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Biomedical subjects

Mark C Fishman

Publications and source records attributed to Mark C Fishman.

10 recordsLinked to original sources
PubMed2003

Nil per os encodes a conserved RNA recognition motif protein required for morphogenesis and cytodifferentiation of digestive organs in zebrafish.

Digestive organ development occurs through a sequence of morphologically distinct stages, from overtly featureless endoderm, through organ primordia to, ultimately, adult form. The developmental controls that govern progression from one stage to the next are not well understood. To identify genes required for the formation of vertebrate digestive organs we performed a genetic screen in zebrafish. We isolated the nil per os (npo) mutation, which arrests morphogenesis and cytodifferentiation of the gut and exocrine pancreas in a primordial state. We identified the npo gene by positional cloning. It encodes a conserved protein, with multiple RNA recognition motifs, that is related to the yeast protein Mrd1p. During development npo is expressed in a dynamic fashion, functioning cell autonomously to promote organ cytodifferentiation. Antisense-mediated knockdown of npo results in organ hypoplasia, and overexpression of npo causes an overgrowth of gastrointestinal organs. Thus, npo is a gene essential for a key step in the gut morphogenetic sequence.

Amino Acid Motifs↗
PubMed2002

In vivo imaging of proteolytic activity in atherosclerosis.

BACKGROUND: Atherosclerotic plaque rupture, the most important cause of acute cardiovascular incidents, has been strongly associated with vascular inflammation. On the basis of the hypothesis that the inflammatory response and proteolysis lead to plaque rupture, we have examined the role of cathepsin B as a model proteolytic enzyme. METHODS AND RESULTS: Using western-type diet-fed apoE and apoE/endothelial NO synthase double knockout mice as models of atherosclerosis, we show (1) that cathepsin B is upregulated in atherosclerotic lesions characterized by high degrees of inflammation compared with normal aorta or silent lesions, (2) that intravenously injectable novel cathepsin B imaging beacons are highly activated within active atherosclerotic lesions and colocalize with cathepsin B immunoreactivity, and (3) that cathepsin B activity in atherosclerotic lesions can be imaged in whole animals by using a novel near-infrared tomographic imaging system. CONCLUSIONS: These studies indicate that cathepsin B, and potentially other proteases, may serve as a biomarker for vulnerable plaques when probed with beacons. The tomographic in vivo imaging method as well as catheter-based optical sensing methods could be readily adapted to screening and potentially to the molecular profiling of a number of proteases in vulnerable plaque in vivo.

Animals↗
PubMed2002

From Zebrafish to human: modular medical models.

Genetic screens in Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio clarified the logic of metazoan development by revealing critical unitary steps and pathways to embryogenesis. Can genetic screens similarly organize medicine? We here examine human diseases that resemble mutations in Danio rerio, the zebrafish, the one vertebrate species for which large-scale genetic screens have been performed and extensively analyzed. Zebrafish mutations faithfully phenocopy many human disorders. Each mutation, once cloned, provides candidate genes and pathways for evaluation in the human. The collection of mutations in their entirety potentially provides a medical taxonomy, one based in developmental biology and genetics.

Animals↗
PubMed2002

Endothelial signaling in kidney morphogenesis: a role for hemodynamic forces.

The local presence of endothelial cells seems necessary for proper embryonic development of several organs. However, the signals involved are unknown. The glomerulus is generated by the coalescence of podocytes around an ingrowing capillary and is the site of blood ultrafiltration. In the absence of vessels, glomerular assembly does not occur. We describe mutations in the zebrafish that prevent glomerulogenesis. All mutants display cardiac dysfunction. Pharmacological interference with cardiac output and focal laser occlusion of the vessel similarly prevent glomerular formation. The unifying feature of all these perturbations is absence of blood flow. We find that expression of matrix metalloproteinase-2 (MMP-2), known in other systems to be regulated in a stretch-responsive manner, is in renal endothelial cells and is regulated by flow, suggesting that an MMP-2-sensitive event may be downstream of the flow-related signal. In support of this, blockade of MMP-2 activity by injection of TIMP-2 does not perturb circulation but does prevent glomerular assembly. Thus, vascular flow is required for glomerular assembly, most probably acting via a stretch-responsive signaling system in the vessel wall.

Animals↗
PubMed2002

Cardiomyopathy in zebrafish due to mutation in an alternatively spliced exon of titin.

The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwickm171 (pikm171), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pikm242, pikm740, pikm186; ref. 3) and one allele (pikmVO62H) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscle. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue.

Alternative Splicing↗
PubMed2002

Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels.

