in gastrulation that present with a decrease in fibronectin matrix assembly, an increased in mesodermal cell migration and decrease in endodermal cell cohesion. We further show that the technique is also effective on a second abundant maternal protein PACSIN2. We discuss potential advantages and limit of the technique in Xenopus embryos as well as the mechanism of gastrulation inhibition.

The endoderm: a divergent cell lineage with many commonalities.

The endoderm is a progenitor tissue that, in humans, gives rise to the majority of internal organs. Over the past few decades, genetic studies have identified many of the upstream signals specifying endoderm identity in different model systems, revealing them to be divergent from invertebrates to vertebrates. However, more recent studies of the cell behaviours driving endodermal morphogenesis have revealed a surprising number of shared features, including cells undergoing epithelial-to-mesenchymal transitions (EMTs), collective cell migration, and mesenchymal-to-epithelial transitions (METs). In this Review, we highlight how cross-organismal studies of endoderm morphogenesis provide a useful perspective that can move our understanding of this fascinating tissue forward.

Type I interferon response impairs differentiation potential of pluripotent stem cells.

Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I-stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system.

Pharyngeal epithelial deletion of Tbx1 causes caudal pharyngeal arch defect but not cardiac conotruncal anomaly.

TBX1 is a major disease gene of 22q11.2 deletion syndrome (22q11.2DS). It is expressed in all three germ layers of pharyngeal apparatus to control the complicated morphogenesis. The haploinsufficiency of pharyngeal endodermal or ectodermal, but not mesodermal Tbx1 causes aortic arch patterning defect. However, the mesodermal deletion of Tbx1 causes much severer pharyngeal and cardiovascular defect than either pharyngeal endodermal or ectodermal Tbx1 deletion does. It is inconsistent with the conventional thought that the invagination of pharyngeal epithelia drives pharyngeal segmentation. Therefore, we asked whether pharyngeal ectodermal and ectodermal Tbx1 can compensate the loss of each other. Here we carefully characterized pharyngeal epithelia-specific Fgf15Cre and Fgf15HspCre lines and used them to perform pharyngeal epithelia-specific deletion. Our data showed that the percentage of E18.5 Fgf15Cre;Tbx1flox/+ embryos with aortic arch patterning defects was similar to that of E10.5 Fgf15Cre;Tbx1flox/+ embryos with the 4th pharyngeal arch artery (PAA) defect, indicating that there is no significant recovery from the initial PAA defect, in contrast to germ line haploinsufficiency. Fgf15Cre;Tbx1flox/flox embryos had hypoplastic caudal pharyngeal arch and defective derivatives, but cardiac OFT development was not affected. The phenotypic spectrum of simultaneous Tbx1 deletion in both pharyngeal ectoderm and endoderm is strikingly similar to what presents with single pharyngeal endoderm or ectoderm-specific deletion of Tbx1. The absence of synergistic effect indicates intimate topographic interactions among pharyngeal endoderm and ectoderm, through which deletion of a gene in one tissue may disrupt the development of adjacent tissues and thereby lead to similar morphological phenotypes in either tissue-specific deletion.

Mechanical and signaling roles for keratin intermediate filaments in the assembly and morphogenesis of Xenopus mesendoderm tissue at gastrulation.

The coordination of individual cell behaviors is a crucial step in the assembly and morphogenesis of tissues. Xenopus mesendoderm cells migrate collectively along a fibronectin (FN) substrate at gastrulation, but how the adhesive and mechanical forces required for these movements are generated and transmitted is unclear. Traction force microscopy (TFM) was used to establish that traction stresses are limited primarily to leading edge cells in mesendoderm explants, and that these forces are balanced by intercellular stresses in follower rows. This is further reflected in the morphology of these cells, with broad lamellipodial protrusions, mature focal adhesions and a gradient of activated Rac1 evident at the leading edge, while small protrusions, rapid turnover of immature focal adhesions and lack of a Rac1 activity gradient characterize cells in following rows. Depletion of keratin (krt8) with antisense morpholinos results in high traction stresses in follower row cells, misdirected protrusions and the formation of actin stress fibers anchored in streak-like focal adhesions. We propose that maintenance of mechanical integrity in the mesendoderm by keratin intermediate filaments is required to balance stresses within the tissue to regulate collective cell movements.

GATA4 and GATA6 regulate pancreatic endoderm identity through inhibition of hedgehog signaling.

