Publications by authors named "Ryuma Haraguchi"

45 Publications

New Insights into Development of Female Reproductive Tract-Hedgehog-Signal Response in Wolffian Tissues Directly Contributes to Uterus Development.

Int J Mol Sci 2021 Jan 26;22(3). Epub 2021 Jan 26.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

The reproductive tract in mammals emerges from two ductal systems during embryogenesis: Wolffian ducts (WDs) and Mullerian ducts (MDs). Most of the female reproductive tract (FRT) including the oviducts, uterine horn and cervix, originate from MDs. It is widely accepted that the formation of MDs depends on the preformed WDs within the urogenital primordia. Here, we found that the WD mesenchyme under the regulation of Hedgehog (Hh) signaling is closely related to the developmental processes of the FRT during embryonic and postnatal periods. Deficiency of Sonic hedgehog (Shh), the only Hh ligand expressed exclusively in WDs, prevents the MD mesenchyme from affecting uterine growth along the radial axis. The in vivo cell tracking approach revealed that after WD regression, distinct cells responding to WD-derived Hh signal continue to exist in the developing FRT and gradually contribute to the formation of various tissues such as smooth muscle, endometrial stroma and vascular vessel, in the mouse uterus. Our study thus provides a novel developmental mechanism of FRT relying on WD.
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http://dx.doi.org/10.3390/ijms22031211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865753PMC
January 2021

Localization of DLL1- and NICD-positive osteoblasts in cortical bone during postnatal growth in rats.

Biochem Biophys Res Commun 2020 08 22;529(2):186-190. Epub 2020 Jun 22.

Laboratory of Veterinary Anatomy, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino City, Tokyo, 180-8602, Japan.

The long bone midshaft expands by forming primary osteons at the periosteal surface of cortical bone in humans and rodents. Osteoblastic bone formation in the vascular cavity in the center of primary osteons is delayed during cortical bone development. The mechanisms of the formation of primary osteons is not fully understood, however. Focusing on NOTCH1 signaling, an inhibitory signaling on osteoblastic bone formation, our immunohistochemical analysis revealed Delta like1 (DLL1), a ligand of NOTCH1, and the NOTCH1 intracellular domain (NICD, an activated form of NOTCH1) immunoreactivity, in the cuboidal osteoblasts lining the bone surface in the vascular cavity of primary osteons during postnatal growth in rats. Interestingly, five days after treatment of primary osteoblasts with ascorbic acid and β glycerophosphate, protein levels of both DLL1 and NICD increased transiently, indicating that DLL1 activates NOTCH1 in primary cultured osteoblasts. Thus, the results imply that DLL1-NOTCH1 signaling in osteoblasts is associated with primary osteonal bone formation.
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http://dx.doi.org/10.1016/j.bbrc.2020.06.039DOI Listing
August 2020

New Insights into the Pathogenesis of Diabetic Nephropathy: Proximal Renal Tubules Are Primary Target of Oxidative Stress in Diabetic Kidney.

Acta Histochem Cytochem 2020 Apr 25;53(2):21-31. Epub 2020 Apr 25.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

Diabetic nephropathy is a major source of end-stage renal failure, affecting about one-third cases of diabetes mellitus. It has long been accepted that diabetic nephropathy is mainly characterized by glomerular defects, while clinical observations have implied that renal tubular damage is closely linked to kidney dysfunction at the early stages of diabetic nephropathy. In this study, we conducted pathohistological analyses focusing on renal tubular lesions in the early-stage diabetic kidney with the use of a streptozotocin (STZ)-induced diabetes mellitus mouse model. The results revealed that histological alterations in renal tubules, shown by a vacuolar nucleic structure, accumulations of PAS-positive substance, and accelerated restoration stress, occur initially without the presence of glomerular lesions in the early-stage diabetic kidney, and that these tubular defects are localized mainly in proximal renal tubules. Moreover, enhanced expression of RAGE, suggesting an aberrant activation of AGEs-RAGE signaling pathway, and accumulation of oxidative modified mitochondria through the impaired autophagy/lysosome system, were also seen in the damaged diabetic proximal renal tubules. Our findings indicate that proximal tubular defects are the initial pathological events increasingly linked to the progression of diabetic nephropathy, and that controlling renal tubular damage could be an effective therapeutic strategy for the clinical treatment of diabetic nephropathy.
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http://dx.doi.org/10.1267/ahc.20008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212204PMC
April 2020

Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss.

