Biomedical subjects

Yuji Mishina

Publications and source records attributed to Yuji Mishina.

At least 19 recordsLinked to original sources

PubMed2007

BMP type I receptor ALK2 is essential for proper patterning at late gastrulation during mouse embryogenesis.

Bone morphogenetic proteins (BMPs) have multiple functions during vertebrate development. Previously, it was shown that BMP type I receptor ALK2 (also known as ACVRI, ActRI, or ActRIA) was important for normal mouse gastrulation by deleting exon 4 or exon 5 of Alk2. Recently, flanking exon 7 by loxP sites generated a conditional allele for Alk2. To assess whether the deletion of exon 7 causes functional null of ALK2, and does not produce a dominant negative form or a partially functional form of ALK2, we performed a comparative analysis between Alk2 homozygous mutant embryos with an exon 5 deletion (Alk2(Delta5/Delta5)) and embryos with an exon 7 deletion (Alk2(Delta7/Delta7)). Both Alk2(Delta5/Delta5) and Alk2(Delta7/Delta7) mutants showed identical morphological gastrulation defects. Histological examinations and molecular marker analyses revealed identical abnormal gastrulation phenotypes in Alk2(Delta5/Delta5) and Alk2(Delta7/Delta7) mutants. Although Fgf8 was expressed in the primitive streak of Alk2(Delta5/Delta5) and Alk2(Delta7/Delta7) mutants, Brachyury, Wnt3a, and Tbx6 were dramatically downregulated in Alk2(Delta5/Delta5) and Alk2(Delta7/Delta7) mutants. These results indicate that deletion of exon 7 for Alk2 leads to a functionally null mutation in vivo, and Alk2 is crucial for sustaining the proper gastrulation events in early mouse embryogenesis.

Activin Receptors, Type I↗

PubMed2006

BMPs regulate multiple aspects of growth-plate chondrogenesis through opposing actions on FGF pathways.

Bone morphogenetic protein (BMP) signaling pathways are essential regulators of chondrogenesis. However, the roles of these pathways in vivo are not well understood. Limb-culture studies have provided a number of essential insights, including the demonstration that BMP pathways are required for chondrocyte proliferation and differentiation. However, limb-culture studies have yielded contradictory results; some studies indicate that BMPs exert stimulatory effects on differentiation, whereas others support inhibitory effects. Therefore, we characterized the skeletal phenotypes of mice lacking Bmpr1a in chondrocytes (Bmpr1a(CKO)) and Bmpr1a(CKO);Bmpr1b+/- (Bmpr1a(CKO);1b+/-) in order to test the roles of BMP pathways in the growth plate in vivo. These mice reveal requirements for BMP signaling in multiple aspects of chondrogenesis. They also demonstrate that the balance between signaling outputs from BMP and fibroblast growth factor (FGF) pathways plays a crucial role in the growth plate. These studies indicate that BMP signaling is required to promote Ihh expression, and to inhibit activation of STAT and ERK1/2 MAPK, key effectors of FGF signaling. BMP pathways inhibit FGF signaling, at least in part, by inhibiting the expression of FGFR1. These results provide a genetic in vivo demonstration that the progression of chondrocytes through the growth plate is controlled by antagonistic BMP and FGF signaling pathways.

Animals↗

PubMed2006

Nonsteroidal anti-inflammatory drug-activated gene-1 over expression in transgenic mice suppresses intestinal neoplasia.

