Cloche Mutants Research Articles

Endoderm-derived islet1-expressing cells differentiate into endothelial cells to function as the vascular HSPC niche in zebrafish.

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

Pharyngeal pouches provide a niche microenvironment for arch artery progenitor specification

ABSTRACTThe paired pharyngeal arch arteries (PAAs) are transient blood vessels connecting the heart with the dorsal aorta during embryogenesis. Although PAA malformations often occur along with pharyngeal pouch defects, the functional interaction between these adjacent tissues remains largely unclear. Here, we report that pharyngeal pouches are essential for PAA progenitor specification in zebrafish embryos. We reveal that the segmentation of pharyngeal pouches coincides spatiotemporally with the emergence of PAA progenitor clusters. These pouches physically associate with pharyngeal mesoderm in discrete regions and provide a niche microenvironment for PAA progenitor commitment by expressing BMP proteins. Specifically, pouch-derived BMP2a and BMP5 are the primary niche cues responsible for activating the BMP/Smad pathway in pharyngeal mesoderm, thereby promoting progenitor specification. In addition, BMP2a and BMP5 play an inductive function in the expression of the cloche gene npas4l in PAA progenitors. cloche mutants exhibit a striking failure to specify PAA progenitors and display ectopic expression of head muscle markers in the pharyngeal mesoderm. Therefore, our results support a crucial role for pharyngeal pouches in establishing a progenitor niche for PAA morphogenesis via BMP2a/5 expression.

The hyaloid vasculature facilitates basement membrane breakdown during choroid fissure closure in the zebrafish eye

A critical aspect of vertebrate eye development is closure of the choroid fissure (CF). Defects in CF closure result in colobomas, which are a significant cause of childhood blindness worldwide. Despite the growing number of mutated loci associated with colobomas, we have a limited understanding of the cell biological underpinnings of CF closure. Here, we utilize the zebrafish embryo to identify key phases of CF closure and regulators of the process. Utilizing Laminin-111 as a marker for the basement membrane (BM) lining the CF, we determine the spatial and temporal patterns of BM breakdown in the CF, a prerequisite for CF closure. Similarly, utilizing a combination of in vivo time-lapse imaging, β-catenin immunohistochemistry and F-actin staining, we determine that tissue fusion, which serves to close the fissure, follows BM breakdown closely. Periocular mesenchyme (POM)-derived endothelial cells, which migrate through the CF to give rise to the hyaloid vasculature, possess distinct actin foci that correlate with regions of BM breakdown. Disruption of talin1, which encodes a regulator of the actin cytoskeleton, results in colobomas and these correlate with structural defects in the hyaloid vasculature and defects in BM breakdown. cloche mutants, which entirely lack a hyaloid vasculature, also possess defects in BM breakdown in the CF. Taken together, these data support a model in which the hyaloid vasculature and/or the POM-derived endothelial cells that give rise to the hyaloid vasculature contribute to BM breakdown during CF closure.

Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.

Renin expression in developing zebrafish is associated with angiogenesis and requires the Notch pathway and endothelium.

Although renin is a critical regulatory enzyme of the cardiovascular system, its roles in organogenesis and the establishment of cardiovascular homeostasis remain unclear. Mammalian renin-expressing cells are widespread in embryonic kidneys but are highly restricted, specialized endocrine cells in adults. With a functional pronephros, embryonic zebrafish are ideal for delineating the developmental functions of renin-expressing cells and the mechanisms governing renin transcription. Larval zebrafish renin expression originates in the mural cells of the juxtaglomerular anterior mesenteric artery and subsequently at extrarenal sites. The role of renin was determined by assessing responses to renin-angiotensin system blockade, salinity variation, and renal perfusion ablation. Renin expression did not respond to renal flow ablation but was modulated by inhibition of angiotensin-converting enzyme and altered salinity. Our data in larval fish are consistent with conservation of renin's physiological functions. Using transgenic renin reporter fish, with mindbomb and cloche mutants, we show that Notch signaling and the endothelium are essential for developmental renin expression. After inhibition of angiogenesis, renin-expressing cells precede angiogenic sprouts. Arising from separate lineages, but relying on mutual interplay with endothelial cells, renin-expressing cells are among the earliest mural cells observed in larval fish, performing both endocrine and paracrine functions.

Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.

Neurovascular development in the embryonic zebrafish hindbrain

The brain is made of billions of highly metabolically active neurons whose activities provide the seat for cognitive, affective, sensory and motor functions. The cerebral vasculature meets the brain's unusually high demand for oxygen and glucose by providing it with the largest blood supply of any organ. Accordingly, disorders of the cerebral vasculature, such as congenital vascular malformations, stroke and tumors, compromise neuronal function and survival and often have crippling or fatal consequences. Yet, the assembly of the cerebral vasculature is a process that remains poorly understood. Here we exploit the physical and optical accessibility of the zebrafish embryo to characterize cerebral vascular development within the embryonic hindbrain. We find that this process is primarily driven by endothelial cell migration and follows a two-step sequence. First, perineural vessels with stereotypical anatomies are formed along the ventro-lateral surface of the neuroectoderm. Second, angiogenic sprouts derived from a subset of perineural vessels migrate into the hindbrain to form the intraneural vasculature. We find that these angiogenic sprouts reproducibly penetrate into the hindbrain via the rhombomere centers, where differentiated neurons reside, and that specific rhombomeres are invariably vascularized first. While the anatomy of intraneural vessels is variable from animal to animal, some aspects of the connectivity of perineural and intraneural vessels occur reproducibly within particular hindbrain locales. Using a chemical inhibitor of VEGF signaling we determine stage-specific requirements for this pathway in the formation of the hindbrain vasculature. Finally, we show that a subset of hindbrain vessels is aligned and/or in very close proximity to stereotypical neuron clusters and axon tracts. Using endothelium-deficient cloche mutants we show that the endothelium is dispensable for the organization and maintenance of these stereotypical neuron clusters and axon tracts in the early hindbrain. However, the cerebellum's upper rhombic lip and the optic tectum are abnormal in clo. Overall, this study provides a detailed, multi-stage characterization of early zebrafish hindbrain neurovascular development with cellular resolution up to the third day of age. This work thus serves as a useful reference for the neurovascular characterization of mutants, morphants and drug-treated embryos.

Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish

BackgroundEarly events in vertebrate liver development have been the major focus in previous studies, however, late events of liver organogenesis remain poorly understood. Liver vasculogenesis in vertebrates occurs through the interaction of endoderm-derived liver epithelium and mesoderm-derived endothelial cells (ECs). In zebrafish, although it has been found that ECs are not required for liver budding, how and when the spatio-temporal pattern of liver growth is coordinated with ECs remains to be elucidated.ResultsTo study the process of liver development and vasculogenesis in vivo, a two-color transgenic zebrafish line Tg(lfabf:dsRed; elaA:EGFP) was generated and named LiPan for liver-specific expression of DsRed RFP and exocrine pancreas-specific expression of GFP. Using the LiPan line, we first followed the dynamic development of liver from live embryos to adult and showed the formation of three distinct yet connected liver lobes during development. The LiPan line was then crossed with Tg(fli1:EGFP)y1 and vascular development in the liver was traced in vivo. Liver vasculogenesis started at 55–58 hpf when ECs first surrounded hepatocytes from the liver bud surface and then invaded the liver to form sinusoids and later the vascular network. Using a novel non-invasive and label-free fluorescence correction spectroscopy, we detected blood circulation in the liver starting at ~72 hpf. To analyze the roles of ECs and blood circulation in liver development, both cloche mutants (lacking ECs) and Tnnt2 morphants (no blood circulation) were employed. We found that until 70 hpf liver growth and morphogenesis depended on ECs and nascent sinusoids. After 72 hpf, a functional sinusoidal network was essential for continued liver growth. An absence of blood circulation in Tnnt2 morphants caused defects in liver vasculature and small liver.ConclusionThere are two phases of liver development in zebrafish, budding and growth. In the growth phase, there are three distinct stages: avascular growth between 50–55 hpf, where ECs are not required; endothelium-dependent growth, where ECs or sinusoids are required for liver growth between 55–72 hpf before blood circulation in liver sinusoids; and circulation-dependent growth, where the circulation is essential to maintain vascular network and to support continued liver growth after 72 hpf.