GATA4 and GATA6 are zinc finger transcription factors that have important functions in several mesodermal and endodermal organs, including heart, liver and pancreas. In humans, heterozygous mutations of either factor are associated with pancreatic agenesis; however, homozygous deletion of both Gata4 and Gata6 is necessary to disrupt pancreas development in mice. In this study, we demonstrate that arrested pancreatic development in Gata4(fl/fl); Gata6(fl/fl); Pdx1:Cre (pDKO) embryos is accompanied by the transition of ventral and dorsal pancreatic fates into intestinal or stomach lineages, respectively. These results indicate that GATA4 and GATA6 play essential roles in maintaining pancreas identity by regulating foregut endodermal fates. Remarkably, pancreatic anlagen derived from pDKO embryos also display a dramatic upregulation of hedgehog pathway components, which are normally absent from the presumptive pancreatic endoderm. Consistent with the erroneous activation of hedgehog signaling, we demonstrate that GATA4 and GATA6 are able to repress transcription through the sonic hedgehog (Shh) endoderm-specific enhancer MACS1 and that GATA-binding sites within this enhancer are necessary for this repressive activity. These studies establish the importance of GATA4/6-mediated inhibition of hedgehog signaling as a major mechanism regulating pancreatic endoderm specification during patterning of the gut tube.

Axis formation in hydra.

A hydra has a simple structure consisting of a head, body column, and foot along a single axis called the oral-aboral axis. The tissue dynamics of a hydra consist of a steady state of production and loss of tissue involving the entire animal. Axis formation and its maintenance is controlled by the head organizer, which is located at the apex of the animal. The head organizer produces two signals, the head activator and head inhibitor, which are transmitted to, and are distributed in, descending gradients among the epithelial cells along the body column. The two gradients control axial patterning along the oral-aboral axis. In the context of the tissue dynamics of the adult hydra, these three elements controlling axis formation and axial patterning are in a steady state of production and loss. The canonical Wnt pathway plays a major role in setting up and maintaining the head organizer.

Transcription factors Mix1 and VegT, relocalization of vegt mRNA, and conserved endoderm and dorsal specification in frogs.

Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi, Epipedobates machalilla, Gastrotheca riobambae, and Eleutherodactylus coqui, as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt (zvegt) mRNA was detected as a minor isoform besides the major maternal (mvegt) isoform of the X. laevis egg. In addition, α-amanitin-insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1, have been evolutionary conserved in frogs.

Functional and phenotypic distinction of the first two trophoblast subdivisions and identification of the border between them during early postimplantation: A prerequisite for understanding early patterning during placentogenesis.

The early stages of mouse placentogenesis (placenta formation) involve poorly understood patterning events within polar trophectoderm-derived trophoblast, the progenitor of all placental trophoblast cell types. By early postimplantation [embryonic day 5.5 (E5.5)], this patterning causes early trophoblast to become subdivided into extraembryonic ectoderm (ExE) and ectoplacental cone (EPC). A prerequisite to understanding this patterning requires knowing the location of ExE-EPC border and being able to distinguish the entire ExE from EPC at E5.5/E6.5, a time when the proamnioitic cavity within ExE is not fully established. However, these issues are unknown, as they have not been directly addressed. Here, we directly addressed these using trophoblast explant culture to functionally test for the location of ExE-EPC border, combined with phenotypic characterization of trophoblast proximal and distal to it. We show for the first time that the proximal-distal level of ExE-EPC border within E5.5/E6.5 trophoblast coincides with where Reichert's membrane (outermost basement membrane of conceptus) inserts into early trophoblast and with the proximal limit of extraembryonic visceral endoderm (primitive endoderm derivative covering part of early trophoblast). Based on these novel findings, we discovered that (a) the entire E5.5/E6.5 ExE can be distinguished from EPC because it is epithelial and specifically expresses Erf and Claudin4 and (b) at E5.5/E6.5, the entire EPC differs from ExE in that it is not epithelial and specifically expresses Snail. This work is expected to contribute to understanding the cellular and molecular basis of early trophoblast patterning during placentogenesis.

Sox17 is essential for proper formation of the marginal zone of extraembryonic endoderm adjacent to a developing mouse placental disk.