BACKGROUND & AIMS: The nonsteroidal anti-inflammatory drug-activated gene (NAG-1) was identified as a proapoptotic, antitumorigenic protein in vitro, induced by many antitumorigenic and chemopreventive drugs including cyclooxygenase inhibitors. However, its antitumorigenic activity has not been elucidated in vivo. METHODS: Transgenic mice were generated that ubiquitously overexpress human NAG-1 under the control of a chicken beta-actin promoter (CAG). The NAG-1 transgenic mice (NAG-(Tg+)) were characterized, and then the antitumorigenic activity was evaluated with 2 colorectal carcinogenesis models: chemical induction with azoxymethane and genetic induction using the Apc(Min+) mutation. RESULTS: NAG-(Tg+) showed no apparent phenotype other than a reduction in body weight, particularly in males. To examine whether NAG-1 expression would suppress intestinal tumorigenesis, the NAG-(Tg+) mice were treated with the colorectal carcinogen azoxymethane. NAG-(Tg+) mice developed 50% fewer aberrant crypt foci and no tumors, in comparison with nontransgenic littermates. This result demonstrates that expression of this human protein in vivo can suppress chemically induced carcinogenesis in the colon. The NAG-(Tg+) mice were also crossed with Apc(Min+) mice to determine the effect of the transgene on intestinal polyp formation. NAG-(Tg+) mice heterozygous for the Apc(Min+) mutation had a significantly reduced polyp load (60%) compared with nontransgenic Apc(Min+) littermates. CONCLUSIONS: Our results support NAG-1 as an important regulator of intestinal adenoma growth in vivo and suggest that NAG-1 may act as a tumor suppressor gene.

Animals↗

PubMed2006

Bone morphogenetic protein signaling inhibits hair follicle anagen induction by restricting epithelial stem/progenitor cell activation and expansion.

Epithelial stem cells (EP-SCs) located in the bulge region of a hair follicle (HF) have the potential to give rise to hair follicle stem/progenitor cells that migrate down to regenerate HFs. Bone morphogenetic protein (BMP) signaling has been shown to regulate the HF cycle by inhibiting anagen induction. Here we show that active BMP signaling functions to prevent EP-SC activation and expansion. Dynamic expression of Noggin, a BMP antagonist, releases EP-SCs from BMP-mediated restriction, leading to EP-SC activation and initiation of the anagen phase. Experimentally induced conditional inactivation of the BMP type IA receptor (Bmpr1a) in EP-SCs leads to overproduction of HF stem/progenitor cells and the eventual formation of matricomas. This genetic manipulation of the BMP signaling pathway also reveals unexpected activation of beta-catenin, a major mediator of Wnt signaling. We propose that BMP activity controls the HF cycle by antagonizing Wnt/beta-catenin activity. This is at least partially achieved by BMP-mediated enhancement of transforming growth factor-beta-regulated epithelial cell-specific phosphatase (PTEN) function. Subsequently, PTEN, through phosphatidyl inositol 3-kinase-Akt, inhibits the activity of beta-catenin, the convergence point of the BMP and Wnt signaling pathways.

Animals↗

PubMed2006

BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites.

Bmpr1a encodes the BMP type IA receptor for bone morphogenetic proteins (BMPs), including 2 and 4. Here, we use mosaic inactivation of Bmpr1a in the epiblast of the mouse embryo (Bmpr-MORE embryos) to assess functions of this gene in mesoderm development. Unlike Bmpr1a-null embryos, which fail to gastrulate, Bmpr-MORE embryos initiate gastrulation, but the recruitment of prospective paraxial mesoderm cells to the primitive streak is delayed. This delay causes a more proximal distribution of cells with paraxial mesoderm character within the primitive streak, resulting in a lateral expansion of somitic mesoderm to form multiple columns. Inhibition of FGF signaling restores the normal timing of recruitment of prospective paraxial mesoderm and partially rescues the development of somites. This suggests that BMP and FGF signaling function antagonistically during paraxial mesoderm development.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

Bone morphogenetic protein receptor 1A signaling is dispensable for hematopoietic development but essential for vessel and atrioventricular endocardial cushion formation.