αA-crystallin expression prevents γ-crystallin insolubility and cataract formation in the zebrafishclochemutant lens

Cataracts, the loss of lens transparency, are the leading cause of human blindness. The zebrafish embryo, with its transparency and relatively large eyes, is an excellent model for studying ocular disease in vivo. We found that the zebrafish cloche mutant, both the cloche(m39) and cloche(S5) alleles, which have defects in hematopoiesis and blood vessel development, also have lens cataracts. Quantitative examination of the living zebrafish lens by confocal microscopy showed significant increases in lens reflectance. Histological analysis revealed retention of lens fiber cell nuclei owing to impeded terminal differentiation. Proteomics identified gamma-crystallin as a protein that was substantially diminished in cloche mutants. Crystallins are the major structural proteins in mouse, human and zebrafish lens. Defects in crystallins have previously been shown in mice and humans to contribute to cataracts. The loss of gamma-crystallin protein in cloche was not due to lowered mRNA levels but rather to gamma-crystallin protein insolubility. AlphaA-crystallin is a chaperone that protects proteins from misfolding and becoming insoluble. The cloche lens is deficient in both alphaA-crystallin mRNA and protein during development from 2-5 dpf. Overexpression of exogenous alphaA-crystallin rescued the cloche lens phenotype, including solubilization of gamma-crystallin, increased lens transparency and induction of lens fiber cell differentiation. Taken together, these results indicate that alphaA-crystallin expression is required for normal lens development and demonstrate that cataract formation can be prevented in vivo. In addition, these results show that proteomics is a valuable tool for detecting protein alterations in zebrafish.

Ets1-Related Protein Is a Key Regulator of Vasculogenesis in Zebrafish

During embryonic development, multiple signaling pathways control specification, migration, and differentiation of the vascular endothelial cell precursors, angioblasts. No single gene responsible for the commitment of mesenchymal cells to the angioblast cell fate has been identified as yet. Here we report characterization and functional studies of Etsrp, a novel zebrafish ETS domain protein. etsrp embryonic expression is only restricted to vascular endothelial cells and their earliest precursors. Morpholino knockdown of Etsrp protein function resulted in the complete absence of circulation in zebrafish embryos. Angioblasts in etsrp–morpholino-injected embryos (morphants) failed to undergo migration and differentiation and did not coalesce into functional blood vessels. Expression of all vascular endothelial molecular markers tested was severely reduced in etsrp morphants, whereas hematopoietic markers were not affected. Overexpression of etsrp RNA caused multiple cell types to express vascular endothelial markers. etsrp RNA restored expression of vascular markers in cloche mutants, defective in hematopoietic and endothelial cell formation, arguing that etsrp functions downstream of cloche in angioblast formation. etsrp gene function was also required for endothelial marker induction by the vascular endothelial growth factor (vegf) and stem cell leukemia (scl/tal1). These results demonstrate that Etsrp is necessary and sufficient for the initiation of vasculogenesis.

Gene Expression Profiling of Hematopoietic Stem and Progenitor Cells in Zebrafish.

