The Wnt Effector TCF7l2 Promotes Oligodendroglial Differentiation by Repressing Autocrine BMP4-Mediated Signaling.

Promoting oligodendrocyte (OL) differentiation represents a promising option for remyelination therapy for treating the demyelinating disease multiple sclerosis (MS). The Wnt effector transcription factor 7-like 2 (TCF7l2) was upregulated in MS lesions and had been proposed to inhibit OL differentiation. Recent data suggest the opposite yet underlying mechanisms remain elusive. Here, we unravel a previously unappreciated function of TCF7l2 in controlling autocrine bone morphogenetic protein (BMP)4-mediated signaling. Disrupting TCF7l2 in mice of both sexes results in oligodendroglial-specific BMP4 upregulation and canonical BMP4 signaling activation in vivo Mechanistically, TCF7l2 binds to Bmp4 gene regulatory element and directly represses its transcriptional activity. Functionally, enforced TCF7l2 expression promotes OL differentiation by reducing autocrine BMP4 secretion and dampening BMP4 signaling. Importantly, compound genetic disruption demonstrates that oligodendroglial-specific BMP4 deletion rescues arrested OL differentiation elicited by TCF7l2 disruption in vivo Collectively, our study reveals a novel connection between TCF7l2 and BMP4 in oligodendroglial lineage and provides new insights into augmenting TCF7l2 for promoting remyelination in demyelinating disorders such as MS.SIGNIFICANCE STATEMENT Incomplete or failed myelin repairs, primarily resulting from the arrested differentiation of myelin-forming oligodendrocytes (OLs) from oligodendroglial progenitor cells, is one of the major reasons for neurologic progression in people affected by multiple sclerosis (MS). Using in vitro culture systems and in vivo animal models, this study unraveled a previously unrecognized autocrine regulation of bone morphogenetic protein (BMP)4-mediated signaling by the Wnt effector transcription factor 7-like 2 (TCF7l2). We showed for the first time that TCF7l2 promotes oligodendroglial differentiation by repressing BMP4-mediated activity, which is dysregulated in MS lesions. Our study suggests that elevating TCF7l2 expression may be possible in overcoming arrested oligodendroglial differentiation as observed in MS patients.

Sox2 Is Essential for Oligodendroglial Proliferation and Differentiation during Postnatal Brain Myelination and CNS Remyelination.

In the CNS, myelination and remyelination depend on the successful progression and maturation of oligodendroglial lineage cells, including proliferation and differentiation of oligodendroglial progenitor cells (OPCs). Previous studies have reported that Sox2 transiently regulates oligodendrocyte (OL) differentiation in the embryonic and perinatal spinal cord and appears dispensable for myelination in the postnatal spinal cord. However, the role of Sox2 in OL development in the brain has yet to be defined. We now report that Sox2 is an essential positive regulator of developmental myelination in the postnatal murine brain of both sexes. Stage-specific paradigms of genetic disruption demonstrated that Sox2 regulated brain myelination by coordinating upstream OPC population supply and downstream OL differentiation. Transcriptomic analyses further supported a crucial role of Sox2 in brain developmental myelination. Consistently, oligodendroglial Sox2-deficient mice developed severe tremors and ataxia, typical phenotypes indicative of hypomyelination, and displayed severe impairment of motor function and prominent deficits of brain OL differentiation and myelination persisting into the later CNS developmental stages. We also found that Sox2 was required for efficient OPC proliferation and expansion and OL regeneration during remyelination in the adult brain and spinal cord. Together, our genetic evidence reveals an essential role of Sox2 in brain myelination and CNS remyelination, and suggests that manipulation of Sox2 and/or Sox2-mediated downstream pathways may be therapeutic in promoting CNS myelin repair.SIGNIFICANCE STATEMENT Promoting myelin formation and repair has translational significance in treating myelin-related neurological disorders, such as periventricular leukomalacia and multiple sclerosis in which brain developmental myelin formation and myelin repair are severely affected, respectively. In this report, analyses of a series of genetic conditional knock-out systems targeting different oligodendrocyte stages reveal a previously unappreciated role of Sox2 in coordinating upstream proliferation and downstream differentiation of oligodendroglial lineage cells in the mouse brain during developmental myelination and CNS remyelination. Our study points to the potential of manipulating Sox2 and its downstream pathways to promote oligodendrocyte regeneration and CNS myelin repair.