Int J Mol Sci 2020 Apr 15;21(8). Epub 2020 Apr 15.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0-48 h (early stage of osteoclast differentiation) or 48-96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.
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http://dx.doi.org/10.3390/ijms21082745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216259PMC
April 2020

Knockdown of Lrp1 in RAW264 cells inhibits osteoclast differentiation and osteoclast-osteoblast interactions in vitro.

Biochem Biophys Res Commun 2020 03 19;523(4):961-965. Epub 2020 Jan 19.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Low density lipoprotein receptor-related protein 1 (LRP1), a multifunctional cell surface protein, is expressed in bone marrow-derived macrophages. While LRP1 is thought to be a suppressor of osteoclast differentiation at late stages, its function at early stages remains unclear. Here we demonstrate that Lrp1 stable knockdown by lentiviral short hairpin RNA in macrophage cell line RAW264 cells inhibited RANKL-induced osteoclast formation and osteoclastic master transcription factor Nfatc1 mRNA expression as assessed by quantitative RT-PCR. Furthermore, knockdown of the Lrp1 gene suppressed not only differentiation, but also proliferation, and inhibitory effects on osteoblastic ALP activity by osteoclast-derived humoral factors. Thus, we propose that LRP1 in macrophages is required for both differentiation into osteoclasts and osteoclast-osteoblast interactions.
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http://dx.doi.org/10.1016/j.bbrc.2020.01.065DOI Listing
March 2020

Hedgehog Signaling for Urogenital Organogenesis and Prostate Cancer: An Implication for the Epithelial-Mesenchyme Interaction (EMI).

Int J Mol Sci 2019 Dec 20;21(1). Epub 2019 Dec 20.

Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University, Kimiidera 811-1, Wakayama 641-8509, Japan.

Hedgehog (Hh) signaling is an essential growth factor signaling pathway especially in the regulation of epithelial-mesenchymal interactions (EMI) during the development of the urogenital organs such as the bladder and the external genitalia (EXG). The Hh ligands are often expressed in the epithelia, affecting the surrounding mesenchyme, and thus constituting a form of paracrine signaling. The development of the urogenital organ, therefore, provides an intriguing opportunity to study EMI and its relationship with other pathways, such as hormonal signaling. Cellular interactions of prostate cancer (PCa) with its neighboring tissue is also noteworthy. The local microenvironment, including the bone metastatic site, can release cellular signals which can affect the malignant tumors, and vice versa. Thus, it is necessary to compare possible similarities and divergences in Hh signaling functions and its interaction with other local growth factors, such as BMP (bone morphogenetic protein) between organogenesis and tumorigenesis. Additionally, this review will discuss two pertinent research aspects of Hh signaling: (1) the potential signaling crosstalk between Hh and androgen signaling; and (2) the effect of signaling between the epithelia and the mesenchyme on the status of the basement membrane with extracellular matrix structures located on the epithelial-mesenchymal interface.
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http://dx.doi.org/10.3390/ijms21010058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982176PMC
December 2019

Recent Insights into Long Bone Development: Central Role of Hedgehog Signaling Pathway in Regulating Growth Plate.

Int J Mol Sci 2019 Nov 20;20(23). Epub 2019 Nov 20.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City 791-0295, Japan.