The differentiation of hematopoietic stem cells (HSCs) into distinct blood lineages is orchestrated by a tight regulation of critical genes during development. To describe the genetic program during the early steps of blood and blood vessel formation in greater detail, we analyzed the expression profiles of zebrafish cloche mutants (clo), in which blood and endothelial cells are completely abrogated, and of isolated hematopoietic and vascular progenitor cell populations from FAC-sorted transgenic lmo2-GFP zebrafish embryos. Consistent with a defect in hemangioblast differentiation, clo mutants at the 5 somite stage were characterized by a prominent downregulation of transcription factors, such as scl and lmo2. As a novel finding, clo mutants at this stage strongly upregulated muscle-related genes, such as myosin, actin, or creatine kinase. FAC-sorted lmo2-GFP cells from 14 somite stage embryos expressed similar hematopoietic transcription factors including scl, lmo2, and gata1. In addition, socs genes, tgfb, fos and jun related genes, and heat shock proteins were also strongly expressed. Comparison with the expression profile of human and mouse fetal liver HSCs revealed a similar regulation between fish and mammals: Expression of 102/168 genes from lmo2-GFP positive cells were also present in mammalian fetal HSCs, indicating an evolutionary highly conserved genetic program of blood formation. In sum, gene expression profiling of zebrafish mutants and of sorted progenitor cells from transgenic zebrafish represent a powerful and versatile tool to define the genetic regulation of vertebrate hematopoietic development.

Identification of novel vascular endothelial–specific genes by the microarray analysis of the zebrafish cloche mutants

AbstractThe zebrafish cloche (clo) mutation affects the earliest known step in differentiation of blood and endothelial cells in vertebrates. We established clo/gata1-GFP transgenic line with erythroid-specific green fluorescent protein (GFP) expression, which allowed differentiation of clo and wild-type siblings at the midsomitogenesis stages before morphologically visible phenotypes appeared. To discover novel genes potentially involved in hematopoietic and vascular development, we performed microarray analysis of more than 15 000 zebrafish genes or expressed sequence tags (ESTs) in clo mutant embryos. We isolated the full-length sequences and determined the expression patterns for 8 novel cDNAs that were significantly down-regulated in clo–/– embryos. Dual specificity phosphatase 5 (dusp5), cadherin 5 (cdh5; VE-cadherin), aquaporin 8 (aqp8), adrenomedullin receptor (admr), complement receptor C1qR-like (crl), scavenger receptor class F, member 1 (scarf1), and ETS1-like protein (etsrp) were specifically expressed in the vascular endothelial cells, while retinol binding protein 4 (rbp4) was expressed in the yolk syncytial layer and the hypochord. Further functional studies of these novel genes should help to elucidate critical early steps leading to the formation of vertebrate blood vessels.

Isolation and characterization of runxa and runxb, zebrafish members of the runt family of transcriptional regulators.

The AML/RUNX family of transcription factors plays important roles in hematopoiesis, neurogenesis, bone development, and segmentation in vertebrate embryos. The aim of this study was to isolate runt-related genes in a genetically and embryologically exploitable system, the zebrafish, and characterize their function during hematopoietic development. Two runt-related genes were isolated by degenerate PCR and standard library screening, and a radiation hybrid panel, T51 RH, was used to resolve their chromosomal localization. In situ hybridization demonstrated their expression whereas their transcriptional activity was assessed using an AML1-responsive reporter gene in the MLA 144 T-cell line. We isolated the zebrafish runxa and runxb cDNAs, which encode proteins highly homologous to the human and murine Runx1 (AML1) and Runx3 (AML2) proteins. In contrast to a recent report, we detected runxa expression in both hematopoietic and neural tissues of the developing zebrafish. runxa transcripts first appear during segmentation in bilateral mesodermal cells that coexpress one of the earliest blood and endothelial cell markers, scl/tal-1. By 24 hours postfertilization (hpf), runxa transcripts are seen in the ventral wall of the dorsal aorta. Hematopoietic runxa expression is lost in cloche mutants, which are defective in blood and endothelial cell formation. runxb transcripts are seen in nonhematopoietic domains. Both Runxa and Runxb transactivate an AML1-responsive human promoter in hematopoietic cells. Genomic localization studies demonstrate that runxa is located on linkage group 1 (LG1), and the runxb gene is located on LG13. Our gene expression analysis strongly suggests that both the functional and spatial aorta-gonad-mesonephros (AGM) region has been conserved throughout evolution. Our runxa spatiotemporal expression data shed light on the role of vertebrate Runx1/AML1 in primitive vs definitive hematopoietic development.