Parkin deficiency accelerates consequences of mitochondrial DNA deletions and Parkinsonism.

Parkinson's disease (PD) is a neurodegenerative condition caused by age-related death of dopaminergic (DA) neurons in the substantia nigra (SN). Mitochondrial DNA (mtDNA) deletions rise exponentially with age in humans and reach their highest levels approaching 60% in dopaminergic neurons of the substantia nigra and overlap with dying neurons. Parkin deletion causes Parkinsonism in humans, presumably through a decrease in mitochondrial quality control, but Parkin knockout mice do not have DA neurodegeneration. We crossed Parkin knockouts to the Twinkle-TG mouse in which mtDNA deletions are increased specifically in substantia nigra to determine the effect of increased deletion mutagenesis in the absence of mitochondrial quality control. These double-mutant 'TwinkPark' mice had 1, the highest mtDNA deletion concentration in SN; 2, the lowest mitochondrial function and membrane potential; 3, the most severe neurobehavioral deficits at 19months; 4, the least dopaminergic neurons in the SN and lowest dopamine levels, i.e. Parkinsonism. This mouse model could provide novel insights into the pathomechanism by which a specific increase in mtDNA deletions with age contribute to dopaminergic neurodegeneration and Parkinson's disease.

Mitochondrial complex I deficiency leads to inflammation and retinal ganglion cell death in the Ndufs4 mouse.

Mitochondrial complex I (NADH dehydrogenase) is a major contributor to neuronal energetics, and mutations in complex I lead to vision loss. Functional, neuroanatomical and transcriptional consequences of complex I deficiency were investigated in retinas of the Ndufs4 knockout mouse. Whole-eye ERGs and multielectrode arrays confirmed a major retinal ganglion cell functional loss at P32, and retinal ganglion cell loss at P42. RNAseq demonstrated a mild and then sharp increase in innate immune and inflammatory retinal transcripts at P22 and P33, respectively, which were confirmed with QRT-PCR. Intraperitoneal injection of the inflammogen lipopolysaccharide further reduced retinal ganglion cell function in Ndufs4 KO, supporting the connection between inflammatory activation and functional loss. Complex I deficiency in the retina clearly caused innate immune and inflammatory markers to increase coincident with loss of vision, and RGC functional loss. How complex I incites inflammation and functional loss is not clear, but could be the result of misfolded complex I generating a 'non-self' response, and induction of innate immune response transcripts was observed before functional loss at P22, including ß-2 microglobulin and Cx3cr1, and during vision loss at P31 (B2m, Tlr 2, 3, 4, C1qa, Cx3cr1 and Fas). These data support the hypothesis that mitochondrial complex I dysfunction in the retina triggers an innate immune and inflammatory response that results in loss of retinal ganglion cell function and death, as in Leber's hereditary Optic Neuropathy and suggests novel therapeutic routes to counter mitochondrial defects that contribute to vision loss.

Mutant Twinkle increases dopaminergic neurodegeneration, mtDNA deletions and modulates Parkin expression.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in the developed world, and is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Somatic mitochondrial DNA (mtDNA) deletions reach their highest concentration with age in the SN in humans, and may contribute to PD; yet whether mtDNA deletions cause DA neuron degeneration remains unclear. Inherited mutations of Twinkle helicase involved in mtDNA replication causes a dominant increase in mtDNA deletions in humans. We constructed a mouse model expressing mutant Twinkle in DA neurons. Mutant mice had an increase in age-related mtDNA deletions, reduction of DA neuron number in SN at 17-22 months and displayed abnormalities in rota-rod behavior. Functional analysis of midbrain indicated a slight reduction in mitochondrial state II respiration in mutants, but no decrease in maximal respiration. Also, Parkin expression was significantly decreased in DA neurons in the SN of 22-month-old mutant mice, and in PC12 cells after 48 h transfection of mutant Twinkle. Both confocal imaging and coimmunoprecipitation indicated interaction of Twinkle with Parkin in the mitochondria. Parkin overexpression rescued the reduction of proteasome activity caused by mutant Twinkle in PC12 cells. In addition, the autophagy marker LC3 was increased in the SN of 22-month transgenics, and this increase was similarly mutant Twinkle-dependent in PC12 cells. Collectively, our data demonstrate that mammalian Twinkle is important for mitochondrial integrity in DA neurons and provide a novel mouse model in which increased mtDNA deletions may lead to DA neuron degeneration and parkinsonism.

