Hepatic maturation in differentiating embryonic stem cells in vitro

We investigated the potential of mouse embryonic stem (ES) cells to differentiate into hepatocytes in vitro. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, transthyretin, alpha 1-anti-trypsin and albumin, when cultured without additional growth factors and late differential markers of hepatic development, such as tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6P), when cultured in the presence of growth factors critical for late embryonic liver development. Further, induction of TAT and G6P expression was induced regardless of expression of the functional SEK1 gene, which is thought to provide a survival signal for hepatocytes during an early stage of liver morphogenesis. The data indicate that the in vitro ES differentiation system has a potential to generate mature hepatocytes. The system has also been found useful in analyzing the role of growth factors and intracellular signaling molecules in hepatic development.     

Understanding dioxin developmental toxicity using the zebrafish model

Zebrafish (Danio rerio) have advantages over mammals as an animal model for investigating developmental toxicity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin, TCDD), a persistent global contaminant, is the most comprehensively studied developmental toxicant in zebrafish. The hallmark responses of TCDD developmental toxicity manifested in zebrafish larvae include edema, anemia, hemorrhage, and ischemia associated with arrested growth and development. Heart and vasculature development and function are severely impaired, and jaw malformations occur secondary to inhibited chondrogenesis. The swim bladder fails to inflate, and the switch from embryonic to adult erythropoiesis is blocked. This profile of developmental toxicity responses, commonly referred to as "blue sac syndrome" because the edematous yolk sac appears blue, is observed in the larval form of all freshwater fish species exposed to TCDD at the embryonic stage of development. Components of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AHR/ARNT) signaling pathway in zebrafish have been identified and functionally characterized. Their role in mediating TCDD toxicity has been determined using morpholinos to specifically knockdown the translation of zfAHR1, zfAHR2, zfARNT1, and zfARNT2 mRNAs, respectively, and a line of zfARNT2 null mutant zebrafish has provided further insight. These studies have shown that zfAHR2 and zfARNT1 mediate TCDD developmental toxicity. In addition, the growing use of molecular and genomic tools for research on zebrafish have led to advances in our understanding of the mechanism of TCDD developmental toxicity at the molecular level, including the recent finding that toxicity is not mediated by increased cytochrome P4501A (zfCYP1A) expression.     

VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis

Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial-mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis.     

Expression and regulation by thyroid hormone (TH) of zebrafish IGF-I gene and amphioxus IGFl gene with implication of the origin of TH/IGF signaling pathway

Thyroid hormone (TH)/insulin-like growth factor (IGF) signaling pathway has been identified in all the vertebrates, but its evolutionary origin remains elusive. In this study we examined the expression profiles in vitro as well as in vivo of the IGF-I gene of fish Danio rerio (vertebrate) and the IGF-like gene (IGFl) of amphioxus Branchiostoma japonicum (protochordate) following T₃ treatment. Our results showed that T₃ was able to enhance hepatic IGF-I/IGFl gene expression in vitro in both zebrafish and amphioxus in a dose-dependent manner. This T₃-induced hepatic expression of IGF-I/IGFl genes in both species was significantly inhibited by the T₃-specific inhibitor DEA, indicating the specificity of IGF-I/IGFl gene regulation by T₃. At 100 nM T₃, in both the long (42 h) and short (8 h) time course experiments, the IGF-I/IGFl gene expression profiles following T₃ treatment in the tissue cultures of both species exhibited closely similar pattern and trend. Moreover, exposure of zebrafish and amphioxus to T₃in vivo for 72 h induced a significant increase in the expression of IGF-I/IGFl genes in both the liver and the hepatic caecum. These data together suggest that amphioxus and zebrafish both share a similar regulatory mechanism of IGF gene expression in response to T₃, providing an evidence for the presence of a vertebrate-like TH/IGF signaling pathway in the protochordate amphioxus.     

Focal adhesion kinase is essential for cardiac looping and multichamber heart formation

Focal adhesion kinase (FAK) is a critical mediator of matrix‐ and growth factor‐induced signaling during development. Myocyte‐restricted FAK deletion in mid‐gestation mice results in impaired ventricular septation and cardiac compaction. However, whether FAK regulates early cardiogenic steps remains unknown. To explore a role for FAK in multi‐chambered heart formation, we utilized anti‐sense morpholinos to deplete FAK in Xenopus laevis. Xenopus FAK morphants exhibited impaired cardiogenesis, pronounced pericardial edema, and lethality by tadpole stages. Spatial‐temporal assessment of cardiac marker gene expression revealed that FAK was not necessary for midline migration, differentiation, fusion of cardiac precursors, or linear heart tube formation. However, myocyte proliferation was significantly reduced in FAK morphant heart tubes and these tubes failed to undergo proper looping morphogenesis. Collectively our data imply that FAK plays an essential role in chamber outgrowth and looping morphogenesis likely stimulated by fibroblast growth factors (and possibly other) cardiotrophic factors. genesis 48:492–504, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular insights of Hippo signaling in the chick developing lung

