Heterogeneity within a population of cells of the same type is a common theme in metazoan biology. Dissecting complex developmental and physiological processes crucially relies on our ability to probe the expression profile of these cell subpopulations. Current strategies rely on cell enrichment based on sequential or simultaneous use of multiple intersecting markers starting from a heterogeneous cell suspension. The extensive tissue manipulations required to generate single-cell suspensions, as well as the complexity of the required equipment, inherently complicate these approaches. Here, we propose an alternative methodology based on a genetically encoded system in the model organism Danio rerio (zebrafish). In transgenic fish, we take advantage of the combinatorial biotin transfer system, where polysome-associated mRNAs are selectively recovered from cells expressing both a tagged ribosomal subunit, Rpl10a, and the bacterial biotin ligase BirA. We have applied this technique to skeletal muscle development and identified new genes with interesting temporal expression patterns. Through this work we have thus developed additional tools for highly specific gene expression profiling.
Dilated cardiomyopathy is a leading cause of congestive heart failure and a debilitating complication of antineoplastic therapies. Despite disparate causes for dilated cardiomyopathy, maladaptive cardiac remodeling and decreased systolic function are common clinical consequences, begging an investigation of in vivo contractile dynamics in development and disease, one that has been impossible to date.
Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.
Morpholino oligomers have been used widely and for many years in the zebrafish community to transiently knock down the function of target genes. It has often been difficult, however, to reliably discriminate between specific and non-specific effects, and thus generally accepted guidelines to control for morpholino side effects do not exist. In light of recent methodologies to generate mutant lines in virtually any zebrafish gene, we discuss these different approaches with a specific focus on how the first description of a loss-of-function phenotype in zebrafish should be accomplished.
Valvular heart disease is responsible for considerable morbidity and mortality. Cardiac valves develop as the heart contracts, and they function throughout the lifetime of the organism to prevent retrograde blood flow. Their precise morphogenesis is crucial for cardiac function. Zebrafish is an ideal model to investigate cardiac valve development as it allows these studies to be carried out in vivo through non-invasive imaging. Accumulating evidence suggests a role for contractility and intracardiac flow dynamics in cardiac valve development. However, these two factors have proved difficult to uncouple, especially since altering myocardial function affects the intracardiac flow pattern.
Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.001.
Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.
Over the course of development, the vertebrate heart undergoes a series of complex morphogenetic processes that transforms it from a simple myocardial epithelium to the complex 3D structure required for its function. One of these processes leads to the formation of trabeculae to optimize the internal structure of the ventricle for efficient conduction and contraction. Despite the important role of trabeculae in the development and physiology of the heart, little is known about their mechanism of formation. Using 3D time-lapse imaging of beating zebrafish hearts, we observed that the initiation of cardiac trabeculation can be divided into two processes. Before any myocardial cell bodies have entered the trabecular layer, cardiomyocytes extend protrusions that invade luminally along neighboring cell-cell junctions. These protrusions can interact within the trabecular layer to form new cell-cell contacts. Subsequently, cardiomyocytes constrict their abluminal surface, moving their cell bodies into the trabecular layer while elaborating more protrusions. We also examined the formation of these protrusions in trabeculation-deficient animals, including erbb2 mutants, tnnt2a morphants, which lack cardiac contractions and flow, and myh6 morphants, which lack atrial contraction and exhibit reduced flow. We found that, compared with cardiomyocytes in wild-type hearts, those in erbb2 mutants were less likely to form protrusions, those in tnnt2a morphants formed less stable protrusions, and those in myh6 morphants extended fewer protrusions per cell. Thus, through detailed 4D imaging of beating hearts, we have identified novel cellular behaviors underlying cardiac trabeculation.
Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic ? cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying ?-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated ?-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3,5-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into ? cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived ? cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of ?-cell differentiation and its application to a cost-effective production of functional ? cells for cell therapy.
In a forward genetic screen for regulators of pancreas development in zebrafish, we identified donut(s908) , 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.
Visceral organs, including the liver and pancreas, adopt asymmetric positions to ensure proper function. Yet the molecular and cellular mechanisms controlling organ laterality are not well understood. We identified a mutation affecting zebrafish laminin ?1a (lamb1a) that disrupts left-right asymmetry of the liver and pancreas. In these mutants, the liver spans the midline and the ventral pancreatic bud remains split into bilateral structures. We show that lamb1a regulates asymmetric left-right gene expression in the lateral plate mesoderm (LPM). In particular, lamb1a functions in Kupffers vesicle (KV), a ciliated organ analogous to the mouse node, to control the length and function of the KV cilia. Later during gut-looping stages, dynamic expression of Lamb1a is required for the bilayered organization and asymmetric migration of the LPM. Loss of Lamb1a function also results in aberrant protrusion of LPM cells into the gut. Collectively, our results provide cellular and molecular mechanisms by which extracellular matrix proteins regulate left-right organ morphogenesis.
