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1.
Development ; 151(13)2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38940292

ABSTRACT

During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall. Llgl1 regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation. We found that llgl1 mutant zebrafish embryos exhibit aberrant apical extrusion of ventricular cardiomyocytes. While investigating apical cardiomyocyte extrusion, we identified a basal-to-apical shift in laminin deposition from the internal to the external ventricular wall. We find that epicardial cells express several laminin subunits as they adhere to the ventricle, and that the epicardium is required for laminin deposition on the ventricular surface. In llgl1 mutants, timely establishment of the epicardial layer is disrupted due to delayed emergence of epicardial cells, resulting in delayed apical deposition of laminin on the ventricular surface. Together, our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the ventricular myocardial wall.


Subject(s)
Heart Ventricles , Laminin , Pericardium , Zebrafish Proteins , Zebrafish , Animals , Laminin/metabolism , Laminin/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Pericardium/metabolism , Pericardium/embryology , Pericardium/cytology , Heart Ventricles/metabolism , Heart Ventricles/embryology , Zebrafish Proteins/metabolism , Zebrafish Proteins/genetics , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Cell Polarity , Mutation/genetics
2.
Curr Top Dev Biol ; 156: 121-156, 2024.
Article in English | MEDLINE | ID: mdl-38556421

ABSTRACT

During human embryonic development the early establishment of a functional heart is vital to support the growing fetus. However, forming the embryonic heart is an extremely complex process, requiring spatiotemporally controlled cell specification and differentiation, tissue organization, and coordination of cardiac function. These complexities, in concert with the early and rapid development of the embryonic heart, mean that understanding the intricate interplay between these processes that help shape the early heart remains highly challenging. In this review I focus on recent insights from animal models that have shed new light on the earliest stages of heart development. This includes specification and organization of cardiac progenitors, cell and tissue movements that make and shape the early heart tube, and the initiation of the first beat in the developing heart. In addition I highlight relevant in vitro models that could support translation of findings from animal models to human heart development. Finally I discuss challenges that are being addressed in the field, along with future considerations that together may help move us towards a deeper understanding of how our hearts are made.


Subject(s)
Heart , Animals , Cell Differentiation
3.
Br J Pharmacol ; 179(5): 900-917, 2022 03.
Article in English | MEDLINE | ID: mdl-33788282

ABSTRACT

Mammalian models including non-human primates, pigs and rodents have been used extensively to study the mechanisms of cardiovascular disease. However, there is an increasing desire for alternative model systems that provide excellent scientific value while replacing or reducing the use of mammals. Here, we review the use of zebrafish, Danio rerio, to study cardiovascular development and disease. The anatomy and physiology of zebrafish and mammalian cardiovascular systems are compared, and we describe the use of zebrafish models in studying the mechanisms of cardiac (e.g. congenital heart defects, cardiomyopathy, conduction disorders and regeneration) and vascular (endothelial dysfunction and atherosclerosis, lipid metabolism, vascular ageing, neurovascular physiology and stroke) pathologies. We also review the use of zebrafish for studying pharmacological responses to cardiovascular drugs and describe several features of zebrafish that make them a compelling model for in vivo screening of compounds for the treatment cardiovascular disease. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.


Subject(s)
Cardiovascular Agents , Cardiovascular Diseases , Stroke , Aging , Animals , Cardiovascular Agents/therapeutic use , Cardiovascular Diseases/drug therapy , Humans , Mammals , Swine , Zebrafish
4.
Cardiovasc Res ; 118(1): 226-240, 2022 01 07.
Article in English | MEDLINE | ID: mdl-33616638