The canonical Wnt/ß-catenin signaling pathway regulates Fgf signaling for early facial development.

The canonical Wnt/ß-catenin signaling pathway has implications in early facial development; yet, its function and signaling mechanism remain poorly understood. We report here that the frontonasal and upper jaw primordia cannot be formed after conditional ablation of ß-catenin with Foxg1-Cre mice in the facial ectoderm and the adjacent telencephalic neuroepithelium. Gene expression of several cell-survival and patterning factors, including Fgf8, Fgf3, and Fgf17, is dramatically diminished in the anterior neural ridge (ANR, a rostral signaling center) and/or the adjacent frontonasal ectoderm of the ß-catenin conditional mutant mice. In addition, Shh expression is diminished in the ventral telencephalon of the mutants, while Tcfap2a expression is less affected in the facial primordia. Apoptosis occurs robustly in the rostral head tissues following inactivation of Fgf signaling in the conditional mutants. Consequently, the upper jaw, nasal, ocular and telencephalic structures are absent, but the tongue and mandible are relatively developed in the conditional mutants at birth. Using molecular biological approaches, we demonstrate that the Fgf8 gene is transcriptionally targeted by Wnt/ß-catenin signaling during early facial and forebrain development. Furthermore, we show that conditional gain-of-function of ß-catenin signaling causes drastic upregulation of Fgf8 mRNA in the ANR and the entire facial ectoderm, which also arrests facial and forebrain development. Taken together, our results suggest that canonical Wnt/ß-catenin signaling is required for early development of the mammalian face and related head structures, which mainly or partly acts through the initiation and modulation of balanced Fgf signaling activity.

Lrp6-mediated canonical Wnt signaling is required for lip formation and fusion.

Neither the mechanisms that govern lip morphogenesis nor the cause of cleft lip are well understood. We report that genetic inactivation of Lrp6, a co-receptor of the Wnt/beta-catenin signaling pathway, leads to cleft lip with cleft palate. The activity of a Wnt signaling reporter is blocked in the orofacial primordia by Lrp6 deletion in mice. The morphological dynamic that is required for normal lip formation and fusion is disrupted in these mutants. The expression of the homeobox genes Msx1 and Msx2 is dramatically reduced in the mutants, which prevents the outgrowth of orofacial primordia, especially in the fusion site. We further demonstrate that Msx1 and Msx2 (but not their potential regulator Bmp4) are the downstream targets of the Wnt/beta-catenin signaling pathway during lip formation and fusion. By contrast, a ;fusion-resistant' gene, Raldh3 (also known as Aldh1a3), that encodes a retinoic acid-synthesizing enzyme is ectopically expressed in the upper lip primordia of Lrp6-deficient embryos, indicating a region-specific role of the Wnt/beta-catenin signaling pathway in repressing retinoic acid signaling. Thus, the Lrp6-mediated Wnt signaling pathway is required for lip development by orchestrating two distinctively different morphogenetic movements.

Cardiac neural crest and outflow tract defects in Lrp6 mutant mice.

The role of a key Wnt coreceptor Lrp6 during heart development remains unclear. Here we show that ablation of Lrp6 in mice causes conotruncal anomalies including double-outlet right ventricle (DORV), outflow tract (OFT) cushion hypoplasia, and ventricular septal defect (VSD). Cardiac neural crest cells are specifically lost in the dorsal neural tube and caudal pharyngeal arches of the mutant embryos. We also demonstrate that Lrp6 is required for proliferation and survival of cardiac progenitors and for the expression of Isl1 in the secondary heart field. Other known cardiogenic regulators such as Msx1, Msx2, and Fgf8 are also significantly diminished in the mutant pharyngeal arches and/or OFT. Unexpectedly, the myocardium differentiation factors Mef2c and Myocardin are upregulated in the mutant OFT. Our results indicate that Lrp6 is essential for cardiac neural crest and OFT development upstream of multiple important cardiogenic genes in different cardiac lineage cells during early cardiogenesis.

Generation of Lrp6 conditional gene-targeting mouse line for modeling and dissecting multiple birth defects/congenital anomalies.