Although the liver and ventral pancreas are thought to arise from a common multipotent progenitor pool, it is unclear whether these progenitors of the hepatopancreas system are specified by a common genetic mechanism. Efforts to determine the role of Hnf1b and Wnt signaling in this crucial process have been confounded by a combination of factors, including a narrow time frame for hepatopancreas specification, functional redundancy among Wnt ligands, and pleiotropic defects caused by either severe loss of Wnt signaling or Hnf1b function. Using a novel hypomorphic hnf1ba zebrafish mutant that exhibits pancreas hypoplasia, as observed in HNF1B monogenic diabetes, we show that hnf1ba plays essential roles in regulating ?-cell number and pancreas specification, distinct from its function in regulating pancreas size and liver specification, respectively. By combining Hnf1ba partial loss of function with conditional loss of Wnt signaling, we uncover a crucial developmental window when these pathways synergize to specify the entire ventrally derived hepatopancreas progenitor population. Furthermore, our in vivo genetic studies demonstrate that hnf1ba generates a permissive domain for Wnt signaling activity in the foregut endoderm. Collectively, our findings provide a new model for HNF1B function, yield insight into pancreas and ?-cell development, and suggest a new mechanism for hepatopancreatic specification.
Despite current treatment regimens, heart failure remains the leading cause of morbidity and mortality in the developed world due to the limited capacity of adult mammalian ventricular cardiomyocytes to divide and replace ventricular myocardium lost from ischaemia-induced infarct. Hence there is great interest to identify potential cellular sources and strategies to generate new ventricular myocardium. Past studies have shown that fish and amphibians and early postnatal mammalian ventricular cardiomyocytes can proliferate to help regenerate injured ventricles; however, recent studies have suggested that additional endogenous cellular sources may contribute to this overall ventricular regeneration. Here we have developed, in the zebrafish (Danio rerio), a combination of fluorescent reporter transgenes, genetic fate-mapping strategies and a ventricle-specific genetic ablation system to discover that differentiated atrial cardiomyocytes can transdifferentiate into ventricular cardiomyocytes to contribute to zebrafish cardiac ventricular regeneration. Using in vivo time-lapse and confocal imaging, we monitored the dynamic cellular events during atrial-to-ventricular cardiomyocyte transdifferentiation to define intermediate cardiac reprogramming stages. We observed that Notch signalling becomes activated in the atrial endocardium following ventricular ablation, and discovered that inhibiting Notch signalling blocked the atrial-to-ventricular transdifferentiation and cardiac regeneration. Overall, these studies not only provide evidence for the plasticity of cardiac lineages during myocardial injury, but more importantly reveal an abundant new potential cardiac resident cellular source for cardiac ventricular regeneration.
Nonalcoholic fatty liver disease is the most common liver disease in both adults and children. The earliest stage of this disease is hepatic steatosis, in which triglycerides are deposited as cytoplasmic lipid droplets in hepatocytes. Through a forward genetic approach in zebrafish, we found that guanosine monophosphate (GMP) synthetase mutant larvae develop hepatic steatosis. We further demonstrate that activity of the small GTPase Rac1 and Rac1-mediated production of reactive oxygen species (ROS) are down-regulated in GMP synthetase mutant larvae. Inhibition of Rac1 activity or ROS production in wild-type larvae by small molecule inhibitors was sufficient to induce hepatic steatosis. More conclusively, treating larvae with hydrogen peroxide, a diffusible ROS that has been implicated as a signaling molecule, alleviated hepatic steatosis in both GMP synthetase mutant and Rac1 inhibitor-treated larvae, indicating that homeostatic production of ROS is required to prevent hepatic steatosis. We further found that ROS positively regulate the expression of the triglyceride hydrolase gene, which is responsible for the mobilization of stored triglycerides in hepatocytes. Consistently, inhibition of triglyceride hydrolase activity in wild-type larvae by a small molecule inhibitor was sufficient to induce hepatic steatosis. Conclusion: De novo GMP synthesis influences the activation of the small GTPase Rac1, which controls hepatic lipid dynamics through ROS-mediated regulation of triglyceride hydrolase expression in hepatocytes.
Biliary epithelial cells (BECs) are considered to be a source of regenerating hepatocytes when hepatocyte proliferation is compromised. However, there is still controversy about the extent to which BECs can contribute to the regenerating hepatocyte population, and thereby to liver recovery. To investigate this issue, we established a zebrafish model of liver regeneration in which the extent of hepatocyte ablation can be controlled.
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.
In a significant fraction of breast cancer patients, distant metastases emerge after years or even decades of latency. How disseminated tumour cells (DTCs) are kept dormant, and what wakes them up, are fundamental problems in tumour biology. To address these questions, we used metastasis assays in mice and showed that dormant DTCs reside on 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-?1 and periostin as tumour-promoting factors derived from endothelial tip cells. Our work reveals that stable microvasculature constitutes a dormant niche, whereas sprouting neovasculature sparks micrometastatic outgrowth.
