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1.  Hepatic stellate cells in liver development, regeneration, and cancer 
The Journal of Clinical Investigation  2013;123(5):1902-1910.
Hepatic stellate cells are liver-specific mesenchymal cells that play vital roles in liver physiology and fibrogenesis. They are located in the space of Disse and maintain close interactions with sinusoidal endothelial cells and hepatic epithelial cells. It is becoming increasingly clear that hepatic stellate cells have a profound impact on the differentiation, proliferation, and morphogenesis of other hepatic cell types during liver development and regeneration. In this Review, we summarize and evaluate the recent advances in our understanding of the formation and characteristics of hepatic stellate cells, as well as their function in liver development, regeneration, and cancer. We also discuss how improved knowledge of these processes offers new perspectives for the treatment of patients with liver diseases.
PMCID: PMC3635734  PMID: 23635788
2.  The perivascular niche regulates breast tumor dormancy 
Nature cell biology  2013;15(7):10.1038/ncb2767.
In a significant fraction of breast cancer patients, distant metastases emerge after years or even decades of latency. How disseminated tumor cells (DTCs) are kept dormant, and what ‘wakes them up’, are fundamental problems in tumor biology. To address these questions, we utilized metastasis assays in mice to show that dormant DTCs reside upon microvasculature of lung, bone marrow and brain. We then engineered organotypic microvascular niches to determine whether endothelial cells directly influence breast cancer cell (BCC) growth. These models demonstrated that endothelial-derived thrombospondin-1 induces sustained BCC quiescence. This suppressive cue was lost in sprouting neovasculature; time-lapse analysis showed that sprouting vessels not only permit, but accelerate BCC outgrowth. We confirmed this surprising result in dormancy models and in zebrafish, and identified active TGF-β1 and periostin as tumor-promoting, endothelial tip cell-derived factors. Our work reveals that stable microvasculature constitutes a ‘dormant niche,’ whereas sprouting neovasculature sparks micrometastatic outgrowth.
PMCID: PMC3826912  PMID: 23728425
3.  miR-10 Regulates the Angiogenic Behavior of Zebrafish and Human Endothelial Cells by Promoting VEGF Signaling 
Circulation research  2012;111(11):1421-1433.
Formation and remodeling of the vasculature during development and disease involves a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their post-transcriptional dose-titration by miRNAs is poorly understood.
To identify miRNAs that regulate angiogenesis.
Methods and Results
We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating pro-angiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth (ISV) in the trunk of zebrafish larvae, while overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells (HUVECs). We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters VEGF, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in HUVECs inhibited the angiogenic behavior of endothelial cells largely by antagonizing VEGF receptor-2 signaling.
Our study provides insights into how FLT1 and VEGF receptor-2 signaling is titrated in a miRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis.
PMCID: PMC3525481  PMID: 22955733
microRNA; angiogenesis; developmental biology; VEGF; fms-related tyrosine kinase 1
4.  The bHLH Transcription Factor Hand2 Marks Hepatic Stellate Cells in Zebrafish: Analysis of Stellate Cell Entry into the Developing Liver 
Hepatology (Baltimore, Md.)  2012;56(5):1958-1970.
Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells that play vital roles in liver development and injury. Our knowledge of HSC biology is limited by the paucity of in vivo data. HSCs and sinusoidal endothelial cells (SECs) reside in close proximity and interactions between these two cell types are potentially critical for their development and function. Here we introduce a transgenic zebrafish line, Tg(hand2:EGFP), that labels HSCs. We find that zebrafish HSCs share many similarities with their mammalian counterparts, including morphology, location, lipid storage, gene expression profile, and increased proliferation and matrix production in response to an acute hepatic insult. Using the Tg(hand2:EGFP) line, we conducted time course analyses during development to reveal that HSCs invade the liver after SECs do. However, HSCs still enter the liver in mutants that lack most endothelial cells including SECs, indicating that SECs are not required for HSC differentiation or their entry into the liver. In the absence of SECs, HSCs become abnormally associated with hepatic biliary cells, suggesting that SECs influence HSC localization during liver development. We analyzed factors that regulate HSC development and show that inhibition of vascular endothelial growth factor signaling significantly reduces the number of HSCs that enter the liver. We also performed a pilot chemical screen and identified two compounds that affect HSC numbers during development.