ABSTRACT

AIMS: Vertebrate heart development requires the complex morphogenesis of a linear tube to form the mature organ, a process essential for correct cardiac form and function, requiring coordination of embryonic laterality, cardiac growth, and regionalized cellular changes. While previous studies have demonstrated broad requirements for extracellular matrix (ECM) components in cardiac morphogenesis, we hypothesized that ECM regionalization may fine tune cardiac shape during heart development. METHODS AND RESULTS: Using live in vivo light sheet imaging of zebrafish embryos, we describe a left-sided expansion of the ECM between the myocardium and endocardium prior to the onset of heart looping and chamber ballooning. Analysis using an ECM sensor revealed the cardiac ECM is further regionalized along the atrioventricular axis. Spatial transcriptomic analysis of gene expression in the heart tube identified candidate genes that may drive ECM expansion. This approach identified regionalized expression of hapln1a, encoding an ECM cross-linking protein. Validation of transcriptomic data by in situ hybridization confirmed regionalized hapln1a expression in the heart, with highest levels of expression in the future atrium and on the left side of the tube, overlapping with the observed ECM expansion. Analysis of CRISPR-Cas9-generated hapln1a mutants revealed a reduction in atrial size and reduced chamber ballooning. Loss-of-function analysis demonstrated that ECM expansion is dependent upon Hapln1a, together supporting a role for Hapln1a in regionalized ECM modulation and cardiac morphogenesis. Analysis of hapln1a expression in zebrafish mutants with randomized or absent embryonic left-right asymmetry revealed that laterality cues position hapln1a-expressing cells asymmetrically in the left side of the heart tube. CONCLUSION: We identify a regionalized ECM expansion in the heart tube which promotes correct heart development, and propose a novel model whereby embryonic laterality cues orient the axis of ECM asymmetry in the heart, suggesting these two pathways interact to promote robust cardiac morphogenesis.


Subject(s)
Extracellular Matrix Proteins/metabolism , Extracellular Matrix/metabolism , Heart/embryology , Morphogenesis , Myocardium/metabolism , Proteoglycans/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Body Patterning , Extracellular Matrix/genetics , Extracellular Matrix Proteins/genetics , Gene Expression Regulation, Developmental , Hyaluronic Acid/metabolism , Mutation , Proteoglycans/genetics , Signal Transduction , Transcriptome , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics
5.
J Matern Fetal Neonatal Med ; 35(25): 7353-7359, 2022 Dec.
Article in English | MEDLINE | ID: mdl-34304671

ABSTRACT

BACKGROUND: Postpartum hemorrhage (PPH) is the most common cause of maternal mortality worldwide. Predicting PPH remains difficult, and risk factors vary among populations. We aimed to determine prevalence, risk factors, and causes for PPH in our obstetric population in South-Central Louisiana. METHODS: We conducted a retrospective cohort study utilizing medical records for deliveries between October 2015 and September 2020 at Woman's Hospital, a tertiary hospital in South-Central Louisiana. PPH was defined by the current American College of Obstetricians and Gynecologists' (ACOG) criteria as cumulative blood loss greater than or equal to 1000 mL within 24 h after the birth process regardless of route of delivery. Logistic regression assessed the association of PPH and possible risk factors: anemia at the time of delivery, race, parity, delivery mode, body mass index, age, and health insurance. An additional logistic regression also investigated risk factors within our cohort for severe maternal morbidity among patients who experienced PPH including the same covariates. RESULTS: A total of 30,674 deliveries were included in our cohort, among which PPH occurred in 12.3% (n = 3773). Patients experiencing PPH were more likely to be of Black race, Medicaid-eligible, deliver via cesarean section, and have lower hemoglobin and hematocrit at time of delivery compared to patients without PPH (all p < .001). Anemia at delivery (aOR = 1.28; 95%CI = 1.154-1.419), cesarean delivery (aOR = 8.796; 95%CI = 7.731-10.007), BMI > 40kg/m2 (aOR = 1.363; 95%CI = 1.186-1.567), and Black race (aOR = 1.233; 95%CI = 1.099-1.383) were the strongest predictors of PPH. Among cesarean cases (n = 10,888), Black race and BMI > 40 kg/m2 were the strongest predictors for PPH. Among patients who experienced PPH, anemia was associated with a higher likelihood of experiencing a severe maternal morbidity event (aOR = 2.587; 95%CI = 1.990-3.364). CONCLUSION: Consistent with literature in the United States, Black race, increased BMI, cesarean delivery, and anemia were associated with risk of PPH. Anemia at delivery increased the risk for severe maternal morbidity among patients experiencing PPH.