Obese individuals exhibit an increase in pancreatic ? cell mass; conversely, scarce nutrition during pregnancy has been linked to ? cell insufficiency in the offspring [reviewed in 1, 2]. These phenomena are thought to be mediated mainly through effects on ? cell proliferation, given that a nutrient-sensitive ? cell progenitor population in the pancreas has not been identified. Here, we employed the fluorescent ubiquitination-based cell-cycle indicator system to investigate ? cell replication in real time and found that high nutrient concentrations induce rapid ? cell proliferation. Importantly, we found that high nutrient concentrations also stimulate ? cell differentiation from progenitors in the intrapancreatic duct (IPD). Furthermore, using a new zebrafish line where ? cells are constitutively ablated, we show that ? cell loss and high nutrient intake synergistically activate these progenitors. At the cellular level, this activation process causes ductal cell reorganization as it stimulates their proliferation and differentiation. Notably, we link the nutrient-dependent activation of these progenitors to a downregulation of Notch signaling specifically within the IPD. Furthermore, we show that the nutrient sensor mechanistic target of rapamycin (mTOR) is required for endocrine differentiation from the IPD under physiological conditions as well as in the diabetic state. Thus, this study reveals critical insights into how cells modulate their plasticity in response to metabolic cues and identifies nutrient-sensitive progenitors in the mature pancreas.
Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.
Vegf signaling specifies arterial fate during early vascular development by inducing the transcription of Delta-like 4 (Dll4), the earliest Notch ligand gene expressed in arterial precursor cells. Dll4 expression precedes that of Notch receptors in arteries, and factors that direct its arterial-specific expression are not known. To identify the transcriptional program that initiates arterial Dll4 expression, we characterized an arterial-specific and Vegf-responsive enhancer of Dll4. Our findings demonstrate that Notch signaling is not required for initiation of Dll4 expression in arteries and suggest that Notch instead functions as a maintenance factor. Importantly, we find that Vegf signaling activates MAP kinase (MAPK)-dependent E26 transformation-specific sequence (ETS) factors in the arterial endothelium to drive expression of Dll4 and Notch4. These findings identify a Vegf/MAPK-dependent transcriptional pathway that specifies arterial identity by activating Notch signaling components and illustrate how signaling cascades can modulate broadly expressed transcription factors to achieve tissue-specific transcriptional outputs.
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.
Much of the effort in understanding the dynamic process of development has focused on dissecting biochemical pathways. Recent studies illustrate that simple physical forces are also important in patterning organs.
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.
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.
Rapid electrical conduction in the His-Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His-Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction-forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.
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, biliary epithelial cells undergo apoptosis, leading to 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 a protein complex that regulates small nuclear RNA (snRNA) transcription. The snapc4(s445) 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 snapc4(s445) mutant larvae. snapc2 knockdown also disrupts the intrahepatic biliary network in a similar fashion as in snapc4(s445) 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.
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.
Members of the Slit family of secreted ligands interact with Roundabout (Robo) receptors to provide guidance cues for many cell types. For example, Slit/Robo signaling elicits repulsion of axons during neural development, whereas in endothelial cells this pathway inhibits or promotes angiogenesis depending on the cellular context. Here, we show that miR-218 is intronically encoded in slit2 and slit3 and that it suppresses Robo1 and Robo2 expression. Our data indicate that miR-218 and multiple Slit/Robo signaling components are required for heart tube formation in zebrafish and that this network modulates the previously unappreciated function of Vegf signaling in this process. These findings suggest a new paradigm for microRNA-based control of ligand-receptor interactions and provide evidence for a novel signaling pathway regulating vertebrate heart tube assembly.
Hepatic competence, or the ability to respond to hepatic-inducing signals, is regulated by a number of transcription factors broadly expressed in the endoderm. However, extrinsic signals might also regulate hepatic competence, as suggested by tissue explant studies. Here, we present genetic evidence that Fgf signaling regulates hepatic competence in zebrafish. We first show that the endoderm posterior to the liver-forming region retains hepatic competence: using transgenic lines that overexpress hepatic inducing signals following heat-shock, we found that at late somitogenesis stages Wnt8a, but not Bmp2b, overexpression could induce liver gene expression in pancreatic and intestinal bulb cells. These manipulations resulted in the appearance of ectopic hepatocytes in the intestinal bulb. Second, by overexpressing Wnt8a at various stages, we found that as embryos develop, the extent of the endodermal region retaining hepatic competence is gradually reduced. Most significantly, we found, using gain- and loss-of-function approaches, that Fgf10a signaling regulates this gradual reduction of the hepatic-competent domain. These data provide in vivo evidence that endodermal cells outside the liver-forming region retain hepatic competence and show that an extrinsic signal, Fgf10a, negatively regulates hepatic competence.
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 ? cell lineage, which is destroyed in individuals with autoimmune diabetes. Some cell types can be reprogrammed using a single factor, whereas other cell types require continuous activity of transcriptional regulators to repress alternate cell fates. Thus, we hypothesized that a transcriptional network continuously maintains the pancreatic acinar cell fate. We found that postembryonic antagonism of Ptf1a, a master regulator of pancreatic development and acinar cell fate specification, 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 microenvironment is sufficient to promote endocrine differentiation. Thus, severe experimental manipulations may not be required to potentiate pancreatic transdifferentiation. These data indicate that targeted postembryonic disruption of the acinar cell fate can restore the developmental plasticity that is lost during development.