Our work provides the first comprehensive description of HSC development in zebrafish and reveals the requirement of SECs in HSC localization. The Tg(hand2:EGFP) line represents a unique tool for in vivo analysis and molecular dissection of HSC behavior.
PMCID: PMC3407311  PMID: 22488653
sinusoidal endothelial cells; liver development; cloche; VEGF; biliary cells; acute alcohol exposure
5.  Abnormal Nuclear Pore Formation Triggers Apoptosis in the Intestinal Epithelium of elys-Deficient Zebrafish 
Gastroenterology  2008;136(3):902-911.
Background & Aims
Zebrafish mutants generated by ethylnitrosourea (ENU)-mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, “flotte lotte” (flo), displays striking defects in intestinal, liver, pancreas and eye formation at 78hpf. In this study we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype.
Positional cloning was employed to map the flo mutation. Sub-cellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, confocal and electron microscopy.
The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, ELYS has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Though elys mRNA is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally-expressed elys mRNA until 24hpf. From 72hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately intestinal epithelial cells undergo apoptosis.
Our results demonstrate that Elys regulates digestive organ formation.
PMCID: PMC3804769  PMID: 19073184
6.  Whole-organism screening for gluconeogenesis identifies activators of fasting metabolism 
Nature chemical biology  2012;9(2):97-104.
Improving the control of energy homeostasis can lower cardiovascular risk in metabolically compromised individuals. To identify new regulators of whole-body energy control, we conducted a high-throughput screen in transgenic reporter zebrafish for small molecules that modulate the expression of the fasting-inducible gluconeogenic gene pck1. We show that this in vivo strategy identified several drugs that impact gluconeogenesis in humans, as well as metabolically uncharacterized compounds. Most notably, we find that the Translocator Protein (TSPO) ligands PK 11195 and Ro5-4864 are glucose lowering agents despite a strong inductive effect on pck1 expression. We show that these drugs are activators of a fasting-like energy state, and importantly that they protect high-fat diet induced obese mice from hepatosteatosis and glucose intolerance, two pathological manifestations of metabolic dysregulation. Thus, using a whole-organism screening strategy, this study has identified new small molecule activators of fasting metabolism.
PMCID: PMC3552031  PMID: 23201900
7.  Hepatocyte Growth Factor Signaling in Intrapancreatic Ductal Cells Drives Pancreatic Morphogenesis 
PLoS Genetics  2013;9(7):e1003650.
In a forward genetic screen for regulators of pancreas development in zebrafish, we identified donuts908, a mutant which exhibits failed outgrowth of the exocrine pancreas. The s908 mutation leads to a leucine to arginine substitution in the ectodomain of the hepatocyte growth factor (HGF) tyrosine kinase receptor, Met. This missense mutation impedes the proteolytic maturation of the receptor, its trafficking to the plasma membrane, and diminishes the phospho-activation of its kinase domain. Interestingly, during pancreatogenesis, met and its hgf ligands are expressed in pancreatic epithelia and mesenchyme, respectively. Although Met signaling elicits mitogenic and migratory responses in varied contexts, normal proliferation rates in donut mutant pancreata together with dysmorphic, mislocalized ductal cells suggest that met primarily functions motogenically in pancreatic tail formation. Treatment with PI3K and STAT3 inhibitors, but not with MAPK inhibitors, phenocopies the donut pancreatic defect, further indicating that Met signals through migratory pathways during pancreas development. Chimera analyses showed that Met-deficient cells were excluded from the duct, but not acinar, compartment in the pancreatic tail. Conversely, wild-type intrapancreatic duct and “tip cells” at the leading edge of the growing pancreas rescued the donut phenotype. Altogether, these results reveal a novel and essential role for HGF signaling in the intrapancreatic ducts during exocrine morphogenesis.
Author Summary
The pancreas functions as an endocrine and exocrine gland that secretes hormones regulating blood glucose homeostasis, and pancreatic juice that aids the digestion and absorption of nutrients, respectively. Contrary to endocrine tissue development, that of the exocrine pancreas has received less attention. We conducted a forward genetic screen in zebrafish and identified HGF/Met signaling as a key regulator of exocrine development. We called the mutant donut because the body of the pancreas fails to elongate and thus remains rounded. The mutation leading to this phenotype affects the extracellular domain of Met, the hepatocyte growth factor (HGF) receptor, impairing its maturation, plasma membrane localization and phospho-activation. Although HGF/Met signaling may elicit many context-dependant cellular responses, our data indicate that HGF/Met signaling triggers the migration, but not the proliferation, of the pancreatic ductal cells to drive the extension of the pancreatic tail.
