Wnt2 signaling is necessary and sufficient to activate the airway smooth muscle program in the lung by regulating myocardin/Mrtf-B and Fgf10 expression.

Smooth muscle in the lung is thought to derive from the developing lung mesenchyme. Smooth muscle formation relies upon coordination of both autocrine and paracrine signaling between the budding epithelium and adjacent mesenchyme to govern its proliferation and differentiation. However, the pathways initiating the earliest aspects of smooth muscle specification and differentiation in the lung are poorly understood. Here, we identify the Wnt2 ligand as a critical regulator of the earliest aspects of lung airway smooth muscle development. Using Wnt2 loss and gain of function models, we show that Wnt2 signaling is necessary and sufficient for activation of a transcriptional and signaling network critical for smooth muscle specification and differentiation including myocardin/Mrtf-B and the signaling factor Fgf10. These studies place Wnt2 high in a hierarchy of signaling molecules that promote the earliest aspects of lung airway smooth muscle development.

Ash2l interacts with Tbx1 and is required during early embryogenesis.

TBX1 encodes a DNA binding transcription factor that is commonly deleted in human DiGeorge syndrome and plays an important role in heart development. Mechanisms of Tbx1 function, such as Tbx1 interacting regulatory proteins and transcriptional target specificity, are largely unknown. Ash2l is the mammalian homolog of Drosophila Ash2 (absent small homeotic 2) and is a core component of a multimeric histone methyltransferase complex that epigenetically regulates transcription via methylation of histone lysine residues. We undertook an unbiased yeast two-hybrid screen to look for functionally relevant Tbx1-interacting proteins and report a physical and functional interaction between Tbx1 and Ash2l. Tbx1 interacts with Ash2l in both yeast and mammalian cells and Ash2l acts as a transcriptional co-activator in luciferase reporter assays. Expression analysis shows that Tbx1 and Ash2l have overlapping mRNA and protein expression patterns during development. By generating an Ash2l knockout mouse utilizing gene-trap technology, we show that although Ash2l heterozygous mice are normal, Ash2l-null embryos die early during gestation. Thus, Ash2l is required for the earliest stages of embryogenesis. Furthermore, our finding of a physical interaction between Tbx1 and Ash2l suggest that at least some functions of Tbx1 may be mediated by direct interactions with a histone methyltransferase complex.

Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.

Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK-SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre-mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76-dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.

Characterization and in vivo pharmacological rescue of a Wnt2-Gata6 pathway required for cardiac inflow tract development.

Little is understood about the molecular mechanisms underlying the morphogenesis of the posterior pole of the heart. Here we show that Wnt2 is expressed specifically in the developing inflow tract mesoderm, which generates portions of the atria and atrio-ventricular canal. Loss of Wnt2 results in defective development of the posterior pole of the heart, resulting in a phenotype resembling the human congenital heart syndrome complete common atrio-ventricular canal. The number and proliferation of posterior second heart field progenitors is reduced in Wnt2(-/-) mutants. Moreover, these defects can be rescued in a temporally restricted manner through pharmacological inhibition of Gsk-3beta. We also show that Wnt2 works in a feedforward transcriptional loop with Gata6 to regulate posterior cardiac development. These data reveal a molecular pathway regulating the posterior cardiac mesoderm and demonstrate that cardiovascular defects caused by loss of Wnt signaling can be rescued pharmacologically in vivo.

Distinct enhancers at the Pax3 locus can function redundantly to regulate neural tube and neural crest expressions.

Pax3 is a transcription factor expressed in somitic mesoderm, dorsal neural tube and pre-migratory neural crest during embryonic development. We have previously identified cis-acting enhancer elements within the proximal upstream genomic region of Pax3 that are sufficient to direct functional expression of Pax3 in neural crest. These elements direct expression of a reporter gene to pre-migratory neural crest in transgenic mice, and transgenic expression of a Pax3 cDNA using these elements is sufficient to rescue neural crest development in mice otherwise lacking endogenous Pax3. We show here that deletion of these enhancer sequences by homologous recombination is insufficient to abrogate neural crest expression of Pax3 and results in viable mice. We identify a distinct enhancer in the fourth intron that is also capable of mediating neural crest expression in transgenic mice and zebrafish. Our analysis suggests the existence of functionally redundant neural crest enhancer modules for Pax3.