CTCF nuclear factor regulates many aspects of gene expression, largely as a transcriptional repressor or via insulator function. Its roles in cellular differentiation are not clear. Here we show an unexpected role for CTCF in myogenesis. Ctcf is expressed in myogenic structures during mouse and zebrafish development. Gain- and loss-of-function approaches in C2C12 cells revealed CTCF as a modulator of myogenesis by regulating muscle-specific gene expression. We addressed the functional connection between CTCF and myogenic regulatory factors (MRFs). CTCF enhances the myogenic potential of MyoD and myogenin and establishes direct interactions with MyoD, indicating that CTCF regulates MRF-mediated muscle differentiation. Indeed, CTCF modulates functional interactions between MyoD and myogenin in co-activation of muscle-specific gene expression and facilitates MyoD recruitment to a muscle-specific promoter. Finally, ctcf loss-of-function experiments in zebrafish embryos revealed a critical role of CTCF in myogenic development and linked CTCF to broader aspects of development via regulation of Wnt signaling. We conclude that CTCF modulates MRF functional interactions in the orchestration of myogenesis.
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.
The cardiac pacemaker controls the rhythmicity of heart contractions and can be substituted by a battery-operated device as a last resort. We created a genetically encoded, optically controlled pacemaker by expressing halorhodopsin and channelrhodopsin in zebrafish cardiomyocytes. Using patterned illumination in a selective plane illumination microscope, we located the pacemaker and simulated tachycardia, bradycardia, atrioventricular blocks, and cardiac arrest. The pacemaker converges to the sinoatrial region during development and comprises fewer than a dozen cells by the time the heart loops. Perturbation of the activity of these cells was entirely reversible, demonstrating the resilience of the endogenous pacemaker. Our studies combine optogenetics and light-sheet microscopy to reveal the emergence of organ function during development.
Cardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cell-autonomously regulates this process.
Electrical cardiac forces have been previously hypothesized to play a significant role in cardiac morphogenesis and remodeling. In response to electrical forces, cultured cardiomyocytes rearrange their cytoskeletal structure and modify their gene expression profile. To translate such in vitro data to the intact heart, we used a collection of zebrafish cardiac mutants and transgenics to investigate whether cardiac conduction could influence in vivo cardiac morphogenesis independent of contractile forces. We show that the cardiac mutant dco(s226) develops heart failure and interrupted cardiac morphogenesis following uncoordinated ventricular contraction. Using in vivo optical mapping/calcium imaging, we determined that the dco cardiac phenotype was primarily due to aberrant ventricular conduction. Because cardiac contraction and intracardiac hemodynamic forces can also influence cardiac development, we further analyzed the dco phenotype in noncontractile hearts and observed that disorganized ventricular conduction could affect cardiomyocyte morphology and subsequent heart morphogenesis in the absence of contraction or flow. By positional cloning, we found that dco encodes Gja3/Cx46, a gap junction protein not previously implicated in heart formation or function. Detailed analysis of the mouse Cx46 mutant revealed the presence of cardiac conduction defects frequently associated with human heart failure. Overall, these in vivo studies indicate that cardiac electrical forces are required to preserve cardiac chamber morphology and may act as a key epigenetic factor in cardiac remodeling.
Liver regeneration has traditionally been investigated in mammalian models. Recent technological developments in mouse genetics have greatly enhanced the resolving power of these studies. In addition, the zebrafish system has emerged as a complementary genetic system to study liver regeneration. One of the most promising attributes of the zebrafish system is its amenability to large-scale screens including genetic and chemical screens. Also, as our understanding of liver development is becoming more detailed, it is important to evaluate the commonalities and differences between organ development and regeneration.
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 [1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine, and vas deferens. Conversely, uncontrolled 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  as well as polycystic kidney disease [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 uncontrolled activation of the CFTR channel. Analyses in zebrafish larvae 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.
Understanding liver development should lead to greater insights into liver diseases and improve therapeutic strategies. In a forward genetic screen for genes regulating liver development in zebrafish, we identified a mutant--oliver--that exhibits liver-specific defects. In oliver mutants, the liver is specified, bile ducts form and hepatocytes differentiate. However, the hepatocytes die shortly after their differentiation, and thus the resulting mutant liver consists mainly of biliary tissue. We identified a mutation in the gene encoding translocase of the outer mitochondrial membrane 22 (Tomm22) as responsible for this phenotype. Mutations in tomm genes have been associated with mitochondrial dysfunction, but most studies on the effect of defective mitochondrial protein translocation have been carried out in cultured cells or unicellular organisms. Therefore, the tomm22 mutant represents an important vertebrate genetic model to study mitochondrial biology and hepatic mitochondrial diseases. We further found that the temporary knockdown of Tomm22 levels by morpholino antisense oligonucleotides causes a specific hepatocyte degeneration phenotype that is reversible: new hepatocytes repopulate the liver as Tomm22 recovers to wild-type levels. The specificity and reversibility of hepatocyte ablation after temporary knockdown of Tomm22 provides an additional model to study liver regeneration, under conditions where most hepatocytes have died. We used this regeneration model to analyze the signaling commonalities between hepatocyte development and regeneration.