Subject(s)
Anemia , Postpartum Hemorrhage , Humans , Pregnancy , Female , Postpartum Hemorrhage/epidemiology , Postpartum Hemorrhage/etiology , Cesarean Section/adverse effects , Retrospective Studies , Tertiary Care Centers , Risk Factors , Anemia/epidemiology , Anemia/complications
6.
Development ; 148(20)2021 10 15.
Article in English | MEDLINE | ID: mdl-34568948

ABSTRACT

During early vertebrate heart development, the heart transitions from a linear tube to a complex asymmetric structure, a morphogenetic process that occurs simultaneously with growth of the heart. Cardiac growth during early heart morphogenesis is driven by deployment of cells from the second heart field (SHF) into both poles of the heart. Laminin is a core component of the extracellular matrix and, although mutations in laminin subunits are linked with cardiac abnormalities, no role for laminin has been identified in early vertebrate heart morphogenesis. We identified tissue-specific expression of laminin genes in the developing zebrafish heart, supporting a role for laminins in heart morphogenesis. Analysis of heart development in lamb1a zebrafish mutant embryos reveals mild morphogenetic defects and progressive cardiomegaly, and that Lamb1a functions to limit heart size during cardiac development by restricting SHF addition. lamb1a mutants exhibit hallmarks of altered haemodynamics, and blocking cardiac contractility in lamb1a mutants rescues heart size and atrial SHF addition. Together, these results suggest that laminin mediates interactions between SHF deployment and cardiac biomechanics during heart morphogenesis and growth in the developing embryo.


Subject(s)
Heart Atria/metabolism , Heart/physiology , Laminin/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Cell Lineage/physiology , Gene Expression Regulation, Developmental/physiology , Heart Defects, Congenital/metabolism , Morphogenesis/physiology , Myocardium/metabolism , Organogenesis/physiology
7.
Development ; 148(5)2021 03 05.
Article in English | MEDLINE | ID: mdl-33674261

ABSTRACT

The developing heart is formed of two tissue layers separated by an extracellular matrix (ECM) that provides chemical and physical signals to cardiac cells. While deposition of specific ECM components creates matrix diversity, the cardiac ECM is also dynamic, with modification and degradation playing important roles in ECM maturation and function. In this Review, we discuss the spatiotemporal changes in ECM composition during cardiac development that support distinct aspects of heart morphogenesis. We highlight conserved requirements for specific ECM components in human cardiac development, and discuss emerging evidence of a central role for the ECM in promoting heart regeneration.


Subject(s)
Extracellular Matrix/metabolism , Heart/growth & development , Animals , Extracellular Matrix Proteins/metabolism , Heart/physiology , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Humans , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Organogenesis , Pericardium/metabolism , Regeneration
9.
Elife ; 62017 11 28.
Article in English | MEDLINE | ID: mdl-29179812

ABSTRACT

Computational modelling of the heart tube during development reveals the interplay between tissue asymmetry and growth that helps our hearts take shape.


Subject(s)
Body Patterning , Imaging, Three-Dimensional , Animals , Embryonic Development , Heart , Mice , Morphogenesis , Organogenesis
10.
Biol Open ; 5(10): 1461-1472, 2016 Oct 15.
Article in English | MEDLINE | ID: mdl-27612508

ABSTRACT

Cadherin complexes mediate cell-cell adhesion and are crucial for embryonic development. Besides their structural function, cadherin complexes also transduce tension across the junction-actomyosin axis into proportional biochemical responses. Central to this mechanotransduction is the stretching of the cadherin-F-actin-linker α-catenin, which opens its central domain for binding to effectors such as vinculin. Mechanical unfolding of α-catenin leads to force-dependent reinforcement of cadherin-based junctions as studied in cell culture. The importance of cadherin mechanotransduction for embryonic development has not been studied yet. Here we used TALEN-mediated gene disruption to perturb endogenous αE-catenin in zebrafish development. Zygotic α-catenin mutants fail to maintain their epithelial barrier, resulting in tissue rupturing. We then specifically disrupted mechanotransduction, while maintaining cadherin adhesion, by expressing an αE-catenin construct in which the mechanosensitive domain was perturbed. Expression of either wild-type or mechano-defective α-catenin fully rescues barrier function in α-catenin mutants; however, expression of mechano-defective α-catenin also induces convergence and extension defects. Specifically, the polarization of cadherin-dependent, lamellipodia-driven cell migration of the lateral mesoderm was lost. These results indicate that cadherin mechanotransduction is crucial for proper zebrafish morphogenesis, and uncover one of the essential processes affected by its perturbation.