Bone morphogenetic protein 4 (BMP4) is crucial for the formation of FLK1-expressing (FLK1(+)) mesodermal cells. To further define the requirement for BMP signaling in the differentiation of blood, endothelial and smooth muscle cells from FLK1(+) mesoderm, we inactivated Alk3 (Bmpr1a) in FLK1(+) cells by crossing Alk3(floxed/floxed) and Flk1(+/Cre)Alk3(+/floxed) mice. Alk3 conditional knockout (CKO) mice died between E10.5 and E11.5. Unexpectedly, Alk3 CKO embryos did not show any hematopoietic defects. However, Alk3 CKO embryos displayed multiple abnormalities in vascular development, including vessel remodeling and maturation, which contributed to severe abdominal hemorrhage. Alk3 CKO embryos also displayed defects in atrioventricular canal (AVC) endocardial cushion formation in the heart. Collectively, our studies indicate a crucial role for ALK3 in vessel remodeling, vessel integrity and endocardial cushion formation during the development of the circulation system.

Animals↗

PubMed2006

BMP receptor type IA in limb bud mesenchyme regulates distal outgrowth and patterning.

The mesenchyme of the developing vertebrate limb responds to inductive signals, giving rise to skeletal elements that define limb shape and size. Several signals emanate from the limb ectoderm and in particular from the specialized epithelium of the apical ectodermal ridge (AER), including three members of the bone morphogenetic protein (BMP) family of signaling molecules, BMP2, BMP4 and BMP7. Using the Cre/loxP system in mice, we rendered limb bud mesenchyme insensitive to BMP signals through the type I receptor, BMPR-IA. Conditional mutants had shortened limbs and almost complete agenesis of the autopod because of reduced cell proliferation. Reduced expression of downstream BMP signaling targets, Msx1, Msx2 and gremlin in the distal mesenchyme (progress zone) correlated with decreased levels of cyclin D1 and Wnt5a. Ectopic anterior activation of sonic hedgehog (SHH) signaling and Hox expression revealed alterations in anterior-posterior (AP) patterning. Abnormal localization of Lmx1b-expressing cells in the ventral mesenchyme, along with histological alterations and an abnormal melanization pattern of the limb, indicate altered dorsal-ventral (DV) boundaries. These findings suggest that signaling through BMPR-IA in limb mesenchyme is essential for distal outgrowth and also influences AP and DV patterning.

Animals↗

PubMed2006

Evidence that autocrine signaling through Bmpr1a regulates the proliferation, survival and morphogenetic behavior of distal lung epithelial cells.

Lung development requires reciprocal epithelial/mesenchymal interactions, mediated by signaling factors such as Bmps made in both cell populations. To address the role of Bmp signaling in the epithelium, we have exploited the fact that Bmp receptor type Ia (Alk3) is expressed in the epithelium during branching morphogenesis. Deletion of Bmpr1a in the epithelium with an Sftpc-cre transgene leads to dramatic defects in lung development. There is reduced epithelial proliferation, extensive apoptosis, changes in cell morphology and extrusion of cells into the lumen. By E18.5, there are fewer Type II cells than normal, and the lung contains large fluid-filled spaces. If cell death is prevented by making embryos homozygous null for the proapoptotic gene, Bax, the epithelial cells that are rescued can apparently differentiate, but normal morphogenesis is not restored. To determine whether Bmps made by the epithelium can function in an autocrine manner, mesenchyme-free endoderm was cultured in Matrigel with Fgfs. Under these conditions, the mutant epithelium fails to undergo secondary budding. Abnormal development was also seen when Bmp4 was specifically deleted in the epithelium using the Sftpc-cre transgene. Our results support a model in which Bmp signaling primarily regulates the proliferation, survival and morphogenetic behavior of distal lung epithelial cells.

Animals↗

PubMed2006

Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2.

BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines.

Activin Receptors, Type I↗

PubMed2006

Optimized beta-galactosidase staining method for simultaneous detection of endogenous gene expression in early mouse embryos.

beta-Galactosidase (beta-gal) is one of the popular reporters for detecting the expression of endogenous or exogenous genes. Here we report 6-chloro-3-indoxyl-beta-D-galactopyranoside (S-gal) is more sensitive for beta-gal activity than 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal), particularly during the early developmental stages of mouse embryos. Further, we successfully combined beta-gal staining with S-gal and in situ hybridization using DIG-labeled probes in both whole and sections of early stage embryos due to the sensitivity and color compatibility of S-gal.