The Hedgehog (Hh) signaling pathway differentially utilizes the primary cilium in mammals and fruit flies. Recent work, including a study in BMC Biology, demonstrates that Hh signals through the cilium in zebrafish, clarifying the evolution of Hh signal transduction. See research article: http://www.biomedcentral.com/1741-7007/8/65.
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 a genetic, 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.
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.
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.
All metazoans use insulin to control energy metabolism, but they secrete it from different cells: neurons in the central nervous system in invertebrates and endocrine cells in the gut or pancreas in vertebrates. Despite their origins in different germ layers, all of these insulin-producing cells share common functional features and gene expression patterns. In this study, we tested the role in insulin-producing cells of the vertebrate homologues of Dachshund, a transcriptional regulator that marks the earliest committed progenitors of the neural insulin-producing cells in Drosophila. Both zebrafish and mice expressed a single dominant Dachshund homologue in the pancreatic endocrine lineage, and in both species loss of this homologue reduced the numbers of all islet cell types including the insulin-producing ?-cells. In mice, Dach1 gene deletion left the pancreatic progenitor cells unaltered, but blocked the perinatal burst of proliferation of differentiated ?-cells that normally generates most of the ?-cell mass. In ?-cells, Dach1 bound to the promoter of the cell cycle inhibitor p27Kip1, which constrains ?-cell proliferation. Taken together, these data demonstrate a conserved role for Dachshund homologues in the production of insulin-producing cells.
Extracellular matrix (ECM) remodeling is critical for organogenesis, yet its molecular regulation is poorly understood. In zebrafish, asymmetric migration of the epithelial lateral plate mesoderm (LPM) displaces the gut leftward, allowing correct placement of the liver and pancreas. To observe LPM migration at cellular resolution, we transgenically expressed EGFP under the control of the regulatory sequences of the bHLH transcription factor gene hand2. We found that laminin is distributed along the LPM/gut boundary during gut looping, and that it appears to become diminished by the migrating hand2-expressing cells. Laminin diminishment is necessary for LPM migration and is dependent on matrix metalloproteinase (MMP) activity. Loss of Hand2 function causes reduced MMP activity and prolonged laminin deposition at the LPM/gut boundary, leading to failed asymmetric LPM migration and gut looping. Our study reveals an unexpected role for Hand2, a key regulator of cell specification and differentiation, in modulating ECM remodeling during organogenesis.
A major goal of regenerative medicine is to instruct formation of multipotent, tissue-specific stem cells from induced pluripotent stem cells (iPSCs) for cell replacement therapies. Generation of haematopoietic stem cells (HSCs) from iPSCs or embryonic stem cells (ESCs) is not currently possible, however, necessitating a better understanding of how HSCs normally arise during embryonic development. We previously showed that haematopoiesis occurs through four distinct waves during zebrafish development, with HSCs arising in the final wave in close association with the dorsal aorta. Recent reports have suggested that murine HSCs derive from haemogenic endothelial cells (ECs) lining the aortic floor. Additional in vitro studies have similarly indicated that the haematopoietic progeny of ESCs arise through intermediates with endothelial potential. Here we have used the unique strengths of the zebrafish embryo to image directly the generation of HSCs from the ventral wall of the dorsal aorta. Using combinations of fluorescent reporter transgenes, confocal time-lapse microscopy and flow cytometry, we have identified and isolated the stepwise intermediates as aortic haemogenic endothelium transitions to nascent HSCs. Finally, using a permanent lineage tracing strategy, we demonstrate that the HSCs generated from haemogenic endothelium are the lineal founders of the adult haematopoietic system.
Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life. Yet, there is little or no significant cardiac muscle regeneration after an injury such as acute myocardial infarction. By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced. In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Using new genetic fate-mapping approaches, here we identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through the 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 labelled most 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 fibroblast growth factor receptor inhibition to arrest cardiac regeneration and enable scar formation, experimental release of the signalling 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. These findings have implications for promoting regeneration of the injured human heart.
Bmp signaling has been shown to regulate early aspects of pancreas development, but its role in endocrine, and especially beta-cell, differentiation remains unclear. Taking advantage of the ability in zebrafish embryos to cell-autonomously modulate Bmp signaling in single cells, we examined how Bmp signaling regulates the ability of individual endodermal cells to differentiate into beta-cells. We find that specific temporal windows of Bmp signaling prevent beta-cell differentiation. Thus, future dorsal bud-derived beta-cells are sensitive to Bmp signaling specifically during gastrulation and early somitogenesis stages. In contrast, ventral pancreatic cells, which require an early Bmp signal to form, do not produce beta-cells when exposed to Bmp signaling at 50 hpf, a stage when the ventral bud-derived extrapancreatic duct is the main source of new endocrine cells. Importantly, inhibiting Bmp signaling within endodermal cells via genetic means increased the number of beta-cells, at early and late stages. Moreover, inhibition of Bmp signaling in the late stage embryo using dorsomorphin, a chemical inhibitor of Bmp receptors, significantly increased beta-cell neogenesis near the extrapancreatic duct, demonstrating the feasibility of pharmacological approaches to increase beta-cell numbers. Our in vivo single-cell analyses show that whereas Bmp signaling is necessary initially for formation of the ventral pancreas, differentiating endodermal cells need to be protected from exposure to Bmps during specific stages to permit beta-cell differentiation. These results provide important unique insight into the intercellular signaling environment necessary for in vivo and in vitro generation of beta-cells.
Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexisting vessels (angiogenesis). With high-resolution imaging of zebrafish vascular development, we uncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.
Pancreatic beta-cells are critical regulators of glucose homeostasis, and they vary dramatically in their glucose stimulated metabolic response and levels of insulin secretion. It is unclear whether these parameters are influenced by the developmental origin of individual beta-cells. Using HOTcre, a Cre-based genetic switch that uses heat-induction to precisely control the temporal expression of transgenes, we labeled two populations of beta-cells within the developing zebrafish pancreas. These populations originate in distinct pancreatic buds and exhibit gene expression profiles suggesting distinct functions during development. We find that the dorsal bud derived beta-cells are quiescent and exhibit a marked decrease in insulin expression postembryonically. In contrast, ventral bud derived beta-cells proliferate actively, and maintain high levels of insulin expression compared with dorsal bud derived beta-cells. Therapeutic strategies to regulate beta-cell proliferation and function are required to cure pathological states that result from excessive beta-cell proliferation (e.g., insulinoma) or insufficient beta-cell mass (e.g., diabetes mellitus). Our data reveal the existence of distinct populations of beta-cells in vivo and should help develop better strategies to regulate beta-cell differentiation and proliferation.
Seryl-transfer RNA synthetase (Sars) is one of the 20 aminoacyl-transfer RNA synthetases that are enzymes essential for protein synthesis; however, the developmental function of Sars has not been elucidated. In zebrafish, impairment of zygotic Sars function leads to a significant dilatation of the aortic arch vessels and aberrant branching of cranial and intersegmental vessels. This abnormal vascular branching in sars mutants can be suppressed by a form of Sars that lacks canonical function, indicating that a noncanonical activity of Sars regulates vascular development. Inhibition or knockdown of vascular endothelial growth factor (Vegf) signaling, which plays pivotal roles in the establishment of the vascular network, suppresses the abnormal vascular branching observed in sars mutants. Here, we discuss the possible functional relationship between Sars function and Vegf signaling.
Selective plane illumination microscopy (SPIM) and other fluorescence microscopy techniques in which a focused sheet of light serves to illuminate the sample have become increasingly popular in developmental studies. Fluorescence light-sheet microscopy bridges the gap in image quality between fluorescence stereomicroscopy and high-resolution imaging of fixed tissue sections. In addition, high depth penetration, low bleaching and high acquisition speeds make light-sheet microscopy ideally suited for extended time-lapse experiments in live embryos. This review compares the benefits and challenges of light-sheet microscopy with established fluorescence microscopy techniques such as confocal microscopy and discusses the different implementations and applications of this easily adaptable technology.
Epigenetic regulation of transcriptional silencing is essential for normal development. Despite its importance, in vivo systems for examining gene silencing at cellular resolution have been lacking in developing vertebrates. We describe a transgenic approach that allows monitoring of an epigenetically regulated fluorescent reporter in developing zebrafish and their progeny. Using a self-reporting Gal4-VP16 gene/enhancer trap vector, we isolated tissue-specific drivers that regulate expression of the green fluorescent protein (GFP) gene through a multicopy, upstream activator sequence (UAS). Transgenic larvae initially exhibit robust fluorescence (GFP(high)); however, in subsequent generations, gfp expression is mosaic (GFP(low)) or entirely absent (GFP(off)), despite continued Gal4-VP16 activity. We find that transcriptional repression is heritable and correlated with methylation of the multicopy UAS. Silenced transgenes can be reactivated by increasing Gal4-VP16 levels or in DNA methyltransferase-1 (dnmt1) mutants. Strikingly, in dnmt1 homozygous mutants, reactivation of gfp expression occurs in a reproducible subset of cells, raising the possibility of different sensitivities or alternative silencing mechanisms in discrete cell populations. The results demonstrate the power of the zebrafish system for in vivo monitoring of epigenetic processes using a genetic approach.
In a recent genetic screen, we identified mutations in genes important for vascular development and maintenance in zebrafish (Jin et al. Dev Biol. 2007;307:29-42). Mutations [corrected] at the adrasteia (adr) locus cause a pronounced dilatation of the aortic arch vessels as well as aberrant patterning of the hindbrain capillaries and, to a lesser extent, intersomitic vessels. This dilatation of the aortic arch vessels does not appear to be caused by increased cell proliferation but is dependent on vascular endothelial growth factor (Vegf) signaling. By positional cloning, we isolated seryl-tRNA synthetase (sars) as the gene affected by the adr mutations. Small interfering RNA knockdown experiments in human umbilical vein endothelial cell cultures indicate that SARS also regulates endothelial sprouting. These analyses of zebrafish and human endothelial cells reveal a new noncanonical function of Sars in endothelial development.