Gata4 and Gata5 cooperatively regulate cardiac myocyte proliferation in mice.

GATA5 is a member of the zinc finger transcription factor GATA family (GATA1-6) that plays a wide variety of roles in embryonic and adult development. Experiments in multiple model systems have emphasized the importance of the GATA family members 4-6 in the development of the endoderm and mesoderm. Yet despite overlapping expression patterns, there is little evidence of an important role for GATA5 in mammalian cardiac development. We have generated a new Gata5 mutant allele lacking exons 2 and 3 that encodes both zinc finger domains (Gata5(tm)(2)(Eem)), and we show that although Gata5(-/-) mice are viable, Gata4(+/-)5(-/-) mutants die at mid-gestation and exhibit profound cardiovascular defects, including abnormalities of cardiomyocyte proliferation and cardiac chamber maturation. These results demonstrate functional redundancy between Gata4 and Gata5 during cardiac development and implicate Gata5 as a candidate modifier gene for congenital heart disease.

Inpp5f is a polyphosphoinositide phosphatase that regulates cardiac hypertrophic responsiveness.

RATIONALE: Cardiac hypertrophy occurs in response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has previously been strongly associated with hypertrophic signaling in the heart, and with the control of cell size in multiple contexts. This pathway is tightly regulated by many factors, including a host of kinases and phosphatases that function at multiple steps in the signaling cascade. For example, the PTEN (phosphatase and tensin homolog) tumor suppressor protein is a phosphoinositide 3-phosphatase that, by metabolizing phosphatidylinositol 3,4,5-trisphosphate (PtdIns[3,4,5]P(3), PIP3), acts in direct antagonism to growth factor-stimulated PI3K. Inhibition of PTEN leads to cardiomyocyte hypertrophy. Another polyphoinositide phosphatase, inositol polyphosphate-5-phosphatase F (Inpp5f) has recently been implicated in regulation of cardiac hypertrophy. Like PTEN, this phosphatase can degrade PtdIns(3,4,5)P(3) and thus modulates the PI3K/Akt pathway. OBJECTIVE: To characterize the role of Inpp5f in regulating cardiac hypertrophy. METHODS AND RESULTS: We generated homozygous Inpp5f knockout mice and cardiac specific Inpp5f overexpression transgenic mice. We evaluated their hearts for biochemical, structural and functional changes. Inpp5f knockout mice have augmented hypertrophy and reactivation of the fetal gene program in response to stress when compared to wild-type littermates. Furthermore, cardiac overexpression of Inpp5f in transgenic mice reduces hypertrophic responsiveness. CONCLUSIONS: Our results suggest that Inpp5f is a functionally important endogenous modulator of cardiac myocyte size and of the cardiac response to stress.

Wnt2/2b and beta-catenin signaling are necessary and sufficient to specify lung progenitors in the foregut.

Patterning of the primitive foregut promotes appropriate organ specification along its anterior-posterior axis. However, the molecular pathways specifying foregut endoderm progenitors are poorly understood. We show here that Wnt2/2b signaling is required to specify lung endoderm progenitors within the anterior foregut. Embryos lacking Wnt2/2b expression exhibit complete lung agenesis and do not express Nkx2.1, the earliest marker of the lung endoderm. In contrast, other foregut endoderm-derived organs, including the thyroid, liver, and pancreas, are correctly specified. The phenotype observed is recapitulated by an endoderm-restricted deletion of beta-catenin, demonstrating that Wnt2/2b signaling through the canonical Wnt pathway is required to specify lung endoderm progenitors within the foregut. Moreover, activation of canonical Wnt/beta-catenin signaling results in the reprogramming of esophagus and stomach endoderm to a lung endoderm progenitor fate. Together, these data reveal that canonical Wnt2/2b signaling is required for the specification of lung endoderm progenitors in the developing foregut.

Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous malformation protein pathway.