11.
Dev Cell ; 36(1): 36-49, 2016 Jan 11.
Article in English | MEDLINE | ID: mdl-26748692

ABSTRACT

In contrast to mammals, zebrafish regenerate heart injuries via proliferation of cardiomyocytes located near the wound border. To identify regulators of cardiomyocyte proliferation, we used spatially resolved RNA sequencing (tomo-seq) and generated a high-resolution genome-wide atlas of gene expression in the regenerating zebrafish heart. Interestingly, we identified two wound border zones with distinct expression profiles, including the re-expression of embryonic cardiac genes and targets of bone morphogenetic protein (BMP) signaling. Endogenous BMP signaling has been reported to be detrimental to mammalian cardiac repair. In contrast, we find that genetic or chemical inhibition of BMP signaling in zebrafish reduces cardiomyocyte dedifferentiation and proliferation, ultimately compromising myocardial regeneration, while bmp2b overexpression is sufficient to enhance it. Our results provide a resource for further studies on the molecular regulation of cardiac regeneration and reveal intriguing differential cellular responses of cardiomyocytes to a conserved signaling pathway in regenerative versus non-regenerative hearts.


Subject(s)
Bone Morphogenetic Proteins/genetics , Gene Expression Regulation/genetics , Genome/genetics , Myocytes, Cardiac/metabolism , Regeneration/physiology , Signal Transduction/genetics , Zebrafish/metabolism , Animals , Bone Morphogenetic Proteins/metabolism , Cell Differentiation , Cell Proliferation/genetics , Myocytes, Cardiac/cytology , Zebrafish/genetics , Zebrafish Proteins/metabolism
12.
Hum Mutat ; 37(2): 194-200, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26531781

ABSTRACT

Establishing correct left-right asymmetry during embryonic development is crucial for proper asymmetric positioning of the organs. Congenital heart defects, such as dextrocardia, transposition of the arteries, and inflow or outflow tract malformations, comprise some of the most common birth defects and may be attributed to incorrect establishment of body laterality. Here, we identify new patients with dextrocardia who have mutations in CFAP53, a coiled-coil domain containing protein. To elucidate the mechanism by which CFAP53 regulates embryonic asymmetry, we used genome editing to generate cfap53 zebrafish mutants. Zebrafish cfap53 mutants have specific defects in organ laterality and randomization of asymmetric gene expression. We show that cfap53 is required for cilia rotation specifically in Kupffer's vesicle, the zebrafish laterality organ, providing a mechanism by which patients with CFAP53 mutations develop dextrocardia and heterotaxy, and confirming previous evidence that left-right asymmetry in humans is regulated through cilia-driven fluid flow in a laterality organ.


Subject(s)
Cytoskeletal Proteins/genetics , Dextrocardia/genetics , Heterotaxy Syndrome/genetics , Mutation , Zebrafish Proteins/genetics , Zebrafish/genetics , Animals , Base Sequence , Body Patterning/genetics , Cilia/metabolism , Cilia/pathology , Conserved Sequence , Cytoskeletal Proteins/metabolism , DNA Mutational Analysis , Dextrocardia/metabolism , Dextrocardia/pathology , Embryo, Nonmammalian , Embryonic Development/genetics , Female , Gene Expression , Heterotaxy Syndrome/metabolism , Heterotaxy Syndrome/pathology , Humans , Lateral Line System/embryology , Lateral Line System/metabolism , Male , Molecular Sequence Data , Pedigree , Siblings , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/metabolism
13.
Dev Cell ; 32(5): 631-9, 2015 Mar 09.
Article in English | MEDLINE | ID: mdl-25684355

ABSTRACT

Tissue patterning is established by extracellular growth factors or morphogens. Although different theoretical models explaining specific patterns have been proposed, our understanding of tissue pattern establishment in vivo remains limited. In many animal species, left-right patterning is governed by a reaction-diffusion system relying on the different diffusivity of an activator, Nodal, and an inhibitor, Lefty. In a genetic screen, we identified a zebrafish loss-of-function mutant for the proprotein convertase FurinA. Embryological and biochemical experiments demonstrate that cleavage of the Nodal-related Spaw proprotein into a mature form by FurinA is required for Spaw gradient formation and activation of Nodal signaling. We demonstrate that FurinA is required cell-autonomously for the long-range signaling activity of Spaw and no other Nodal-related factors. Combined in silico and in vivo approaches support a model in which FurinA controls the signaling range of Spaw by cleaving its proprotein into a mature, extracellular form, consequently regulating left-right patterning.