The longitudinal growth of long bone, regulated by an epiphyseal cartilaginous component known as the "growth plate", is generated by epiphyseal chondrocytes. The growth plate provides a continuous supply of chondrocytes for endochondral ossification, a sequential bone replacement of cartilaginous tissue, and any failure in this process causes a wide range of skeletal disorders. Therefore, the cellular and molecular characteristics of the growth plate are of interest to many researchers. Hedgehog (Hh), well known as a mitogen and morphogen during development, is one of the best known regulatory signals in the developmental regulation of the growth plate. Numerous animal studies have revealed that signaling through the Hh pathway plays multiple roles in regulating the proliferation, differentiation, and maintenance of growth plate chondrocytes throughout the skeletal growth period. Furthermore, over the past few years, a growing body of evidence has emerged demonstrating that a limited number of growth plate chondrocytes transdifferentiate directly into the full osteogenic and multiple mesenchymal lineages during postnatal bone development and reside in the bone marrow until late adulthood. Current studies with the genetic fate mapping approach have shown that the commitment of growth plate chondrocytes into the skeletal lineage occurs under the influence of epiphyseal chondrocyte-derived Hh signals during endochondral bone formation. Here, we discuss the valuable observations on the role of the Hh signaling pathway in the growth plate based on mouse genetic studies, with some emphasis on recent advances.
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http://dx.doi.org/10.3390/ijms20235840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928971PMC
November 2019

Modulation of αβ Integrin via Transactivation of β Integrin Gene on Murine Bone Marrow Macrophages by 1,25(OH)D, Retinoic Acid and Interleukin-4.

Acta Histochem Cytochem 2019 Aug 10;52(4):77-83. Epub 2019 Aug 10.

Department of Molecular Pathology, Ehime University Graduate School of Medicine.

The interleukin (IL)-4, 1,25(OH)D and retinoic acid, increase surface expression of functional integrin αβ on murine osteoclast precursors. All three agonists stimulate transcription of the β gene, leading to increased steady-state levels of mRNA this protein. By contrast, mRNA levels of α remain unchanged. In each instance, the increase in the surface expression of the integrin results in increased migration of the cells onto an αβ substrate. Because β subunit, except platelet where β subunit conform a dimer with α, associates solely with α subunit monogamously, while promiscuous α subunit combines with various subunit, our present data support the idea that the β subunit governs the surface-expressed functional integrin complex.
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http://dx.doi.org/10.1267/ahc.19015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773611PMC
August 2019

Activation of protein kinase C accelerates murine osteoclastogenesis partly via transactivation of RANK gene through functional AP-1 responsive element in RANK gene promoter.

Biochem Biophys Res Commun 2019 07 28;515(2):268-274. Epub 2019 May 28.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan; Division of Diagnostic Molecular Pathology, Kobe University Graduate School of Medicine, Kusunoki-cho 7-5-1, Kobe, 650-0017, Japan. Electronic address:

Receptor activator of NF-κB (RANK) expressed on osteoclasts and their precursors is a receptor for RANK ligand (RANKL). Signals transduced by RANKL-RANK interaction induce genes essential for the differentiation and function of osteoclasts. We have cloned a basic promoter region of the mouse RANK gene and have analyzed the transcription machinery by transcription factors such as PU.1 (-480), and MITF (-100). Here, we examined the regulatory mechanisms of RANK gene transcription through AP-1 binding site, agagctca (-240). RANK mRNA expression in pre-osteoclastic RAW264.7 cells was induced by Phorbol12-myristate13-acetate (PMA) and suppressed by protein kinase C (PKC) inhibitor calphostin C. In RAW264.7 cells, Fos knockdown by siRNA blocked the inducible effect of PMA on RANK expression. By EMSA, an oligonucleotide (-246/-238) showed DNA protein binding, the specificity of which was confirmed by block-shift assay with an anti-Fos antibody and by the addition of the excess of a cold consensus probe. Co-transfection with a Fos expression vector showed that Fos increased RANK promoter activity 6-fold in RAW264.7 cells, and the addition of PU.1 and MITF superinduced the activity more than twenty-fold by the addition of PU.1 and MITF. Mutagenesis of the putative AP-1 site (-240) blocked the inducible effect of Fos on promoter activity. Taken together, these results indicate that during the differentiation of bone marrow mono-nucleated cells into osteoclast precursors, RANK transcription is positively regulated by Fos/AP-1 through the binding element of its gene promoter, supporting the concept that Fos activation by continuous CSF-1 stimulation on macrophages triggers initial expression of RANK and, later, a positive feedback loop by RANKL-RANK interaction.
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http://dx.doi.org/10.1016/j.bbrc.2019.05.144DOI Listing
July 2019

Morphology-oriented epigenetic research.