Gap junctions form electrical conduits between adjacent myocardial cells, permitting rapid spatial passage of the excitation current essential to each heartbeat. Arrhythmogenic decreases in gap junction coupling are a characteristic of stressed, failing, and aging myocardium, but the mechanisms of decreased coupling are poorly understood. We previously found that microtubules bearing gap junction hemichannels (connexons) can deliver their cargo directly to adherens junctions. The specificity of this delivery requires the microtubule plus-end tracking protein EB1. We performed this study to investigate the hypothesis that the oxidative stress that accompanies acute and chronic ischemic disease perturbs connexon forward trafficking. We found that EB1 was displaced in ischemic human hearts, stressed mouse hearts, and isolated cells subjected to oxidative stress. As a result, we observed limited microtubule interaction with adherens junctions at intercalated discs and reduced connexon delivery and gap junction coupling. A point mutation within the tubulin-binding domain of EB1 reproduced EB1 displacement and diminished connexon delivery, confirming that EB1 displacement can limit gap junction coupling. In zebrafish hearts, oxidative stress also reduced the membrane localization of connexin and slowed the spatial spread of excitation. We anticipate that protecting the microtubule-based forward delivery apparatus of connexons could improve cell-cell coupling and reduce ischemia-related cardiac arrhythmias.
Development and maturation of the nascent cardiovascular system requires the recruitment of mural cells (MCs) around the vascular tree in a process called vascular myogenesis. Understanding the origin and development of vascular MCs has been hampered by difficulties in observing these cells in vivo and performing defined genetic and experimental manipulations in available model organisms. Here, we investigate the origin of vascular MCs using molecular and genetic tools in zebrafish. We show that vascular MCs are present around the lateral dorsal aortae (LDA) and anterior mesenteric arteries (AMA) of developing animals, and that they also contribute to the outflow tract of the developing heart and ventral aorta (VA). Genetic data indicate that the vascular MCs of the LDA and AMA do not arise from blood or endothelial progenitors but from other derivatives of the lateral plate mesoderm. We further show that zebrafish vascular MCs share many of the morphological, molecular and functional characteristics of vascular smooth muscle cells and pericytes found in higher vertebrates. These data establish the zebrafish as a useful cellular and genetic model to study vascular myogenesis as well as tumor angiogenesis and other MC-associated diseases.
Members of the Hedgehog (Hh) family of secreted proteins function as morphogens to pattern developing tissues and control cell proliferation. The seven-transmembrane domain (7TM) protein Smoothened (Smo) is essential for the activation of all levels of Hh signaling. However, the mechanisms by which Smo differentially activates low- or high-level Hh signaling are not known. Here we show that a newly identified mutation in the extracellular domain (ECD) of zebrafish Smo attenuates Smo signaling. The Smo agonist purmorphamine induces the stabilization, ciliary translocation, and high-level signaling of wild-type Smo. In contrast, purmorphamine induces the stabilization but not the ciliary translocation or high-level signaling of the Smo ECD mutant protein. Surprisingly, a truncated form of Smo that lacks the cysteine-rich domain of the ECD localizes to the cilium but is unable to activate high-level Hh signaling. We also present evidence that cilia may be required for Hh signaling in early zebrafish embryos. These data indicate that the ECD, previously thought to be dispensable for vertebrate Smo function, both regulates Smo ciliary localization and is essential for high-level Hh signaling.
Zebrafish mutants generated by ethylnitrosourea-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 78 hours postfertilization (hpf). In this study, we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype.
Recent studies have begun to elucidate how the endothelial lineage is specified from the nascent mesoderm. However, the molecular mechanisms which regulate this process remain largely unknown. We hypothesized that Notch signaling might play an important role in specifying endothelial progenitors from the mesoderm, given that this pathway acts as a bipotential cell-fate switch on equipotent progenitor populations in other settings. We found that zebrafish embryos with decreased levels of Notch signaling exhibited a significant increase in the number of endothelial cells, whereas embryos with increased levels of Notch signaling displayed a reduced number of endothelial cells. Interestingly, there is a concomitant gain of endothelial cells and loss of erythrocytes in embryos with decreased Notch activity, without an effect on cell proliferation or apoptosis. Lineage-tracing analyses indicate that the ectopic endothelial cells in embryos with decreased Notch activity originate from mesodermal cells that normally produce erythrocyte progenitors. Taken together, our data suggest that Notch signaling negatively regulates the number of endothelial cells by limiting the number of endothelial progenitors within the mesoderm, probably functioning as a cell-fate switch between the endothelial and the hematopoietic lineages.
Developmental mechanisms regulating gene expression and the stable acquisition of cell fate direct cytodifferentiation during organogenesis. Moreover, it is likely that such mechanisms could be exploited to repair or regenerate damaged organs. DNA methyltransferases (Dnmts) are enzymes critical for epigenetic regulation, and are used in concert with histone methylation and acetylation to regulate gene expression and maintain genomic integrity and chromosome structure. We carried out two forward genetic screens for regulators of endodermal organ development. In the first, we screened for altered morphology of developing digestive organs, while in the second we screed for the lack of terminally differentiated cell types in the pancreas and liver. From these screens, we identified two mutant alleles of zebrafish dnmt1. Both lesions are predicted to eliminate dnmt1 function; one is a missense mutation in the catalytic domain and the other is a nonsense mutation that eliminates the catalytic domain. In zebrafish dnmt1 mutants, the pancreas and liver form normally, but begin to degenerate after 84 h post fertilization (hpf). Acinar cells are nearly abolished through apoptosis by 100 hpf, though neither DNA replication, nor entry into mitosis is halted in the absence of detectable Dnmt1. However, endocrine cells and ducts are largely spared. Surprisingly, dnmt1 mutants and dnmt1 morpholino-injected larvae show increased capacity for pancreatic beta cell regeneration in an inducible model of pancreatic beta cell ablation. Thus, our data suggest that Dnmt1 is dispensable for pancreatic duct or endocrine cell formation, but not for acinar cell survival. In addition, Dnmt1 may influence the differentiation of pancreatic beta cell progenitors or the reprogramming of cells toward the pancreatic beta cell fate.