A zebrafish orthologue ( whnb) of the mouse nude gene is expressed in the epithelial compartment of the embryonic thymic rudiment

The cloning and characterization of the zebrafish orthologue of the mouse nude ( Whn/Foxn1) gene, whnb are reported. A previously described Whn-like gene from zebrafish, now designated as whna, is shown to be the orthologue of the mouse Foxn4 gene. The whnb gene is specifically expressed in the thymic rudiment of zebrafish embryos at day 3 after fertilization, whereas the whna gene is expressed in eye and brain structures. whnb expression is maintained in cloche mutants, where endothelial and haematopoietic cell differentiation is defective, but absent in casanova mutants where endoderm formation is impaired. In adult thymi, whnb is expressed throughout cortical and medullary areas, whereas whna expression is observed in rare cell clusters only. Our results provide the first specific marker for the epithelial compartment of the zebrafish thymus.

10.4172/0975-9042.000122

We investigated the contribution of blood vessel formation and neuronal excitability to the development of functional neural circuitry in larval zebrafish by analyzing oculomotor performance in response to visual and vestibular stimuli. To address the dependence of neuronal function on the presence of blood vessels, we compared wild type embryos to reck and cloche mutants that lacked intracerebral blood vessels. To test how neuronal excitability impacts neuronal development and intracerebral vascularization, we blocked neural activity using Tetraodotoxin (TTX) and Tricaine. In reck mutants, we found both slow phase horizontal tracking and fast phase resets with only a slightly reduced amplitude and bandwidth. Spontaneous saccades, eye position holding and vestibular gravitoinertial induced eye rotation were also present. All of these behaviors except for visual tracking were observed in cloche mutants that lacked any head vasculature. Thus, numerous oculomotor neuronal circuits spanning the forebrain, midbrain and hindbrain compartments, ending in motor innervations of the eye muscles, were correctly formed and generated appropriate oculomotor behaviors without blood vessels. However, our observations indicate that beginning at approximately six days, circulation was required for sustained behavioral performance. We further found that blocking neuronal excitability with either TTX or Tricaine up to 4-5 days post fertilization did not noticeably interfere with intracerebral blood vessel formation in wild type larvae. After removal of drug treatments, the oculomotor behaviors returned within hours. Thus, development of neuronal circuits that drive oculomotor performance does not require neuronal spiking or activity. Together these findings demonstrate that neither vascularization nor neuronal excitability are essential for the formation of numerous oculomotor nuclei with intricately designed connectivity and signal processing. We conclude that a genetic blueprint specifies early larval structural and physiological features, and this developmental strategy may be viewed as a unique adaptation required for early survival.

Cell-autonomous and non-autonomous requirements for the zebrafish gene cloche in hematopoiesis.

Vertebrate embryonic hematopoiesis is a complex process that involves a number of cellular interactions, notably those occurring between endothelial and blood cells. The zebrafish cloche mutation affects both the hematopoietic and endothelial lineages from an early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W. R., Zon, L. I. and Fishman, M. C. (1995) Development 121, 3141-3150). cloche mutants lack endocardium, as well as head and trunk endothelium, and nearly all blood cells. Cell transplantation studies have revealed that the endocardial defect in cloche is cell-autonomous: wild-type cells can form endocardium in mutant hosts, but mutant cells never contribute to the endocardium in wild-type or mutant hosts. In this paper, we analyze the cell-autonomy of the blood defect in cloche. The blood cell deficiency in cloche mutants could be an indirect effect of the endothelial defects. Alternatively, cloche could be required cell-autonomously in the blood cells themselves. To distinguish between these possibilities, we cotransplanted wild-type and mutant cells into a single wild-type host in order to compare their respective hematopoietic capacity. We found that transplanted wild-type cells were much more likely than mutant cells to contribute to circulating blood in a wild-type host. Furthermore, in the few cases where both wild-type and mutant donors contributed to blood in a wild-type host, the number of blood cells derived from the wild-type donor was always much greater than the number of blood cells derived from the mutant donor. These data indicate that cloche is required cell-autonomously in blood cells for their differentiation and/or proliferation. When we assessed early expression of the erythropoietic gene gata-1 in transplant recipients, we found that mutant blastomeres were as likely as wild-type blastomeres to give rise to gata-1-expressing cells in a wild-type host. Together, these two sets of data argue that cloche is not required cell-autonomously for the differentiation of red blood cells, as assayed by gata-1 expression, but rather for their proliferation and/or survival, as assayed by their contribution to circulating blood. In addition, we found that transplanted wild-type cells were less likely to express gata-1 in a mutant environment than in a wild-type one, suggesting that cloche also acts non-autonomously in red blood cell differentiation. This non-autonomous function of cloche in red blood cell differentiation may reflect its cell-autonomous requirement in the endothelial lineage. Thus, cloche appears to be required in erythropoiesis cell non-autonomously at a step prior to gata-1 expression, and cell-autonomously subsequently.