In mouse conceptus, two yolk-sac membranes, the parietal endoderm (PE) and visceral endoderm (VE), are involved in protecting and nourishing early-somite-stage embryos prior to the establishment of placental circulation. Both PE and VE membranes are tightly anchored to the marginal edge of the developing placental disk, in which the extraembryonic endoderm (marginal zone endoderm: ME) shows the typical flat epithelial morphology intermediate between those of PE and VE in vivo. However, the molecular characteristics and functions of the ME in mouse placentation remain unclear. Here, we show that SOX17, not SOX7, is continuously expressed in the ME cells, whereas both SOX17 and SOX7 are coexpressed in PE cells, by at least 10.5 days postconception. The Sox17-null conceptus, but not the Sox7-null one, showed the ectopic appearance of squamous VE-like epithelial cells in the presumptive ME region, together with reduced cell density and aberrant morphology of PE cells. Such aberrant ME formation in the Sox17-null extraembryonic endoderm was not rescued by the chimeric embryo replaced with the wild-type gut endoderm by the injection of wild-type ES cells into the Sox17-null blastocyst, suggesting the cell autonomous defects in the extraembryonic endoderm of Sox17-null concepti. These findings provide direct evidence of the crucial roles of SOX17 in proper formation and maintenance of the ME region, highlighting a novel entry point to understand the in vivo VE-to-PE transition in the marginal edge of developing placenta.

Tissue growth pressure drives early blood flow in the chicken yolk sac.

BACKGROUND: Understanding how molecular and physical cues orchestrate vascular morphogenesis is a challenge for developmental biology. Only little attention has been paid to the impact of mechanical stress caused by tissue growth on early blood distribution. Here we study the peripheral accumulation of blood in the chicken embryonic yolk sac, which precedes sinus vein formation. RESULTS: We report that blood accumulation starts before heart-induced blood circulation. We hypothesized that the driving force for the primitive blood flow is a growth-induced gradient of tissue pressure in the yolk sac mesoderm. Therefore, we studied embryos in which heart development was arrested after 2 days of incubation, and found that yolk sac growth and blood peripheral accumulation still occurred. This suggests that tissue growth is sufficient to initiate the flow and the formation of the sinus vein, whereas heart contractions are not required. We designed a simple mathematical model which makes explicit the growth-induced pressure gradient and the subsequent blood accumulation, and show that growth can indeed account for the observed blood accumulation. CONCLUSIONS: This study shows that tissue growth pressure can drive early blood flow, and suggests that the mechanical environment, beyond hemodynamics, can contribute to vascular morphogenesis. Developmental Dynamics 246:573-584, 2017. © 2017 Wiley Periodicals, Inc.

Erythropoietin facilitates definitive endodermal differentiation of mouse embryonic stem cells via activation of ERK signaling.

Artificially generated pancreatic ß-cells from pluripotent stem cells are expected for cell replacement therapy for type 1 diabetes. Several strategies are adopted to direct pluripotent stem cells toward pancreatic differentiation. However, a standard differentiation method for clinical application has not been established. It is important to develop more effective and safer methods for generating pancreatic ß-cells without toxic or mutagenic chemicals. In the present study, we screened several endogenous factors involved in organ development to identify the factor, which induced the efficiency of pancreatic differentiation and found that treatment with erythropoietin (EPO) facilitated the differentiation of mouse embryonic stem cells (ESCs) into definitive endoderm. At an early stage of differentiation, EPO treatment significantly increased Sox17 gene expression, as a marker of the definitive endoderm. Contrary to the canonical function of EPO, it did not affect the levels of phosphorylated JAK2 and STAT5, but stimulated the phosphorylation of ERK1/2 and Akt. The MEK inhibitor U0126 significantly inhibited EPO-induced Sox17 expression. The differentiation of ESCs into definitive endoderm is an important step for the differentiation into pancreatic and other endodermal lineages. This study suggests a possible role of EPO in embryonic endodermal development and a new agent for directing the differentiation into endodermal lineages like pancreatic ß-cells.

A regulatory network controls nephrocan expression and midgut patterning.