Lrp6 is a key coreceptor in the canonical Wnt pathway that is widely involved in tissue/organ morphogenesis. We generated a loxP-floxed Lrp6 mouse line. Crossing with a general Cre deleter, we obtained the Lrp6-floxdel mice, in which the loxP-floxed exon 2 of Lrp6 gene has been deleted ubiquitously. The homozygotes of Lrp6-floxdel mice reproduced typical defects as seen in the conventional Lrp6-deficient mice, such as defects in eye, limb, and neural tube, and die around birth. We also found new phenotypes including cleft palate and agenesis of external genitalia in the Lrp6-floxdel mice. In addition, the Lrp6-deficient embryos are known to be defective in other systems and internal organs including the heart and brain. Thus, by selectively crossing with a lineage-specific or inducible Cre mouse line, the Lrp6 conditional gene-targeting mice will allow us to model specific types of birth defects for mechanism and prevention studies.

Myocardial smad4 is essential for cardiogenesis in mouse embryos.

Congenital heart diseases are the most commonly observed human birth defects and are the leading cause of infant morbidity and mortality. Accumulating evidence indicates that transforming growth factor-beta/bone morphogenetic protein signaling pathways play critical roles during cardiogenesis. Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of transforming growth factor-beta/bone morphogenetic protein signaling. The aim of this work was to investigate the roles of Smad4 during heart development. To overcome the early embryonic lethality of Smad4(-/-) mice, we specifically disrupted Smad4 in the myocardium using a Cre/loxP system. We show that myocardial-specific inactivation of Smad4 caused heart failure and embryonic lethality at midgestation. Histological analysis revealed that mutant mice displayed a hypocellular myocardial wall defect, which is likely the primary cause for heart failure. Both decreased cell proliferation and increased apoptosis contributed to the myocardial wall defect in mutant mice. Data presented in this article contradict a previous report showing that Smad4 is dispensable for heart development. Our further molecular characterization showed that expression of Nmyc and its downstream targets, including cyclin D1, cyclin D2, and Id2, were downregulated in mutant embryos. Reporter analysis indicated that the transcriptional activity of the 351-bp Nmyc promoter can be positively regulated by bone morphogenetic protein stimulation and negatively regulated by transforming growth factor-beta stimulation. Chromatin immunoprecipitation analysis revealed that the Nmyc promoter can form a complex with Smad4, suggesting that Nmyc is a direct downstream target of Smad4. In conclusion, this study provides the first mouse model showing that Smad4 plays essential roles during cardiogenesis.

Bipolar cell reduction precedes retinal ganglion neuron loss in a complex 1 knockout mouse model.

Inherited mitochondrial complex 1 deficiency causes Leber's hereditary Optic Neuropathy (LHON) and retinal ganglion cell (RGC) degeneration, and optic neuropathies are common in many inherited mitochondrial diseases. How mitochondrial defects pathomechanistically trigger optic neuropathy remains unclear. We observe that complex 1-deficient Ndufs4-/- mice present with acute vision loss around p30, and this vision loss is coincident with an 'inflammatory wave'. In order to understand what causes the inflammatory wave we explored retinal pathology that occurs from p20-p30. The results indicated that in the period p20-p30 in Ndufs4-/- retinas, there is: significant reduction in bipolar cells, RGC dendritic atrophy, reduced PSD95, increased oxidative stress as manifested by increased 4HNE, HO1 and Cuzn-SOD, increased mitochondrial biogenesis and increased apoptosis. These precede the major induction of 'inflammatory wave' at p30 shown previously, but occur earlier than frank RGC loss at p42. In general, complex 1 deficiency in retina triggers oxidative stress and mitochondrial respiratory dysfunction that causes death of the most sensitive cells, including bipolar cells and their synaptic contacts and amacrine cells in the early period, 20-24days. The early death of these cells is the likely precursor to the sharp rise in inflammatory molecules that occurs at day 30 and coincides with vision loss, and greatly precedes the death of RGCs that occurs at p42. These data suggest that metabolic antioxidant support of the most sensitive cells in the retina, or anti-inflammatory suppression of the consequences of their death, are both rational strategies for mitochondrial blinding disease.

Synthesis, characterization and catalytic epoxidation properties of lanthanide-stabilized peroxoisopolytungstates.