PMCID: PMC3723531  PMID: 23935514
8.  Mutation of zebrafish Snapc4 is associated with loss of the intrahepatic biliary network 
Developmental biology  2011;363(1):128-137.
Biliary epithelial cells line the intrahepatic biliary network, a complex three-dimensional network of conduits. The loss of differentiated biliary epithelial cells is the primary cause of many congenital liver diseases. We identified a zebrafish snapc4 (small nuclear RNA-activating complex polypeptide 4) mutant in which biliary epithelial cells initially differentiate but subsequently disappear. In these snapc4 mutant larvae, the biliary epithelial cells undergo apoptosis, leading to the degeneration of the intrahepatic biliary network. Consequently, in snapc4 mutant larvae, biliary transport of ingested fluorescent lipids to the gallbladder is blocked. Snapc4 is the largest subunit of the protein complex that regulates small nuclear RNA (snRNA) transcription. The snapc4s445 mutation causes a truncation of the C-terminus thereby deleting the domain responsible for a specific interaction with Snapc2, a vertebrate specific subunit of the SNAP complex. This mutation leads to a hypomorphic phenotype, as only a subset of snRNA transcripts are quantitatively altered in snapc4s445 mutant larvae. snapc2 knockdown also disrupts the intrahepatic biliary network in a similar fashion as in snapc4s445 mutant larvae. These data indicate that the physical interaction between Snapc2 and Snapc4 is important for the expression of a subset of snRNAs and biliary epithelial cell survival in zebrafish.
PMCID: PMC3711868  PMID: 22222761
Liver; snRNA; SNAP190; SNAPC2; biliary epithelial cells; vanishing bile duct
9.  Adenosine signaling promotes regeneration of pancreatic β-cells in vivo 
Cell Metabolism  2012;15(6):885-894.
Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing β-cells is still needed. Using a zebrafish model of diabetes, we screened ~7000 small molecules to identify enhancers of β-cell regeneration. The compounds we identified converge on the adenosine signaling pathway and include exogenous agonists and compounds that inhibit degradation of endogenously produced adenosine. The most potent enhancer of β-cell regeneration was the adenosine agonist 5′-N-Ethylcarboxamidoadenosine (NECA), which acting through the adenosine receptor A2aa increased β-cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating β-cell proliferation during regeneration, NECA had only a modest effect during development. The proliferative and glucose-lowering effect of NECA was confirmed in diabetic mice, suggesting an evolutionarily conserved role for adenosine in β-cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes.
PMCID: PMC3372708  PMID: 22608007
10.  Zebrafish in the Study of Early Cardiac Development 
Circulation Research  2012;110(6):870-874.
Heart development is a complex process that involves cell specification and differentiation, as well as elaborate tissue morphogenesis and remodeling, to generate a functional organ. The zebrafish has emerged as a powerful model system to unravel the basic genetic, molecular and cellular mechanisms of cardiac development and function. Here we summarize and discuss recent discoveries on early cardiac specification and the identification of the second heart field in zebrafish. In addition to the inductive signals regulating cardiac specification, these studies have shown that heart development also requires a repressive mechanism imposed by retinoic acid signaling to select cardiac progenitors from a multipotent population. Another recent advance in the study of early zebrafish cardiac development is the identification of the second heart field (SHF). These studies suggest that the molecular mechanisms that regulate SHF development are conserved between zebrafish and other vertebrates including mammals, and provide insight into the evolution of the SHF and its derivatives.
PMCID: PMC3329892  PMID: 22427324
retinoid acid signaling; Fgf signaling; second heart field; Ltbp3; zebrafish
11.  Nodal signaling regulates endodermal cell motility and actin dynamics via Rac1 and Prex1 
The Journal of Cell Biology  2012;198(5):941-952.
Nodal, acting through Prex1 and Rac1, promotes dynamic actin and random motility in endodermal cells during early gastrulation.
