Abnormal embryonic lymphatic vessel development in Tie1 hypomorphic mice.

Tie1 is an endothelial receptor tyrosine kinase that is essential for development and maintenance of the vascular system; however, the role of Tie1 in development of the lymphatic vasculature is unknown. To address this question, we first documented that Tie1 is expressed at the earliest stages of lymphangiogenesis in Prox1-positive venous lymphatic endothelial cell (LEC) progenitors. LEC Tie1 expression is maintained throughout embryonic development and persists in postnatal mice. We then generated two lines of Tie1 mutant mice: a hypomorphic allele, which has reduced expression of Tie1, and a conditional allele. Reduction of Tie1 levels resulted in abnormal lymphatic patterning and in dilated and disorganized lymphatic vessels in all tissues examined and in impaired lymphatic drainage in embryonic skin. Homozygous hypomorphic mice also exhibited abnormally dilated jugular lymphatic vessels due to increased production of Prox1-positive LECs during initial lymphangiogenesis, indicating that Tie1 is required for the early stages of normal lymphangiogenesis. During later stages of lymphatic development, we observed an increase in LEC apoptosis in the hypomorphic embryos after mid-gestation that was associated with abnormal regression of the lymphatic vasculature. Therefore, Tie1 is required for early LEC proliferation and subsequent survival of developing LECs. The severity of the phenotypes observed correlated with the expression levels of Tie1, confirming a dosage dependence for Tie1 in LEC integrity and survival. No defects were observed in the arterial or venous vasculature. These results suggest that the developing lymphatic vasculature is particularly sensitive to alterations in Tie1 expression.

Ndrg4 is required for normal myocyte proliferation during early cardiac development in zebrafish.

NDRG4 is a novel member of the NDRG family (N-myc downstream-regulated gene). The roles of NDRG4 in development have not previously been evaluated. We show that, during zebrafish embryonic development, ndrg4 is expressed exclusively in the embryonic heart, the central nervous system (CNS) and the sensory system. Ndrg4 knockdown in zebrafish embryos causes a marked reduction in proliferative myocytes and results in hypoplastic hearts. This growth defect is associated with cardiac phenotypes in morphogenesis and function, including abnormal heart looping, inefficient circulation and weak contractility. We reveal that ndrg4 is required for restricting the expression of versican and bmp4 to the developing atrioventricular canal. This constellation of ndrg4 cardiac defects phenocopies those seen in mutant hearts of heartstrings (hst), the tbx5 loss-of-function mutants in zebrafish. We further show that ndrg4 expression is significantly decreased in hearts with reduced tbx5 activities. Conversely, increased expression of tbx5 that is due to tbx20 knockdown leads to an increase in ndrg4 expression. Together, our studies reveal an essential role of ndrg4 in regulating proliferation and growth of cardiomyocytes, suggesting that ndrg4 may function downstream of tbx5 during heart development and growth.

Tgfbeta signaling is required for atrioventricular cushion mesenchyme remodeling during in vivo cardiac development.

The transforming growth factorbeta (Tgfbeta) signaling pathway plays crucial roles in many biological processes. To understand the role(s) of Tgfbeta signaling during cardiogenesis in vivo and to overcome the early lethality of Tgfbr2(-/-) embryos, we applied a Cre/loxp system to specifically inactivate Tgfbr2 in either the myocardium or the endothelium of mouse embryos. Our results show that Tgfbr2 in the myocardium is dispensable for cardiogenesis in most embryos. Contrary to the prediction from results of previous in vitro collagen gel assays, inactivation of Tgfbr2 in the endocardium does not prevent atrioventricular cushion mesenchyme formation, arguing against its essential role in epithelium-mesenchyme transformation in vivo. We further demonstrate that Tgfbeta signaling is required for the proper remodeling of the atrioventricular canal and for cardiac looping, and that perturbation in Tgfbeta signaling causes the double-inlet left ventricle (DILV) defect. Thus, our study provides a unique mouse genetic model for DILV, further characterization of which suggests a potential cellular mechanism for the defect.