A series of new lanthanide-containing peroxoisopolyoxotungstates, K6Na4[H32{Ln4(WO4)(H2O)16[W7O22(O2)2]4}3]·105H2O [Ln = CeIII (1), NdIII (2), SmIII (3), TbIII (4), ErIII (5)], have been successfully synthesized and structurally characterized. All polyanions [Ln(WO4)(H2O)16{W7O22(O2)2}4]14- are isostructural and consist of a central [Ln(WO4)(H2O)16]10+ cluster surrounded by four peripheral [W7O22(O2)2]6- units. They could act as efficient recyclable catalysts for the epoxidation of various alkenes including different cycloalkenes, styrene derivatives, internal and long-chain alkenes. Under optimal conditions, catalyst 2 displays the best catalytic activity for the oxidation of cyclooctene with high cyclooctene conversion (98.3%) and excellent selectivity (up to 99%) and could be reused for three cycles with a negligible decrease in reactivity.

Mycobacterium tuberculosis infection and FHIT gene alterations in lung cancer.

Although it is fairly well accepted that pulmonary tuberculosis is a major risk factor of lung cancer, the exact molecular mechanisms involved in its tumorigenesis are unclear. For this purpose, we have examined the relationship between Mycobacterium tuberculosis (M-TB) infection and FHIT gene alteration in lung cancer. Tumors with M-TB infection had a slightly higher abnormal FHIT protein expression compared with tumors without M-TB infection, although not statistically significant (Fisher's exact test, P=0.248). LOH affecting at least one locus of the FHIT gene was significantly more frequent in lung cancer patients with M-TB infection than in patients without M-TB infection whether assessment by univariate testing methods or logistic regression modeling analysis (Fisher's exact test P=0.025, logistic regression analysis P=0.012). These results indicate that M-TB infection is associated with FHIT gene LOH in lung cancer.

Mitochondrial complex I defects increase ubiquitin in substantia nigra.

Parkinson׳s disease (PD) is the second most common neurodegenerative disorder in the developed world, and is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN) of midbrain. Mitochondrial complex I dysfunction has been implicated in PD pathophysiology, yet the molecular mechanism by which complex I defects may cause DA neurodegeneration remain unclear. Using Ndufs4 mouse model of mitochondrial complex I deficiency, we observed a remarkable ubiquitin protein increase in SN of Ndufs4-/- (KO) mice. By contrast, neurofilaments were significantly decreased in SN of KO mice. Furthermore, mass spectrometry and co-immunoprecipitation (Co-IP) analysis indicated an increase in ubiquitinated neurofilaments in midbrain of KO mice, whereas 20S proteasome activities were decreased, which could potentially explain the buildup of ubiquitin protein. Collectively, these data suggest that mitochondrial complex I defects cause proteasome inhibition, a consequent increase in ubiquitinated neurofilaments and other proteins, and decrease the expression of neurofilaments that could be relevant to the mechanism of DA neuronal death in PD.

[Detection of Mycobacterium tuberculosis in lung cancer tissue by indirect in situ nested PCR].

OBJECTIVE: To localize Mycobacterium tuberculosis DNA (TB-DNA) in lung cancer tissue and to investigate the possible relationship of this bacterial infection with the development of lung cancer. METHODS: A sensitive and specific indirect in situ nested PCR (ISNPCR) was used to identify and localize TB-DNA in 15 formalin fixed paraffin-embedded lung cancer tissue specimens, which had been demonstrated to be positive for TB-DNA by conventional PCR. RESULTS: Positive granules of TB-DNA in brown color was found mainly in the cytoplasm of the alveolar epithelial cells, pulmonary macrophages, inflammatory cells and a few tumor cells within lung cancer tissues. CONCLUSION: Mycobacterium tuberculosis infection may play a role in the pathogenesis of lung cancer.

Critical roles of miRNA-mediated regulation of TGFß signalling during mouse cardiogenesis.

AIMS: MicroRNAs (miRNAs) play critical roles during the development of the cardiovascular system. Blocking miRNA biosynthesis in embryonic hearts through a conditional gene inactivation approach led to differential cardiac defects depending on the Cre drivers used in different studies. The goal of this study is to reveal the cardiogenic pathway that is regulated by the miRNA mechanism at midgestation, a stage that has not been evaluated in previous publications. METHODS AND RESULTS: We specifically inactivated Dicer1, which is essential for generation of functional mature miRNAs, in the myocardium by crossing cTnt-Cre mice with Dicer1(loxP) mice. cTnt-Cre efficiently inactivates target genes in cardiomyocytes at midgestation. All mutants died between E14.5 and E16.5 with severe myocardial wall defects, including reduced cell proliferation, increased cell death, and spongy myocardial wall. Expression of TGFß type I receptor (Tgfbr1), which encodes the Type I receptor of TGFß ligands, was up-regulated in mutant hearts. As expected, TGFß activity was increased in Dicer1-inactivated hearts. Our further molecular analysis suggested that Tgfbr1 is a direct target of three miRNAs. Reducing TGFß activities using a pharmacological inhibitor on in vitro cultured hearts, or through an in vivo genetic approach, partially rescued the cardiac defects caused by Dicer1 inactivation. CONCLUSIONS: We show for the first time that TGFß signalling is directly regulated by the miRNA mechanism during myocardial wall morphogenesis. Increased TGFß activity plays a major role in the cardiac defects caused by myocardial deletion of Dicer1. Thus, miRNA-mediated regulation of TGFß signalling is indispensable for normal cardiogenesis.