Embryo morphogenesis is driven by dynamic cell behaviors, including migration, that are coordinated with fate specification and differentiation, but how such coordination is achieved remains poorly understood. During zebrafish gastrulation, endodermal cells sequentially exhibit first random, nonpersistent migration followed by oriented, persistent migration and finally collective migration. Using a novel transgenic line that labels the endodermal actin cytoskeleton, we found that these stage-dependent changes in migratory behavior correlated with changes in actin dynamics. The dynamic actin and random motility exhibited during early gastrulation were dependent on both Nodal and Rac1 signaling. We further identified the Rac-specific guanine nucleotide exchange factor Prex1 as a Nodal target and showed that it mediated Nodal-dependent random motility. Reducing Rac1 activity in endodermal cells caused them to bypass the random migration phase and aberrantly contribute to mesodermal tissues. Together, our results reveal a novel role for Nodal signaling in regulating actin dynamics and migration behavior, which are crucial for endodermal morphogenesis and cell fate decisions.
PMCID: PMC3432772  PMID: 22945937
12.  Analysis of Sphingosine-1-phosphate signaling mutants reveals endodermal requirements for the growth but not dorsoventral patterning of jaw skeletal precursors 
Developmental Biology  2011;362(2):230-241.
Development of the head skeleton involves reciprocal interactions between cranial neural crest cells (CNCCs) and the surrounding pharyngeal endoderm and ectoderm. Whereas elegant experiments in avians have shown a prominent role for the endoderm in facial skeleton development, the relative functions of the endoderm in growth versus regional identity of skeletal precursors have remained unclear. Here we describe novel craniofacial defects in zebrafish harboring mutations in the Sphingosine-1-phospate (S1P) type 2 receptor (s1pr2) or the S1P transporter Spinster 2 (spns2), and we show that S1P signaling functions in the endoderm for the proper growth and positioning of the jaw skeleton. Surprisingly, analysis of s1pr2 and spns2 mutants, as well as sox32 mutants that completely lack endoderm, reveals that the dorsal-ventral (DV) patterning of jaw skeletal precursors is largely unaffected even in the absence of endoderm. Instead, we observe reductions in the ectodermal expression of Fibroblast growth factor 8a (Fgf8a), and transgenic misexpression of Shha restores fgf8a expression and partially rescues the growth and differentiation of jaw skeletal precursors. Hence, we propose that the S1P-dependent anterior foregut endoderm functions primarily through Shh to regulate the growth but not DV patterning of zebrafish jaw precursors.
PMCID: PMC3265674  PMID: 22185793
Sphingosine-1-phosphate; Pharyngeal Endoderm; Facial Ectoderm; Craniofacial Skeleton; Shh; Zebrafish
13.  Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS Activity by CoQ10 Synthesis 
Cell  2013;152(3):504-518.
Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity.
Graphical Abstract
► UBIAD1 is a Golgi prenyltransferase ► UBIAD1 contributes to the nonmitochondrial pool of CoQ10 ► UBIAD1 protects cardiovascular tissues from NOS-dependent oxidative damage ► UBIAD1 is a target for therapeutic strategies by limiting the side effects of statins
UBIAD1 is identified as a nonmitochondrial CoQ10 biosynthetic enzyme required for oxidative damage protection. Through CoQ10 synthesis, UBIAD1 modulates endothelial nitric oxide synthase activity and nitric oxide signaling necessary for cardiovascular development and homeostasis.
PMCID: PMC3574195  PMID: 23374346
14.  Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b 
Developmental biology  2011;361(1):68-78.
The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.
PMCID: PMC3235368  PMID: 22020048
Claudin; liver development; zebrafish; biliary duct remodeling; biliary cells; biliary duct morphogenesis; tight junctions; cholestasis
15.  Determination of Endothelial Stalk versus Tip Cell Potential during Angiogenesis by H2.0-like Homeobox-1 
Current Biology  2012;22(19):1789-1794.
Tissue branching morphogenesis requires the hierarchical organization of sprouting cells into leading “tip” and trailing “stalk” cells [1, 2]. During new blood vessel branching (angiogenesis), endothelial tip cells (TCs) lead sprouting vessels, extend filopodia, and migrate in response to gradients of the secreted ligand, vascular endothelial growth factor (Vegf) [3]. In contrast, adjacent stalk cells (SCs) trail TCs, generate the trunk of new vessels, and critically maintain connectivity with parental vessels. Here, we establish that h2.0-like homeobox-1 (Hlx1) determines SC potential, which is critical for angiogenesis during zebrafish development. By combining a novel pharmacological strategy for the manipulation of angiogenic cell behavior in vivo with transcriptomic analyses of sprouting cells, we identify the uniquely sprouting-associated gene, hlx1. Expression of hlx1 is almost entirely restricted to sprouting endothelial cells and is excluded from adjacent nonangiogenic cells. Furthermore, Hlx1 knockdown reveals its essential role in angiogenesis. Importantly, mosaic analyses uncover a cell-autonomous role for Hlx1 in the maintenance of SC identity in sprouting vessels. Hence, Hlx1-mediated maintenance of SC potential regulates angiogenesis, a finding that may have novel implications for sprouting morphogenesis of other tissues.