Subject(s)
Left-Right Determination Factors/metabolism , Nodal Protein/metabolism , Proprotein Convertases/metabolism , Signal Transduction , Zebrafish Proteins/metabolism , Zebrafish/growth & development , Zebrafish/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Body Patterning/physiology , Fluorescent Antibody Technique , In Situ Hybridization , Mesoderm/cytology , Mesoderm/metabolism , Molecular Sequence Data , RNA, Messenger/genetics , Sequence Homology, Amino Acid
14.
Cell ; 159(3): 662-75, 2014 Oct 23.
Article in English | MEDLINE | ID: mdl-25417113

ABSTRACT

Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.


Subject(s)
Embryo, Nonmammalian/metabolism , Gene Expression Profiling , Sequence Analysis, RNA , Tomography/methods , Zebrafish/embryology , Animals , Imaging, Three-Dimensional
15.
Development ; 141(9): 1961-70, 2014 May.
Article in English | MEDLINE | ID: mdl-24718990

ABSTRACT

Germline mutations in PTPN11, encoding Shp2, cause Noonan syndrome (NS) and LEOPARD syndrome (LS), two developmental disorders that are characterized by multiple overlapping symptoms. Interestingly, Shp2 catalytic activity is enhanced by NS mutations and reduced by LS mutations. Defective cardiac development is a prominent symptom of both NS and LS, but how the Shp2 variants affect cardiac development is unclear. Here, we have expressed the most common NS and LS Shp2-variants in zebrafish embryos to investigate their role in cardiac development in vivo. Heart function was impaired in embryos expressing NS and LS variants of Shp2. The cardiac anomalies first occurred during elongation of the heart tube and consisted of reduced cardiomyocyte migration, coupled with impaired leftward heart displacement. Expression of specific laterality markers was randomized in embryos expressing NS and LS variants of Shp2. Ciliogenesis and cilia function in Kupffer's vesicle was impaired, likely accounting for the left/right asymmetry defects. Mitogen-activated protein kinase (MAPK) signaling was activated to a similar extent in embryos expressing NS and LS Shp2 variants. Interestingly, inhibition of MAPK signaling prior to gastrulation rescued cilia length and heart laterality defects. These results suggest that NS and LS Shp2 variant-mediated hyperactivation of MAPK signaling leads to impaired cilia function in Kupffer's vesicle, causing left-right asymmetry defects and defective early cardiac development.


Subject(s)
Heart Defects, Congenital/genetics , LEOPARD Syndrome/genetics , Mutation/genetics , Noonan Syndrome/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Animals , Benzamides/pharmacology , Body Patterning/drug effects , Cell Movement/drug effects , Cilia/drug effects , Cilia/metabolism , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/enzymology , Embryo, Nonmammalian/pathology , Heart Defects, Congenital/embryology , Heart Defects, Congenital/physiopathology , Heart Function Tests , Humans , LEOPARD Syndrome/enzymology , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Noonan Syndrome/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Zebrafish/metabolism , Zebrafish Proteins/metabolism
16.
Nat Commun ; 4: 2754, 2013.
Article in English | MEDLINE | ID: mdl-24212328

ABSTRACT

Breaking left-right symmetry in bilateria is a major event during embryo development that is required for asymmetric organ position, directional organ looping and lateralized organ function in the adult. Asymmetric expression of Nodal-related genes is hypothesized to be the driving force behind regulation of organ laterality. Here we identify a Nodal-independent mechanism that drives asymmetric heart looping in zebrafish embryos. In a unique mutant defective for the Nodal-related southpaw gene, preferential dextral looping in the heart is maintained, whereas gut and brain asymmetries are randomized. As genetic and pharmacological inhibition of Nodal signalling does not abolish heart asymmetry, a yet undiscovered mechanism controls heart chirality. This mechanism is tissue intrinsic, as explanted hearts maintain ex vivo retain chiral looping behaviour and require actin polymerization and myosin II activity. We find that Nodal signalling regulates actin gene expression, supporting a model in which Nodal signalling amplifies this tissue-intrinsic mechanism of heart looping.