A zebrafish model of human Barth syndrome reveals the essential role of tafazzin in cardiac development and function.

Barth syndrome is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, neutropenia, organic aciduria, and growth retardation caused by mutations in tafazzin. The sequence similarity of tafazzin to acyltransferases suggests a role in mitochondrial phospholipid metabolism. To study the role of tafazzin in heart function and development, we created a knockdown zebrafish model. Zebrafish tafazzin mRNA is first evident at 7 hours post-fertilization (hpf). At 10 and 24 hpf, tafazzin mRNA is ubiquitous, with highest levels in the head. By 51 hpf, expression becomes cardiac restricted. The tafazzin knockdown created by antisense morpholino yolk injection resulted in dose-dependent lethality, severe developmental and growth retardation, marked bradycardia and pericardial effusions, and generalized edema, signs that resemble human Barth syndrome heart failure. This knockdown phenotype was rescued by concomitant injection of normal tafazzin mRNA. Abnormal cardiac development, with a linear, nonlooped heart, and hypomorphic tail and eye development proves that tafazzin is essential for overall zebrafish development, especially of the heart. The tafazzin knockdown zebrafish provides an animal model similar to Barth syndrome to analyze the severity of human mutants and to test potential treatments.

Bmp signaling is required for intestinal growth and morphogenesis.

Intestinal growth, morphogenesis, differentiation, and homeostasis are regulated by reciprocal interactions between the epithelium and the underlying mesenchymal stroma. The identification of BMPR1A mutations in patients with Juvenile Polyposis implicates Bmp signaling as an important mediator of these interactions. To test this hypothesis, we inhibited Bmp signaling in the mouse proximal intestine by transgenic misexpression of the BMP antagonist, noggin, using regulatory elements of the fatty acid binding protein (Fabp1) gene. This leads to abnormal villus morphogenesis, stromal and epithelial hyperplasia, and ectopic crypt formation. The resulting intestinal histopathology resembles that seen in human Juvenile Polyposis. Misexpression of noggin in the large intestine gives a similar abnormal phenotype in this region of the gut. Analysis of gene expression in the transgenic small intestine raises the possibility that Hedgehog and Pdgf signaling play a role in the development of the Juvenile Polyposis-like phenotype.

An essential role of Bmp4 in the atrioventricular septation of the mouse heart.

Proper septation and valvulogenesis during cardiogenesis depend on interactions between the myocardium and the endocardium. By combining use of a hypomorphic Bone morphogenetic protein 4 (Bmp4) allele with conditional gene inactivation, we here identify Bmp4 as a signal from the myocardium directly mediating atrioventricular septation. Defects in this process cause one of the most common human congenital heart abnormalities, atrioventricular canal defect (AVCD). The spectrum of defects obtained through altering Bmp4 expression in the myocardium recapitulates the range of AVCDs diagnosed in patients, thus providing a useful genetic model with AVCD as the primary defect.

Tissue interactions pattern the mesenchyme of the embryonic mouse lung.

The mechanisms that control proliferation and differentiation of embryonic lung mesenchyme are largely unknown. We describe an explant system in which exogenous recombinant N-Sonic Hedgehog (N-Shh) protein sustains the survival and proliferation of lung mesenchyme in a dose-dependent manner. In addition, Shh upregulates several mesenchymal cell markers, including its target gene Patched (Ptc), intercellular signaling genes Bone Morphogenetic Protein-4 (Bmp4) and Noggin (Nog), and smooth muscle actin and myosin. In explants exposed to N-Shh in the medium, these products are upregulated throughout the mesenchyme, but not in the periphery. This exclusion zone correlates with the presence of an overlying mesothelial layer, which, as in vivo, expresses Fibroblast Growth Factor 9 (Fgf9). Recombinant Fgf9 protein inhibits the differentiation response of the mesenchyme to N-Shh, but does not affect proliferation. We propose a model for how factors made by two epithelial cell populations, the inner endoderm and the outer jacket of mesothelium, coordinately regulate the proliferation and differentiation of the lung mesoderm.