► Expression of hlx1 is associated with angiogenic cell behavior in vivo ► hlx1 selectively marks sprouting endothelial cells during zebrafish development ► Hlx1 is required for intersegmental vessel angiogenesis in zebrafish embryos ► Hlx1 cell-autonomously maintains endothelial stalk cell potential
PMCID: PMC3471071  PMID: 22921365
16.  Molecular control of endothelial cell behaviour during blood vessel morphogenesis 
The vertebrate vasculature forms an extensive branched network of blood vessels that supplies tissues with nutrients and oxygen. During vascular development, coordinated control of endothelial cell behaviour at the levels of cell migration, proliferation, polarity, differentiation and cell–cell communication is critical for functional blood vessel morphogenesis. Recent data uncover elaborate transcriptional, post-transcriptional and post-translational mechanisms that fine-tune key signalling pathways (such as the vascular endothelial growth factor and Notch pathways) to control endothelial cell behaviour during blood vessel sprouting (angiogenesis). These emerging frameworks controlling angiogenesis provide unique insights into fundamental biological processes common to other systems, such as tissue branching morphogenesis, mechanotransduction and tubulogenesis.
PMCID: PMC3319719  PMID: 21860391
17.  Intrinsic and Extrinsic Modifiers of the Regulative Capacity of the Developing Liver 
Mechanisms of development  2012;128(11-12):525-535.
Zebrafish wnt2bb mutants initially fail to form a liver, but surprisingly the liver eventually forms in a majority of these embryos which then develop into fertile adults. This unexpected result raised the possibility that identifying the mechanisms of liver formation in wnt2bb mutants could provide insights into the poorly understood yet general principle of regulative development, a process by which some cells can change fate in order to compensate for a deficiency. Here, we identify two factors that underlie the regulative capacity of endodermal tissues: an intrinsic factor, Sox32, a transcription factor of the SoxF subfamily, and an extrinsic factor, Fgf10a. sox32 is expressed in the extrahepatic duct primordium which is not affected in wnt2bb mutants. Blocking Sox32 function prevented liver formation in most wnt2bb mutants. fgf10a, which is expressed in the mesenchyme surrounding non-hepatic endodermal cells, negatively impacts the regulative capacity of endodermal tissues. In Wnt/β-catenin signaling deficient embryos, in which the liver completely fails to form, the repression of Fgf10a function allowed liver formation. Altogether, these studies reveal that there is more than one way to form a liver, and provide molecular insights into the phenomenon of tissue plasticity.
PMCID: PMC3297115  PMID: 22313811
sox17; sox32; fgf10a; developmental plasticity; endoderm; zebrafish
18.  sox9b Is a Key Regulator of Pancreaticobiliary Ductal System Development 
PLoS Genetics  2012;8(6):e1002754.
The pancreaticobiliary ductal system connects the liver and pancreas to the intestine. It is composed of the hepatopancreatic ductal (HPD) system as well as the intrahepatic biliary ducts and the intrapancreatic ducts. Despite its physiological importance, the development of the pancreaticobiliary ductal system remains poorly understood. The SRY-related transcription factor SOX9 is expressed in the mammalian pancreaticobiliary ductal system, but the perinatal lethality of Sox9 heterozygous mice makes loss-of-function analyses challenging. We turned to the zebrafish to assess the role of SOX9 in pancreaticobiliary ductal system development. We first show that zebrafish sox9b recapitulates the expression pattern of mouse Sox9 in the pancreaticobiliary ductal system and use a nonsense allele of sox9b, sox9bfh313, to dissect its function in the morphogenesis of this structure. Strikingly, sox9bfh313 homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9bfh313 mutant embryos and larvae reveals that the HPD cells appear to mis-differentiate towards hepatic and/or pancreatic fates, resulting in a dysmorphic structure. The intrahepatic biliary cells are specified but fail to assemble into a functional network. Similarly, intrapancreatic duct formation is severely impaired in sox9bfh313 mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9bfh313 mutants worsen during larval and juvenile stages, prompting the adult phenotype. We further show that Sox9b interacts with Notch signaling to regulate intrahepatic biliary network formation: sox9b expression is positively regulated by Notch signaling, while Sox9b function is required to maintain Notch signaling in the intrahepatic biliary cells. Together, these data reveal key roles for SOX9 in the morphogenesis of the pancreaticobiliary ductal system, and they cast human Sox9 as a candidate gene for pancreaticobiliary duct malformation-related pathologies.