Subject(s)
Body Patterning/physiology , Embryo, Nonmammalian/anatomy & histology , Gene Expression Regulation, Developmental/physiology , Heart/embryology , Nodal Protein/metabolism , Zebrafish/embryology , Actins/physiology , Actomyosin/physiology , Animals , Embryo, Nonmammalian/physiology , Mutation , Nodal Protein/genetics , Signal Transduction/physiology
17.
PLoS Genet ; 7(9): e1002289, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21980297

ABSTRACT

In vertebrates, left-right (LR) axis specification is determined by a ciliated structure in the posterior region of the embryo. Fluid flow in this ciliated structure is responsible for the induction of unilateral left-sided Nodal activity in the lateral plate mesoderm, which in turn regulates organ laterality. Bmp signalling activity has been implied in repressing Nodal expression on the right side, however its mechanism of action has been controversial. In a forward genetic screen for mutations that affect LR patterning, we identified the zebrafish linkspoot (lin) mutant, characterized by cardiac laterality and mild dorsoventral patterning defects. Mapping of the lin mutation revealed an inactivating missense mutation in the Bmp receptor 1aa (bmpr1aa) gene. Embryos with a mutation in lin/bmpr1aa and a novel mutation in its paralogue, bmpr1ab, displayed a variety of dorsoventral and LR patterning defects with increasing severity corresponding with a decrease in bmpr1a dosage. In Bmpr1a-deficient embryos we observed bilateral expression of the Nodal-related gene, spaw, coupled with reduced expression of the Nodal-antagonist lefty1 in the midline. Using genetic models to induce or repress Bmp activity in combination with Nodal inhibition or activation, we found that Bmp and Nodal regulate lefty1 expression in the midline independently of each other. Furthermore, we observed that the regulation of lefty1 by Bmp signalling is required for its observed downregulation of Nodal activity in the LPM providing a novel explanation for this phenomenon. From these results we propose a two-step model in which Bmp regulates LR patterning. Prior to the onset of nodal flow and Nodal activation, Bmp is required to induce lefty1 expression in the midline. When nodal flow has been established and Nodal activity is apparent, both Nodal and Bmp independently are required for lefty1 expression to assure unilateral Nodal activation and correct LR patterning.


Subject(s)
Bone Morphogenetic Protein Receptors, Type I/genetics , Bone Morphogenetic Proteins/genetics , Embryonic Development/genetics , Left-Right Determination Factors/genetics , Nodal Protein/genetics , Zebrafish Proteins/metabolism , Zebrafish/growth & development , Activin Receptors, Type I/genetics , Activin Receptors, Type I/metabolism , Animals , Body Patterning/genetics , Bone Morphogenetic Protein Receptors, Type I/metabolism , Bone Morphogenetic Proteins/metabolism , Left-Right Determination Factors/metabolism , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutation, Missense , Nodal Protein/metabolism , Signal Transduction , Zebrafish/genetics , Zebrafish Proteins/genetics
18.
Methods Cell Biol ; 104: 401-28, 2011.
Article in English | MEDLINE | ID: mdl-21924175

ABSTRACT

The generation of complex organisms requires that an initial population of cells with identical gene expression profiles can adopt different cell fates during development by progressively diverging transcriptional programs. These programs depend on the binding of transcritional regulators to specific genomic sites, which in turn is controlled by modifications of the chromatin. Chromatin modifications may occur directly upon DNA by methylation of specific nucleotides, or may involve post-translational modification of histones. Local regulation of histone post-translational modifications regionalizes the genome into euchromatic regions, which are more accessible to DNA-binding factors, and condensed heterochromatic regions, inhibiting the binding of such factors. In addition, these modifications may be required in a genome-wide fashion for processes such as DNA replication or chromosome condensation. From an embryologist's point of view chromatin modifications are intensively studied in the context of imprinting and have more recently received increasing attention in understanding the basis of pluripotency and cellular differentiation. Here, we describe recently uncovered roles of chromatin modifications in zebrafish development and regeneration, as well as available resources and commonly used techniques. We provide a general introduction into chromatin modifications and their respective functions with a focus on gene transcription, as well as key aspects of their roles in the early zebrafish embryo, neural development, formation of the digestive system and tissue regeneration.


Subject(s)
Chromatin Assembly and Disassembly , Gene Expression Regulation, Developmental , Zebrafish/growth & development , Acetylation , Animals , Body Patterning/genetics , Chromatin Immunoprecipitation , DNA Methylation , Digestive System/growth & development , Fish Proteins/genetics , Fish Proteins/metabolism , Histone Acetyltransferases/metabolism , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Larva/genetics , Larva/growth & development , Mutation , Nervous System/growth & development , Regeneration/genetics , Sequence Analysis, DNA/methods , Transcription, Genetic , Zebrafish/genetics , Zebrafish/metabolism , Zygote/growth & development
19.
Gene Expr Patterns ; 10(6): 237-43, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20471496