Animals↗

PubMed2006

Genetically modified bone morphogenetic protein signalling alters traumatic brain injury-induced gene expression responses in the adult mouse.

Three genetic mouse models were examined to define effects of bone morphogenetic protein (BMP) signalling on gene expression in normal and injured adult brain. CaMKII-Cre eliminated the BMP receptor Acvr1 (Alk2) and the common TGFbeta superfamily signal mediator Smad4 or activated a constitutively active Acvr1 in postnatal forebrain neurons. All mutants followed mendelian ratios, with no overt phenotypic changes. In situ hybridization demonstrated normal patterns of the dendritic marker MAP2 (Mtap2) throughout cortex despite neuron-specific losses of Acvr1 or Smad4. However, strong up-regulation of Mtap2 transcript in these mice was found by quantitative RT-PCR (qRT-PCR), indicating that Mtap2 is normally suppressed by BMP. Traumatic brain injury (TBI) resulted in increases of histone-associated DNA fragments in both control and Smad4-deficient cortex. Several cell-type-specific transcripts known to be involved in injury-related responses were measured by qRT-PCR. Gfap mRNA was strongly up-regulated in controls as well as in the loss-of-BMP-signalling mutants. Notably, activated Acvr1 signalling gave significantly lower TBI-induced up-regulations of Gfap and Phox2a mRNA levels, indicating reductions in astroglial and neuronal reactions to injury. Strong impairment in injury-induced Timp1 transcript up-regulation was also seen in these mice. In contrast, osteopontin (Spp1) transcript levels in activated microglia were not reduced by Acvr1 signalling. Altogether, the data suggest that BMP signalling is dispensable in adult cortical neurons but that augmented BMP signalling affects molecular changes associated with neuronal lesions.

Activin Receptors, Type I↗

PubMed2005

Alk3/Bmpr1a receptor is required for development of the atrioventricular canal into valves and annulus fibrosus.

Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein's anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus.

Animals↗

PubMed2005

Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo.

Previous studies have demonstrated the ability of bone morphogenetic proteins (BMPs) to promote chondrogenic differentiation in vitro. However, the in vivo role of BMP signaling during chondrogenesis has been unclear. We report here that BMP signaling is essential for multiple aspects of early chondrogenesis. Whereas mice deficient in type 1 receptors Bmpr1a or Bmpr1b in cartilage are able to form intact cartilaginous elements, double mutants develop a severe generalized chondrodysplasia. The majority of skeletal elements that form through endochondral ossification are absent, and the ones that form are rudimentary. The few cartilage condensations that form in double mutants are delayed in the prechondrocytic state and never form an organized growth plate. The reduced size of mutant condensations results from increased apoptosis and decreased proliferation. Moreover, the expression of cartilage-specific extracellular matrix proteins is severely reduced in mutant elements. We demonstrate that this defect in chondrocytic differentiation can be attributed to lack of Sox9, L-Sox5, and Sox6 expression in precartilaginous condensations in double mutants. In summary, our study demonstrates that BMPR1A and BMPR1B are functionally redundant during early chondrogenesis and that BMP signaling is required for chondrocyte proliferation, survival, and differentiation in vivo.

Animals↗

PubMed2005

Distinct functions for Bmp signaling in lip and palate fusion in mice.