Author Summary
The liver and pancreas function as exocrine glands that secrete bile and pancreatic juice, respectively, to aid the digestion and absorption of nutrients. These fluids reach the intestine via the pancreaticobiliary ductal system, a complex network of ducts. Despite its pivotal role, the development of this ductal system is poorly understood. We have discovered that the zebrafish transcription factor gene sox9b, like its mammalian ortholog, is specifically expressed in the pancreaticobiliary ductal system. The perinatal lethality of Sox9 heterozygous mice makes the analysis of SOX9 function challenging; thus, we turned to the zebrafish to analyze the role of SOX9 in pancreaticobiliary ductal system development. We found that zebrafish sox9b mutants, which survive to adulthood, display defects in the morphogenesis of this ductal network: the intrahepatic and intrapancreatic ducts fail to form a branched network, whereas the ducts connecting the liver and pancreas to the intestine are malformed. These ductal defects affect bile transport and lead to cholestasis in adult mutant fish. At the molecular level, Sox9b interacts with the Notch signaling pathway to regulate the development of the intrahepatic biliary network. Therefore, our work in zebrafish reveals a broad and complex role for SOX9 in pancreaticobiliary ductal system morphogenesis.
PMCID: PMC3375260  PMID: 22719264
19.  Suppression of Ptf1a activity induces acinar to endocrine conversion 
Current biology : CB  2011;21(8):712-717.
Pluripotent embryonic cells become progressively lineage-restricted during development in a process that culminates in the differentiation of stable organ specific cell types that perform specialized functions. Terminally-differentiated pancreatic acinar cells do not have the innate capacity to contribute to the endocrine β-cell lineage, which is destroyed in individuals with autoimmune diabetes [1]. Some cell types can be reprogrammed using a single factor [2, 3], whereas other cell types require continuous activity of transcriptional regulators to repress alternate cell fates [4–6]. Thus, we hypothesized that a transcriptional network continuously maintains the pancreatic acinar cell fate. We found that post-embryonic antagonism of Ptf1a, a master regulator of pancreatic development [7] and acinar cell fate specification [8, 9], induced the expression of endocrine genes including insulin in the exocrine compartment. Using a genetic lineage tracing approach, we show that the induced insulin+ cells are derived from acinar cells. Cellular reprogramming occurred under homeostatic conditions, suggesting that the pancreatic micro-environment is sufficient to promote endocrine differentiation. Thus, severe experimental manipulations [10, 11] may not be required to potentiate pancreatic transdifferentiation. These data indicate that targeted post-embryonic disruption of the acinar cell fate can restore the developmental plasticity that is lost during development.
PMCID: PMC3081919  PMID: 21497092
pancreas; acinar; beta cells; endocrine; zebrafish; diabetes; lineage tracing; ptf1a
20.  Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens 
Developmental biology  2010;350(1):50-63.
DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.
PMCID: PMC3022120  PMID: 21126517
21.  Dynamic glucoregulation and mammalian-like responses to metabolic and developmental disruption in zebrafish 
Zebrafish embryos are emerging as models of glucose metabolism. However, patterns of endogenous glucose levels, and the role of the islet in glucoregulation, are unknown. We measured absolute glucose levels in zebrafish and mouse embryos, and demonstrate similar, dynamic glucose fluctuations in both species. Further, we show that chemical and genetic perturbations elicit mammalian-like glycemic responses in zebrafish embryos. We show that glucose is undetectable in early zebrafish and mouse embryos, but increases in parallel with pancreatic islet formation in both species. In zebrafish, increasing glucose is associated with activation of gluconeogenic phosphoenolpyruvate carboxykinase1 (pck1) transcription. Non-hepatic Pck1 protein is expressed in mouse embryos. We show, using RNA in situ hybridization, that zebrafish pck1 mRNA is similarly expressed in multiple cell types prior to hepatogenesis. Further, we demonstrate that the Pck1 inhibitor 3-mercaptopicolinic acid suppresses normal glucose accumulation in early zebrafish embryos. This shows that pre- and extra-hepatic pck1 is functional, and provides glucose locally to rapidly developing tissues. To determine if the primary islet is glucoregulatory in early fish embryos, we injected pdx1-specific morpholinos into transgenic embryos expressing GFP in beta cells. Most morphant islets were hypomorphic, not agenetic, but embryos still exhibited persistent hyperglycemia. We conclude from these data that the early zebrafish islet is functional, and regulates endogenous glucose. In summary, we identify mechanisms of glucoregulation in zebrafish embryos that are conserved with embryonic and adult mammals. These observations justify use of this model in mechanistic studies of human metabolic disease.