Activation of the Wnt/beta-catenin signaling reporter in developing mouse olfactory nerve layer marks a specialized subgroup of olfactory ensheathing cells.

Wnt reporter TOPgal mice carry a beta-galactosidase (betagal) gene under the control of the Wnt/beta-catenin signaling responsive elements. We found that the intensely immunolabeled betagal+ cells were co-immunolabeled with Nestin and formed a tangentially oriented single-cell layer in the "connecting or docking zone" where the olfactory sensory axons attached to the brain surface during mid-gestation. During early postnatal development, betagal+ cells were located in the inner olfactory nerve layer (ONLi) and co-labeled with olfactory ensheathing cell (OEC) markers S100beta and NPY but not with lineage-specific markers for neurons, oligodendrocytes, astrocytes, and microglia, demonstrating that the TOPgal marked a subpopulation of OECs. By confocal microscopy, we found that TOPgal activated processes extended along the developing glomerulus and formed multiple tunnel-like structures that ensheathe and bridge olfactory sensory axonal bundles from ONLi to the glomerulus, which may play a key role in glomerulus formation and convergent sorting of the peripheral olfactory axons.

Ocular coloboma and dorsoventral neuroretinal patterning defects in Lrp6 mutant eyes.

Coloboma, an ocular birth defect seen in humans and other species, is caused by incomplete closure of the optic fissure. Here, we demonstrate that genetic deletion of Lrp6, a bottleneck coreceptor in the canonical Wnt signaling pathway, results in ocular coloboma and neuroretinal patterning defects in mice. The expression of ventral neuroretinal patterning gene Vax2 was conserved but with dorsally shifted expression domains; however, the dorsal neuroretinal patterning gene Tbx5 was lost in the Lrp6-mutant eyes at embryonic day 10.5. Both Bmp4 and phosphorylated Smad 1/5/8 were also significantly attenuated in the dorsal neuroretina. In addition, the retinoic acid synthesizing enzymes Raldh1 and Raldh3 were significantly changed in the mutant eyes. Our findings suggest that defective retinal patterning causes coloboma in the Lrp6-deficient mice, and that canonical Wnt signaling plays a primary role in dorsal neuroretinal patterning and related morphogenetic movements by regulation of both Bmp and retinoic acid signaling pathways.

Essential functions of Alk3 during AV cushion morphogenesis in mouse embryonic hearts.

Accumulated evidence has suggested that BMP pathways play critical roles during mammalian cardiogenesis and impairment of BMP signaling may contribute to human congenital heart diseases (CHDs), which are the leading cause of infant morbidity and mortality. Alk3 encodes a BMP specific type I receptor expressed in mouse embryonic hearts. To reveal functions of Alk3 during atrioventricular (AV) cushion morphogenesis and to overcome the early lethality of Alk3(-/-) embryos, we applied a Cre/loxp approach to specifically inactivate Alk3 in the endothelium/endocardium. Our studies showed that endocardial depletion of Alk3 severely impairs epithelium-mesenchymal-transformation (EMT) in the atrioventricular canal (AVC) region; the number of mesenchymal cells formed in Tie1-Cre;Alk3(loxp/loxp) embryos was reduced to only approximately 20% of the normal level from both in vivo section studies and in vitro explant assays. We showed, for the first time, that in addition to its functions on mesenchyme formation, Alk3 is also required for the normal growth/survival of AV cushion mesenchymal cells. Functions of Alk3 are accomplished through regulating expression/activation/subcellular localization of multiple downstream genes including Smads and cell-cycle regulators. Taken together, our study supports the notion that Alk3-mediated BMP signaling in AV endocardial/mesenchymal cells plays a central role during cushion morphogenesis.