Although many regulatory networks involved in defining definitive endoderm have been identified, the mechanisms through which these networks interact to pattern the endoderm are less well understood. To explore the mechanisms involved in midgut patterning, we dissected the transcriptional regulatory elements of nephrocan (Nepn), the earliest known midgut specific gene in mice. We observed that Nepn expression is dramatically reduced in Sox17(-/-) and Raldh2(-/-) embryos compared with wild-type embryos. We further show that Nepn is directly regulated by Sox17 and the retinoic acid (RA) receptor via two enhancer elements located upstream of the gene. Moreover, Nepn expression is modulated by Activin signaling, with high levels inhibiting and low levels enhancing RA-dependent expression. In Foxh1(-/-) embryos in which Nodal signaling is reduced, the Nepn expression domain is expanded into the anterior gut region, confirming that Nodal signaling can modulate its expression in vivo. Together, Sox17 is required for Nepn expression in the definitive endoderm, while RA signaling restricts expression to the midgut region. A balance of Nodal/Activin signaling regulates the anterior boundary of the midgut expression domain.

Conserved and divergent expression patterns of markers of axial development in eutherian mammals.

BACKGROUND: Mouse embryos are cup shaped, but most nonrodent eutherian embryos are disk shaped. Extraembryonic ectoderm (ExEc), which may have essential roles in anterior-posterior (A-P) axis formation in mouse embryos, does not develop in many eutherian embryos. To assess A-P axis formation in eutherians, comparative analyses were made on rabbit, porcine, and Suncus embryos. RESULTS: All embryos examined expressed Nodal initially throughout epiblast and visceral endoderm; its expression became restricted to the posterior region before gastrulation. Anterior visceral endoderm (AVE) genes were expressed in Otx2-positive visceral endoderm, with Dkk1 expression being most anterior. The mouse pattern of AVE formation was conserved in rabbit embryos, but had diverged in porcine and Suncus embryos. No structure that was molecularly equivalent to Bmp-positive ExEc, existed in rabbit or pig embryos. In Suncus embryos, A-P axis was determined at prehatching stage, and these embryos attached to uterine wall at future posterior side. CONCLUSIONS: Nodal, but not Bmp, functions in epiblast and visceral endoderm development may be conserved in eutherians. AVE functions may also be conserved, but the pattern of its formation has diverged among eutherians. Roles of BMP and NODAL gradients in AVE formation seem to have been established in a subset of rodents.

Nodal signaling from the visceral endoderm is required to maintain Nodal gene expression in the epiblast and drive DVE/AVE migration.

In the early mouse embryo, a specialized population of extraembryonic visceral endoderm (VE) cells called the distal VE (DVE) arises at the tip of the egg cylinder stage embryo and then asymmetrically migrates to the prospective anterior, recruiting additional distal cells. Upon migration these cells, called the anterior VE (AVE), establish the anterior posterior (AP) axis by restricting gastrulation-inducing signals to the opposite pole. The Nodal-signaling pathway has been shown to have a critical role in the generation and migration of the DVE/AVE. The Nodal gene is expressed in both the VE and in the pluripotent epiblast, which gives rise to the germ layers. Previous findings have provided conflicting evidence as to the relative importance of Nodal signaling from the epiblast vs. VE for AP patterning. Here we show that conditional mutagenesis of the Nodal gene specifically within the VE leads to reduced Nodal expression levels in the epiblast and incomplete or failed DVE/AVE migration. These results support a required role for VE Nodal to maintain normal levels of expression in the epiblast, and suggest signaling from both VE and epiblast is important for DVE/AVE migration.

AVE protein expression and visceral endoderm cell behavior during anterior-posterior axis formation in mouse embryos: Asymmetry in OTX2 and DKK1 expression.

The initial landmark of anterior-posterior (A-P) axis formation in mouse embryos is the distal visceral endoderm, DVE, which expresses a series of anterior genes at embryonic day 5.5 (E5.5). Subsequently, DVE cells move to the future anterior region, generating anterior visceral endoderm (AVE). Questions remain regarding how the DVE is formed and how the direction of the movement is determined. This study compares the detailed expression patterns of OTX2, HHEX, CER1, LEFTY1 and DKK1 by immunohistology and live imaging at E4.5-E6.5. At E6.5, the AVE is subdivided into four domains: most anterior (OTX2, HHEX, CER1-low/DKK1-high), anterior (OTX2, HHEX, CER1-high/DKK1-low), main (OTX2, HHEX, CER1, LEFTY1-high) and antero-lateral and posterior (OTX2, HHEX-low). The study demonstrates how this pattern is established. AVE protein expression in the DVE occurs de novo at E5.25-E5.5. Neither HHEX, LEFTY1 nor CER1 expression is asymmetric. In contrast, OTX2 expression is tilted on the future posterior side with the DKK1 expression at its proximal domain; the DVE cells move in the opposite direction of the tilt.