ABSTRACT

Members of the Albumin/alpha-Fetoprotein/Afamin/Group specific component (Alb/Afp/Afm/Gc) multi-gene family perform physiological functions essential for body homeostasis and are among the earliest genes to be expressed in the fetal liver in mammals. A comprehensive search of the zebrafish genome has led to the isolation of a single member of this gene family, exhibiting close homology to group specific component (gc; also described as vitamin D binding protein (dbp)). Our phylogenetic analyses did not uncover albumin in the genome, indicating its likely absence in zebrafish, whereas the absence of afp and afm is in agreement with previous findings that both genes arose at a later stage of vertebrate evolution. gc mRNA expression is initiated weakly around 55 hours post fertilisation (hpf) in the developing liver, and increases until it reaches a continuously high level from about 72 hpf onwards. Investigation of gc mRNA in hdac1 mutants revealed a severe delay of expression, indicating a defect in progression of hepatic differentiation. This provides further evidence for Hdac1 regulating the precise timely execution of hepatic gene expression programmes. Conversely, onset of gc expression was unaltered in cloche mutant embryos, which lack hepatic vasculature, suggesting that this particular step of hepatic differentiation occurs independently from endothelial cells. Our studies identify gc as the likely only member of the Alb/Afp/Afm/Gc gene family in zebrafish, providing important insights into the evolution of this multi-gene family in vertebrates. Furthermore, the identification of gc adds a valuable temporal marker for investigating progressive hepatic differentiation in zebrafish.


Subject(s)
Albumins/genetics , Liver/embryology , Vitamin D-Binding Protein/genetics , Zebrafish/embryology , alpha-Fetoproteins/genetics , Albumins/analysis , Albumins/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Cell Differentiation/genetics , Embryo, Nonmammalian , Gene Expression Profiling , Liver/metabolism , Models, Biological , Molecular Sequence Data , Multigene Family , Phylogeny , Sequence Homology, Amino Acid , Serum Albumin/analysis , Serum Albumin/genetics , Serum Albumin/metabolism , Vitamin D-Binding Protein/analysis , Vitamin D-Binding Protein/metabolism , Zebrafish/genetics , alpha-Fetoproteins/analysis , alpha-Fetoproteins/metabolism
20.
Dev Biol ; 322(2): 237-50, 2008 Oct 15.
Article in English | MEDLINE | ID: mdl-18687323

ABSTRACT

Liver, pancreas and lung originate from the presumptive foregut in temporal and spatial proximity. This requires precisely orchestrated transcriptional activation and repression of organ-specific gene expression within the same cell. Here, we show distinct roles for the chromatin remodelling factor and transcriptional repressor Histone deacetylase 1 (Hdac1) in endodermal organogenesis in zebrafish. Loss of Hdac1 causes defects in timely liver specification and in subsequent differentiation. Mosaic analyses reveal a cell-autonomous requirement for hdac1 within the hepatic endoderm. Our studies further reveal specific functions for Hdac1 in pancreas development. Loss of hdac1 causes the formation of ectopic endocrine clusters anteriorly to the main islet, as well as defects in exocrine pancreas specification and differentiation. In addition, we observe defects in extrahepatopancreatic duct formation and morphogenesis. Finally, loss of hdac1 results in an expansion of the foregut endoderm in the domain from which the liver and pancreas originate. Our genetic studies demonstrate that Hdac1 is crucial for regulating distinct steps in endodermal organogenesis. This suggests a model in which Hdac1 may directly or indirectly restrict foregut fates while promoting hepatic and exocrine pancreatic specification and differentiation, as well as pancreatic endocrine islet morphogenesis. These findings establish zebrafish as a tractable system to investigate chromatin remodelling factor functions in controlling gene expression programmes in vertebrate endodermal organogenesis.


Subject(s)
Histone Deacetylases/metabolism , Liver/embryology , Pancreas/embryology , Zebrafish Proteins/metabolism , Zebrafish/embryology , Amino Acid Sequence , Animals , Cell Differentiation/physiology , Cell Proliferation , Endoderm/embryology , Hepatocytes/cytology , Hepatocytes/physiology , Histone Deacetylase 1 , Histone Deacetylases/genetics , Liver/enzymology , Lung/embryology , Lung/enzymology , Molecular Sequence Data , Mutation , Organ Specificity , Pancreas/enzymology , Zebrafish/metabolism , Zebrafish Proteins/genetics
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