Hippo signaling pathway and its effector YAP have been recognized as an essential growth regulator during embryonic development. Hippo has been studied in different contexts; nevertheless, its role during chick lung branching morphogenesis remains unknown. Therefore, this work aims to determine Hippo role during early pulmonary organogenesis in the avian animal model. The current study describes the spatial distribution of Hippo signaling members in the embryonic chick lung by in situ hybridization. Overall, their expression is comparable to their mammalian counterparts. Moreover, the expression levels of phosphorylated-YAP (pYAP) and total YAP revealed that Hippo signaling is active in the embryonic chick lung. Furthermore, the presence of pYAP in the cytoplasm demonstrated that the Hippo machinery distribution is maintained in this tissue. In vitro studies were performed to assess the role of the Hippo signaling pathway in lung branching. Lung explants treated with a YAP/TEAD complex inhibitor (verteporfin) displayed a significant reduction in lung size and branching and decreased expression of ctgf (Hippo target gene) compared to the control. This approach also revealed that Hippo seems to modulate the expression of key molecular players involved in lung branching morphogenesis (sox2, sox9, axin2, and gli1). Conversely, when treated with dobutamine, an upstream regulator that promotes YAP phosphorylation, explant morphology was not severely affected. Overall, our data indicate that Hippo machinery is present and active in the early stages of avian pulmonary branching and that YAP is likely involved in the regulation of lung growth.     

fgf17b, a novel member of Fgf family, helps patterning zebrafish embryos

Fibroblast growth factors (Fgfs) play important roles in the pattern formation of early vertebrate embryos. We have identified a zebrafish ortholog of human FGF17, named fgf17b. The first phase of fgf17b expression occurs in the blastodermal margin of late blastulae and in the embryonic shield of early gastrulae. The second phase starts after the onset of segmentation, mainly in the presomitic mesoderm and newly formed somites. Injection of fgf17b mRNA into one-cell embryos induces expression of the mesodermal marker no tail (ntl) and rescues ntl expression suppressed by overexpression of lefty1 (lft1). Overexpression of fgf17b dorsalizes zebrafish gastrulae by enhancing expression of chordin (chd), which is an antagonist of the ventralizing signals BMPs. In addition, overexpression of fgf17b posteriorizes the neuroectoderm. Simultaneous knockdown of fgf17b and fgf8 with antisense morpholinos results in reduction of chd and ntl. Knockdown of fgf17b can alleviate inhibitory effect of ectopic expression of fgf3 on otx1. These data together suggest that Fgf17b plays a role in early embryonic patterning. We also demonstrate that fgf17b and fgf8 have stronger mesoderm inducting activity than fgf3, whereas fgf17b and fgf3 have stronger activity in posteriorizing the neuroectoderm than fgf8. Like fgf8, activation of fgf17b expression depends on Nodal signaling.     

APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development

Developmental signaling pathways hold the keys to unlocking the promise of adult tissue regeneration, and to inhibiting carcinogenesis. Patients with mutations in the Adenomatous Polyposis Coli (APC) gene are at increased risk of developing hepatoblastoma, an embryonal form of liver cancer, suggesting that Wnt affects hepatic progenitor cells. To elucidate the role of APC loss and enhanced Wnt activity in liver development, we examined APC mutant and wnt inducible transgenic zebrafish. APC^+/− embryos developed enlarged livers through biased induction of hepatic gene programs and increased proliferation. Conversely, APC^−/− embryos formed no livers. Blastula transplantations determined that the effects of APC loss were cell autonomous. Induction of wnt modulators confirmed biphasic consequences of wnt activation: endodermal pattern formation and gene expression required suppression of wnt signaling in early somitogenesis; later, increased wnt activity altered endodermal fate by enhancing liver growth at the expense of pancreas formation; these effects persisted into the larval stage. In adult APC^+/− zebrafish, increased wnt activity significantly accelerated liver regeneration after partial hepatectomy. Similarly, liver regeneration was significantly enhanced in APC^Min/+ mice, indicating the conserved effect of Wnt pathway activation in liver regeneration across vertebrate species. These studies reveal an important and time-dependent role for wnt signaling during liver development and regeneration.     