The zebrafish mutant violet beauregarde (vbg) can be identified at two days post-fertilization by an abnormal circulation pattern in which most blood cells flow through a limited number of dilated cranial vessels and fail to perfuse the trunk and tail. This phenotype cannot be explained by caudal vessel abnormalities or by a defect in cranial vessel patterning, but instead stems from an increase in endothelial cell number in specific cranial vessels. We show that vbg encodes activin receptor-like kinase 1 (Acvrl1; also known as Alk1), a TGFbeta type I receptor that is expressed predominantly in the endothelium of the vessels that become dilated in vbg mutants. Thus, vbg provides a model for the human autosomal dominant disorder, hereditary hemorrhagic telangiectasia type 2, in which disruption of ACVRL1 causes vessel malformations that may result in hemorrhage or stroke. Movies available on-line

Activin Receptors↗
PubMed2002

The heartstrings mutation in zebrafish causes heart/fin Tbx5 deficiency syndrome.

Holt-Oram syndrome is one of the autosomal dominant human "heart-hand" disorders, with a combination of upper limb malformations and cardiac defects. Holt-Oram syndrome is caused by mutations in the TBX5 gene, a member of a large family of T-box transcription factors that play important roles in cell-type specification and morphogenesis. In a screen for mutations affecting zebrafish cardiac function, we isolated the recessive lethal mutant heartstrings, which lacks pectoral fins and exhibits severe cardiac dysfunction, beginning with a slow heart rate and progressing to a stretched, non-functional heart. We mapped and cloned the heartstrings mutation and find it to encode the zebrafish ortholog of the TBX5 gene. The heartstrings mutation causes premature termination at amino acid 316. Homozygous mutant embryos never develop pectoral fin buds and do not express several markers of early fin differentiation. The total absence of any fin bud differentiation distinguishes heartstrings from most other mutations that affect zebrafish fin development, suggesting that Tbx5 functions very early in the pectoral fin induction pathway. Moderate reduction of Tbx5 by morpholino causes fin malformations, revealing an additional early requirement for Tbx5 in coordinating the axes of fin outgrowth. The heart of heartstrings mutant embryos appears to form and function normally through the early heart tube stage, manifesting only a slight bradycardia compared with wild-type siblings. However, the heart fails to loop and then progressively deteriorates, a process affecting the ventricle as well as the atrium. Relative to mammals, fish require lower levels of Tbx5 to produce malformed appendages and display whole-heart rather than atrial-predominant cardiac defects. However, the syndromic deficiencies of tbx5 mutation are remarkably well retained between fish and mammals.

Animals↗
PubMed2002

The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10.

A major gap in our knowledge of development is how the growth and identity of tissues and organs are linked during embryogenesis. The vertebrate limb is one of the best models to study these processes. Combining mutant analyses with gain- and loss-of-function approaches in zebrafish and chick embryos, we show that Tbx5, in addition to its role governing forelimb identity, is both necessary and sufficient for limb outgrowth. We find that Tbx5 functions downstream of WNT signaling to regulate Fgf10, which, in turn, maintains Tbx5 expression during limb outgrowth. Furthermore, our results indicate that Tbx5 and Wnt2b function together to initiate and specify forelimb outgrowth and identity. The molecular interactions governed by members of the T-box, Wnt and Fgf gene families uncovered in this study provide a framework for understanding not only limb development, but how outgrowth and identity of other tissues and organs of the embryo may be regulated.

Amino Acid Sequence↗
PubMed2002

Patterning of angiogenesis in the zebrafish embryo.

Little is known about how vascular patterns are generated in the embryo. The vasculature of the zebrafish trunk has an extremely regular pattern. One intersegmental vessel (ISV) sprouts from the aorta, runs between each pair of somites, and connects to the dorsal longitudinal anastomotic vessel (DLAV). We now define the cellular origins, migratory paths and cell fates that generate these metameric vessels of the trunk. Additionally, by a genetic screen we define one gene, out of bounds (obd), that constrains this angiogenic growth to a specific path. We have performed lineage analysis, using laser activation of a caged dye and mosaic construction to determine the origin of cells that constitute the ISV. Individual angioblasts destined for the ISVs arise from the lateral posterior mesoderm (LPM), and migrate to the dorsal aorta, from where they migrate between somites to their final position in the ISVs and dorsal longitudinal anastomotic vessel (DLAV). Cells of each ISV leave the aorta only between the ventral regions of two adjacent somites, and migrate dorsally to assume one of three ISV cell fates. Most dorsal is a T-shaped cell, based in the DLAV and branching ventrally; the second constitutes a connecting cell; and the third an inverted T-shaped cell, based in the aorta and branching dorsally. The ISV remains between somites during its ventral course, but changes to run mid-somite dorsally. This suggests that the pattern of ISV growth ventrally and dorsally is guided by different cues. We have also performed an ENU mutagenesis screen of 750 mutagenized genomes and identified one mutation, obd that disrupts this pattern. In obd mutant embryos, ISVs sprout precociously at abnormal sites and migrate anomalously in the vicinity of ventral somite. The dorsal extent of the ISV is less perturbed. Precocious sprouting can be inhibited in a VEGF morphant, but the anomalous site of origin of obd ISVs remains. In mosaic embryos, obd somite causes adjacent wild-type endothelial cells to assume the anomalous ISV pattern of obd embryos. Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth.

Animals↗