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 affect gluconeogenesis in humans as well as metabolically uncharacterized compounds. Most notably, we find that the translocator protein 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, notably, 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.
Over the past 20 years, the zebrafish has emerged as a powerful model organism for studying cardiac development. Its ability to survive without an active circulation and amenability to forward genetics has led to the identification of numerous mutants whose study has helped elucidate new mechanisms in cardiac development. Furthermore, its transparent, externally developing embryos have allowed detailed cellular analyses of heart development. In this review, we discuss the molecular and cellular processes involved in zebrafish heart development from progenitor specification to development of the valve and the conduction system. We focus on imaging studies that have uncovered the cellular bases of heart development and on zebrafish mutants with cardiac abnormalities whose study has revealed novel molecular pathways in cardiac cell specification and tissue morphogenesis.
Formation and remodeling of the vasculature during development and disease involve a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their posttranscriptional dose titration by microRNAs is poorly understood.
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.
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) . 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.
Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.
scotch tape (sco) is a zebrafish cardiac mutant initially proposed to exhibit a reduced amount of cardiac jelly, the extracellular matrix between the myocardial and endocardial layers. We analyzed sco(te382) mutant hearts in detail using both selective plane illumination microscopy (SPIM) and transmission electron microscopy (TEM), and observed a fascinating endocardial defect. Time-lapse SPIM imaging of wild-type and mutant embryos revealed significant and dynamic gaps between endocardial cells during development. Although these gaps close in wild-type animals, they fail to close in the mutants, ultimately leading to a near complete absence of endocardial cells in the atrial chamber by the heart looping stage. TEM analyses confirm the presence of gaps between endocardial cells in sco mutants, allowing the apparent leakage of cardiac jelly into the lumen. High-resolution mapping places the sco(te382) mutation within the fbn2b locus, which encodes the extracellular matrix protein Fibrillin 2b (OMIM ID: 121050). Complementation and further phenotypic analyses confirm that sco is allelic to puff daddy(gw1) (pfd(gw1)), a null mutant in fbn2b, and that sco(te382) is a hypomorphic allele of fbn2b. fbn2b belongs to a family of genes responsible for the assembly of microfibrils throughout development, and is essential for microfibril structural integrity. In sco(te382) mutants, Fbn2b is disabled by a missense mutation in a highly conserved cbEGF domain, which likely interferes with protein folding. Integrating data obtained from microscopy and molecular biology, we posit that this mutation impacts the rigidity of Fbn2b, imparting a structural defect that weakens endocardial adhesion thereby resulting in perforated endocardium.
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, sox9b(fh313), to dissect its function in the morphogenesis of this structure. Strikingly, sox9b(fh313) homozygous mutants survive to adulthood and exhibit cholestasis associated with hepatic and pancreatic duct proliferation, cyst formation, and fibrosis. Analysis of sox9b(fh313) 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 sox9b(fh313) mutants, while the embryonic endocrine and acinar compartments appear unaffected. The defects in the intrahepatic and intrapancreatic ducts of sox9b(fh313) 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.
Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing ? cells is still needed. Using a zebrafish model of diabetes, we screened ~7,000 small molecules to identify enhancers of ? 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 ? cell regeneration was the adenosine agonist 5-N-ethylcarboxamidoadenosine (NECA), which, acting through the adenosine receptor A2aa, increased ? cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating ? 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 ? cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes.
Genetic studies have implicated Notch signaling in the maintenance of pancreatic progenitors. However, how Notch signaling regulates the quiescent, proliferative or differentiation behaviors of pancreatic progenitors at the single-cell level remains unclear. Here, using single-cell genetic analyses and a new transgenic system that allows dynamic assessment of Notch signaling, we address how discrete levels of Notch signaling regulate the behavior of endocrine progenitors in the zebrafish intrapancreatic duct. We find that these progenitors experience different levels of Notch signaling, which in turn regulate distinct cellular outcomes. High levels of Notch signaling induce quiescence, whereas lower levels promote progenitor amplification. The sustained downregulation of Notch signaling triggers a multistep process that includes cell cycle entry and progenitor amplification prior to endocrine differentiation. Importantly, progenitor amplification and differentiation can be uncoupled by modulating the duration and/or extent of Notch signaling downregulation, indicating that these processes are triggered by distinct levels of Notch signaling. These data show that different levels of Notch signaling drive distinct behaviors in a progenitor population.
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