Bmp and Fgf signaling are essential for liver specification in zebrafish

Based on data from in vitro tissue explant and ex vivo cell/bead implantation experiments, Bmp and Fgf signaling have been proposed to regulate hepatic specification. However, genetic evidence for this hypothesis has been lacking. Here, we provide in vivo genetic evidence that Bmp and Fgf signaling are essential for hepatic specification. We utilized transgenic zebrafish that overexpress dominant-negative forms of Bmp or Fgf receptors following heat-shock induction. These transgenes allow one to bypass the early embryonic requirements for Bmp and Fgf signaling, and also to completely block Bmp or Fgf signaling. We found that the expression of hhex and prox1, the earliest liver markers in zebrafish, was severely reduced in the liver region when Bmp or Fgf signaling was blocked just before hepatic specification. However, hhex and prox1 expression in adjacent endodermal and mesodermal tissues appeared unaffected by these manipulations. Additional genetic studies indicate that the endoderm maintains competence for Bmp-mediated hepatogenesis over an extended window of embryonic development. Altogether, these data provide the first genetic evidence that Bmp and Fgf signaling are essential for hepatic specification, and suggest that endodermal cells remain competent to differentiate into hepatocytes for longer than anticipated.     

New directions in craniofacial morphogenesis

The 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.     

A field of myocardial-endocardial NFAT signaling underlies heart valve morphogenesis

The delicate leaflets that make up vertebrate heart valves are essential for our moment-to-moment existence. Abnormalities of valve formation are the most common serious human congenital defect. Despite their importance, relatively little is known about valve development. We show that the initiation of heart valve morphogenesis in mice requires calcineurin/NFAT to repress VEGF expression in the myocardium underlying the site of prospective valve formation. This repression of VEGF at E9 is essential for endocardial cells to transform into mesenchymal cells. Later, at E11, a second wave of calcineurin/NFAT signaling is required in the endocardium, adjacent to the earlier myocardial site of NFAT action, to direct valvular elongation and refinement. Thus, NFAT signaling functions sequentially from myocardium to endocardium within a valvular morphogenetic field to initiate and perpetuate embryonic valve formation. This mechanism also operates in zebrafish, indicating a conserved role for calcineurin/NFAT signaling in vertebrate heart valve morphogenesis.     

Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish

The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.     

Insulin-like growth factor receptor 1b is required for zebrafish primordial germ cell migration and survival

Insulin-like growth factor (IGF) signaling is a critical regulator of somatic growth during fetal and adult development, primarily through its stimulatory effects on cell proliferation and survival. IGF signaling is also required for development of the reproductive system, although its precise role in this regard remains unclear. We have hypothesized that IGF signaling is required for embryonic germline development, which requires the specification and proliferation of primordial germ cells (PGCs) in an extragonadal location, followed by directed migration to the genital ridges. We tested this hypothesis using loss-of-function studies in the zebrafish embryo, which possesses two functional copies of the Type-1 IGF receptor gene (igf1ra, igf1rb). Knockdown of IGF1Rb by morpholino oligonucleotides (MO) results in mismigration and elimination of primordial germ cells (PGCs), resulting in fewer PGCs colonizing the genital ridges. In contrast, knockdown of IGF1Ra has no effect on PGC migration or number despite inducing widespread somatic cell apoptosis. Ablation of both receptors, using combined MO injections or overexpression of a dominant-negative IGF1R, yields embryos with a PGC-deficient phenotype similar to IGF1Rb knockdown. TUNEL analyses revealed that mismigrated PGCs in IGF1Rb-deficient embryos are eliminated by apoptosis; overexpression of an antiapoptotic gene (Bcl2l) rescues ectopic PGCs from apoptosis but fails to rescue migration defects. Lastly, we show that suppression of IGF signaling leads to quantitative changes in the expression of genes encoding CXCL-family chemokine ligands and receptors involved in PGC migration. Collectively, these data suggest a novel role for IGF signaling in early germline development, potentially via cross-talk with chemokine signaling pathways.     

The homeobox gene Hhex is essential for proper hepatoblast differentiation and bile duct morphogenesis

Hhex is required for early development of the liver. A null mutation of Hhex results in a failure to form the liver bud and embryonic lethality. Therefore, Hhex null mice are not informative as to whether this gene is required during later stages of hepatobiliary morphogenesis. To address this question, we derived Hhex conditional null mice using the Cre-loxP system and two different Cre transgenics (Foxa3-Cre and Alfp-Cre). Deletion of Hhex in the hepatic diverticulum (Foxa3-Cre;Hhex(d2,3/-)) led to embryonic lethality and resulted in a small and cystic liver with loss of Hnf4alpha and Hnf6 expression in early hepatoblasts. In addition, the gall bladder was absent and the extrahepatic bile duct could not be identified. Loss of Hhex in the embryonic liver (Alfp-Cre;Hhex(d2,3/-)) caused irregular development of intrahepatic bile ducts and an absence of Hnf1beta in many (cystic) biliary epithelial cells, which resulted in a slow, progressive form of polycystic liver disease in adult mice. Thus, we have shown that Hhex is required during multiple stages of hepatobiliary development. The altered expression of Hnf4alpha, Hnf6 and Hnf1beta in Hhex conditional null mice suggests that Hhex is an essential component of the genetic networks regulating hepatoblast differentiation and intrahepatic bile duct morphogenesis.