PMCID: PMC3014420  PMID: 20965191
zebrafish; mouse; embryo; glucose; pck1; gluconeogenesis; islet; pdx1
22.  Cse1l is a negative regulator of CFTR-dependent fluid secretion 
Current biology : CB  2010;20(20):1840-1845.
Transport of chloride through the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates[1, 2]. Loss of CFTR function leads to cystic fibrosis (CF)[1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine and vas deferens. Conversely, un-controlled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera[5, 6] and other secretory diarrheas [7] as well as Polycystic Kidney Disease (PKD)[8–10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the un-controlled activation of the CFTR channel. Analyses in zebrafish embryos and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much needed model system to study CFTR regulation in vivo.
PMCID: PMC2963654  PMID: 20933420
23.  Tbx5 and Bmp signaling are essential for proepicardium specification in zebrafish 
Circulation research  2010;106(12):1818-1828.
The proepicardial organ (PE) contributes to the cellular diversity of the developing heart by giving rise to the epicardium as well as vascular smooth muscle cells and fibroblasts. Despite the importance of these cells in cardiac development, function and regeneration, the signals required for the specification of the PE remain largely unexplored.
We aim to identify the signaling molecules and transcription factors that regulate PE specification.
Methods and Results
Here, we present the first genetic evidence that Bmp signaling in conjunction with the T-box transcription factor Tbx5a is essential for PE specification in zebrafish. Specifically, Bmp4 from the cardiac region, but not the liver bud, acting through the type I BMP receptor Acvr1l, is required for PE specification. By overexpressing a dominant-negative form of a Bmp receptor at various embryonic stages, we determined when Bmp signaling was required for PE specification. We also found that overexpression of bmp2b right before PE specification led to the ectopic expression of PE specific markers including tbx18. Furthermore, using loss-of-function approaches, we discovered a previously unappreciated PE specification role for Tbx5a at early somite stages; this role occurs earlier than, and appears to be independent from, the requirement for Bmp signaling in this process.
Altogether, these data lead us to propose that Tbx5a confers anterior lateral plate mesodermal cells the competence to respond to Bmp signals and initiate PE development.
PMCID: PMC2892550  PMID: 20413782
proepicardial organ (PE); tbx18; tcf21; acvr1l; tbx5; zebrafish
24.  Chromatin remodelling complex dosage modulates transcription factor function in heart development 
Nature communications  2011;2:187.
Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2–5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs.
PMCID: PMC3096875  PMID: 21304516
25.  Primary contribution to zebrafish heart regeneration by gata4+ cardiomyocytes 
Nature  2010;464(7288):601-605.
Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life1,2. Yet, there is little or no significant cardiac muscle regeneration after an injury like acute myocardial infarction (MI)3. By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced4,5. In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Here, we used new genetic fate-mapping approaches to identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through use of a transgenic reporter strain, we found that cardiomyocytes throughout the subepicardial ventricular layer trigger expression of the embryonic cardiogenesis gene gata4 within a week of trauma, before expression localizes to proliferating cardiomyocytes surrounding and within the injury site. Cre recombinase-based lineage-tracing of cells expressing gata4 before evident regeneration, or of cells expressing the contractile gene cmlc2 before injury, each labeled a majority of cardiac muscle in the ensuing regenerate. By optical voltage mapping of surface myocardium in whole ventricles, we found that electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury. After injury and prolonged Fgf receptor inhibition to arrest cardiac regeneration and enable scar formation, experimental release of the signaling block led to gata4 expression and morphological improvement of the injured ventricular wall without loss of scar tissue. Our results indicate that electrically coupled cardiac muscle regenerates after resection injury primarily through activation and expansion of cardiomyocyte populations, findings with implications for promoting regeneration of the injured human heart.
PMCID: PMC3040215  PMID: 20336144

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