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: KRIT1, CCM2 and PDCD10. Here we show that the heart of glass (HEG1) receptor, which in zebrafish has been linked to ccm gene function, is selectively expressed in endothelial cells. Heg1(-/-) mice showed defective integrity of the heart, blood vessels and lymphatic vessels. Heg1(-/-); Ccm2(lacZ/+) and Ccm2(lacZ/lacZ) mice had more severe cardiovascular defects and died early in development owing to a failure of nascent endothelial cells to associate into patent vessels. This endothelial cell phenotype was shared by zebrafish embryos deficient in heg, krit1 or ccm2 and reproduced in CCM2-deficient human endothelial cells in vitro. Defects in the hearts of zebrafish lacking heg or ccm2, in the aortas of early mouse embryos lacking CCM2 and in the lymphatic vessels of neonatal mice lacking HEG1 were associated with abnormal endothelial cell junctions like those observed in human CCMs. Biochemical and cellular imaging analyses identified a cell-autonomous pathway in which the HEG1 receptor couples to KRIT1 at these cell junctions. This study identifies HEG1-CCM protein signaling as a crucial regulator of heart and vessel formation and integrity.

Cre reporter mouse expressing a nuclear localized fusion of GFP and beta-galactosidase reveals new derivatives of Pax3-expressing precursors.

A new Cre-reporter strain of mouse has been developed that expresses a fusion protein derived from the lacZ gene fused to GFP with a nuclear localization signal. This construct is expressed from the ROSA26 locus upon Cre-mediated recombination that removes a loxP-flanked PGK-neo cassette, thus allowing for detection of Cre activity in all tissues. This reporter strain, which is similar to prior R26R and R26EGFP strains, has certain advantages related to the nuclear expression and the combined expression of both beta-galactosidase and GFP activities. We show that the use of this newly developed reporter line allows for enhanced resolution, detection and co-localization. Thus, we report a previously unrecognized subset of venous endothelial cells derived from Pax3 expressing precursors.

Klf2 is an essential regulator of vascular hemodynamic forces in vivo.

Hemodynamic responses that control blood pressure and the distribution of blood flow to different organs are essential for survival. Shear forces generated by blood flow regulate hemodynamic responses, but the molecular and genetic basis for such regulation is not known. The transcription factor KLF2 is activated by fluid shear stress in cultured endothelial cells, where it regulates a large number of vasoactive endothelial genes. Here, we show that Klf2 expression during development mirrors the rise of fluid shear forces, and that endothelial loss of Klf2 results in lethal embryonic heart failure due to a high-cardiac-output state. Klf2 deficiency does not result in anemia or structural vascular defects, and it can be rescued by administration of phenylephrine, a catecholamine that raises vessel tone. These findings identify Klf2 as an essential hemodynamic regulator in vivo and suggest that hemodynamic regulation in response to fluid shear stress is required for cardiovascular development and function.

In vivo beta1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines.

Integrins are heterodimeric adhesion receptors associated with bidirectional signaling. In vitro studies support a role for the binding of evolutionarily conserved tyrosine motifs (NPxY) in the beta integrin cytoplasmic tail to phosphotyrosine-binding (PTB) domain-containing proteins, an interaction proposed to be dynamically regulated by tyrosine phosphorylation. Here we show that replacement of both beta1 integrin cytoplasmic tyrosines with alanines, resulting in the loss of all PTB domain interaction, causes complete loss of beta1 integrin function in vivo. In contrast, replacement of beta1 integrin cytoplasmic tyrosines with phenylalanines, a mutation that prevents tyrosine phosphorylation, conserves in vivo integrin function. These results have important implications for the molecular mechanism and regulation of integrin function.

Insertion of Cre into the Pax3 locus creates a new allele of Splotch and identifies unexpected Pax3 derivatives.

Pax3 is a transcription factor expressed in the dorsal neural tube and somite of the developing embryo. It plays critical roles in pre-migratory neural crest cells and in myogenic precursors of skeletal muscle. Pax3-deficient Splotch embryos display neural tube and neural crest defects and lack hypaxial muscles. We have created a new allele of Splotch by replacing the first coding exon with a gene encoding Cre recombinase. This functions as a null allele and no Pax3 protein is detected in homozygous embryos. Heterozygous Pax3(Cre/+) mice display a white belly spot, as do Splotch heterozygotes. Homozygous Pax3(Cre/Cre) embryos are embryonic lethal. We have used Pax3(Cre/+) mice to fate-map Pax3 derivatives in the developing mouse. As expected, neural crest and some somitic derivatives are identified. However, we also detect previously unappreciated derivatives of Pax3-expressing precursors in the colonic epithelium of the hindgut and within the urogenital system.