Previous work suggested that cleft lip with or without cleft palate (CL/P) is genetically distinct from isolated cleft secondary palate (CP). Mutations in the Bmp target gene Msx1 in families with both forms of orofacial clefting has implicated Bmp signaling in both pathways. To dissect the function of Bmp signaling in orofacial clefting, we conditionally inactivated the type 1 Bmp receptor Bmpr1a in the facial primordia, using the Nestin cre transgenic line. Nestin cre; Bmpr1a mutants had completely penetrant, bilateral CL/P with arrested tooth formation. The cleft secondary palate of Nestin cre; Bmpr1a mutant embryos was associated with diminished cell proliferation in maxillary process mesenchyme and defective anterior posterior patterning. By contrast, we observed elevated apoptosis in the fusing region of the Nestin cre; Bmpr1a mutant medial nasal process. Moreover, conditional inactivation of the Bmp4 gene using the Nestin cre transgenic line resulted in isolated cleft lip. Our data uncover a Bmp4-Bmpr1a genetic pathway that functions in lip fusion, and reveal that Bmp signaling has distinct roles in lip and palate fusion.

Animals↗

PubMed2005

Distinct developmental programs require different levels of Bmp signaling during mouse retinal development.

The Bmp family of secreted signaling molecules is implicated in multiple aspects of embryonic development. However, the cell-type-specific requirements for this signaling pathway are often obscure in the context of complex embryonic tissue interactions. To define the cell-autonomous requirements for Bmp signaling, we have used a Cre-loxP strategy to delete Bmp receptor function specifically within the developing mouse retina. Disruption of a Bmp type I receptor gene, Bmpr1a, leads to no detectable eye abnormality. Further reduction of Bmp receptor activity by removing one functional copy of another Bmp type I receptor gene, Bmpr1b, in the retina-specific Bmpr1a mutant background, results in abnormal retinal dorsoventral patterning. Double mutants completely lacking both of these genes exhibit severe eye defects characterized by reduced growth of embryonic retina and failure of retinal neurogenesis. These studies provide direct genetic evidence that Bmpr1a and Bmpr1b play redundant roles during retinal development, and that different threshold levels of Bmp signaling regulate distinct developmental programs such as patterning, growth and differentiation of the retina.

Animals↗

PubMed2005

BMP signaling and early embryonic patterning.

Bone morphogenetic proteins (BMPs) play pleiotropic roles during embryonic development as well as throughout life. Recent genetic approaches especially using the mouse gene knockout system revealed that BMP signaling is greatly involved in early embryonic patterning, which is a dynamic event to establish three-dimensional polarities. The purpose of this review is to describe the diverse function of BMPs through different receptor signaling systems during embryonic patterning including gastrulation and establishment of the left-right asymmetry.

Animals↗

PubMed2005

Conditional transgenic system for mouse aurora a kinase: degradation by the ubiquitin proteasome pathway controls the level of the transgenic protein.

Aurora A is a mitotic kinase that localizes to centrosomes. Expression of this protein is normally limited to the mitotic stage (G(2)-M) of the cell cycle, whereas human cancer cells frequently exhibit overexpression of Aurora A protein regardless of the cell cycle stage. In the present study, Aurora A transgenic mouse lines were generated with a new conditional expression system (cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter-Z-enhanced green fluorescent protein) in order to analyze the function of this protein. Although transcripts for Aurora A were elevated in multiple organs of the transgenic mice, the corresponding protein was not detected in extracts analyzed by immunoblotting. The treatment of transgenic-derived embryonic fibroblasts (MEF) with proteasome inhibitors markedly increased the protein level of transgenic Aurora A, indicating that the transgenic Aurora A protein is readily degraded in normal mouse tissues. Under the exponential growth conditions of MEF cells, transgenic Aurora A was detected within the mitotic stage of the cell cycle and localized to centrosomes. In contrast, the marker of the transgenic promoter (enhanced green fluorescent protein) was continuously expressed throughout the cell cycle, indicating the constitutive transcription of transgenic mRNA. These results indicate that transgenic Aurora A is protected from degradation within G(2)-M but is immediately degraded after translation in the G(1)-S stage of the cell cycle. The findings obtained with this transgenic model and derived cells support that the transition from protection to degradation by the ubiquitin proteasome system at the end of mitosis is an important step in controlling the level of Aurora A protein during the cell cycle.

Animals↗