The cloche and spadetail Genes Differentially Affect Hematopoiesis and Vasculogenesis

In vertebrates, hematopoietic and vascular progenitors develop from ventral mesoderm. The first primitive wave of hematopoiesis yields embryonic red blood cells, whereas progenitor cells of subsequent definitive waves form all hematopoietic cell lineages. In this report we examine the development of hematopoietic and vasculogenic cells in normal zebrafish and characterize defects in cloche and spadetail mutant embryos. The zebrafish homologs of lmo2, c-myb, fli1, flk1, and flt4 have been cloned and characterized in this study. Expression of these genes identifies embryonic regions that contain hematopoietic and vascular progenitor cells. The expression of c-myb also identifies definitive hematopoietic cells in the ventral wall of the dorsal aorta. Analysis of b316 mutant embryos that carry a deletion of the c-myb gene demonstrates that c-myb is not required for primitive erythropoiesis in zebrafish even though it is expressed in these cells. Both cloche and spadetail mutant embryos have defects in primitive hematopoiesis and definitive hematopoiesis. The cloche mutants also have significant decreases in vascular gene expression, whereas spadetail mutants expressed normal levels of these genes. These studies demonstrate that the molecular mechanisms that regulate hematopoiesis and vasculogenesis have been conserved throughout vertebrate evolution and the clo and spt genes are key regulators of these programs.

Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo

As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor coordination between the chambers with regard to the timing of contraction. The mutations identified in this screen point to discrete and critical steps in the formation and function of the heart and vasculature.

Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages

Endothelial and hematopoietic cells appear synchronously on the extra-embryonic membranes of amniotes in structures known as blood islands. This observation has led to the suggestion that these two ventral lineages share a common progenitor. Recently, we have shown in the zebrafish, Danio rerio, that a single cell in the ventral marginal zone of the early blastula can give rise to both endothelial and blood cells as well as to other mesodermal cells (Stainier, D. Y. R., Lee, R. K. and Fishman, M. C. (1993). Development 119, 31-40; Lee, R. K. K., Stainier, D. Y. R., Weinstein, B. M. and Fishman, M. C. (1994). Development 120, 3361-3366). Here we describe a zebrafish mutation, cloche, that affects both the endothelial and hematopoietic lineages at a very early stage. The endocardium, the endothelial lining of the heart, is missing in mutant embryos. This deletion is selective as evidenced by the presence of other endothelial cells, for example those lining the main vessels of the trunk. Early cardiac morphogenesis proceeds normally even in the absence of the endocardium. The myocardial cells form a tube that is demarcated into chambers, beats rhythmically, but exhibits a reduced contractility. This functional deficit is likely due to the absence of the endocardial cells, although it may be a direct effect of the mutation on the myocardial cells. Cell transplantation studies reveal that the endothelial defect, i.e. the endocardial deletion, is a cell-autonomous lesion, consistent with the possibility that cloche is part of a signal transduction pathway. In addition, the number of blood cells is greatly reduced in cloche mutants and the hematopoietic tissues show no expression of GATA-1 or GATA-2, two key hematopoietic transcription factors that are first expressed during early embryogenesis. These results show that cloche is involved in the genesis and early diversification of the endothelial and blood lineages, possibly by affecting a common progenitor cell population.