Histochem Cell Biol 2018 Jul 2;150(1):3-12. Epub 2018 May 2.

Division of Diagnostic Pathology, Ehime University Hospital, Toon City, Ehime, 791-0295, Japan.

Cytosine methylation plays a major role in the regulation of sequential and tissue-specific expression of genes. De novo aberrant DNA methylation and demethylation are also crucial processes in tumorigenesis and tumor progression. The mechanisms of how and when such aberrant methylation and demethylation occur in tumor cells are still obscure, however. To evaluate subtle epigenetic alteration among minor subclonal populations, morphology-oriented epigenetic analysis is requisite, especially where heterogeneity and flexibility are as notable as in the process of cancer progression and cellular differentiation at critical stages. Therefore, establishment of reliable morphology-oriented epigenetic studies has become increasingly important in not only the experimental but also the diagnostic field. By selecting a subset of cells based on characteristic morphological features disclosed by microdissection or in situ hybridization, we discovered how methylation at certain CpG sites outside of CpG islands would play a crucial epigenetic role in the versatility and flexibility of gene expression during cancer progression. In this review, we first introduce technical aspects of two morphology-oriented epigenetic studies: (1) histoendonuclease-linked detection of methylated sites of DNA (HELMET), and (2) padlock probe and rolling circle amplification (RCA) for in situ identification of methylated cytosine in a sequence-dependent manner. We then present our observation of a novel MeCP2-mediated gene-silencing mechanism through the addition of methylation to a single-CpG-locus upstream of the TATA-box of the receptor activator of NF-κB ligand (RANKL) and of secreted frizzled-related protein 4 (SFRP4) gene promoters.
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http://dx.doi.org/10.1007/s00418-018-1675-8DOI Listing
July 2018

Regulation of masculinization: androgen signalling for external genitalia development.

Nat Rev Urol 2018 06;15(6):358-368

Department of Developmental Genetics, Wakayama Medical University, Wakayama City, Wakayama, Japan.

The biology of masculinization is fundamentally important for understanding the embryonic developmental processes that are involved in the development of the male reproductive tract, external genitalia, and also the tumorigenesis of prostate cancer. The molecular mechanisms of masculinization are of interest to many researchers and clinicians involved in varied fields, including molecular developmental biology, cancer research, endocrinology, and urology. Androgen signalling is mediated by the nuclear androgen receptor, which has fundamental roles in masculinization during development. Various modes of androgen signalling, including 5α-dihydrotestosterone-induced regulation of mesenchymal cell proliferation, have been observed in masculinization. Such regulation is essential for regulating urogenital tissue development, including external genitalia development. Androgen-induced genes, such as MAFB, which belongs to the activator protein 1 (AP-1) superfamily of genes, have essential roles in male urethral formation, and disruption of its signalling can interfere with urethral formation, which often results in hypospadias. Another AP-1 superfamily gene, ATF3, could be responsible for some instances of hypospadias in humans. These androgen-dependent signals and downstream events are crucial for not only developmental processes but also processes of diseases such as hypospadias and prostate cancer.
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http://dx.doi.org/10.1038/s41585-018-0008-yDOI Listing
June 2018

Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas.

Diagn Pathol 2018 Jan 3;13(1). Epub 2018 Jan 3.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Background: A proper balance between the activator and the repressor form of GLI3, a zinc-finger transcription factor downstream of hedgehog signaling, is essential for proper development of various organs during development. Mutations in different domains of the GLI3 gene underlie several congenital diseases including Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS).

Case Presentation: Here, we describe the case of an overlapped phenotype of these syndromes with agenesis of the gallbladder and the pancreas, bearing a c.2155 C > T novel likely pathogenic variant of GLI3 gene by missense point mutation causing p.P719S at the proteolytic cleavage site.

Conclusions: Although agenesis of the gallbladder and the pancreas is uncommon in GLI3 morphopathy, a slight difference in the gradient or the balance between activator and repressor in this case may hinder sophisticated spatial and sequential hedgehog signaling that is essential for proper development of gallbladder and pancreas from endodermal buds.
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http://dx.doi.org/10.1186/s13000-017-0682-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389258PMC
January 2018

Pathologic conditions of hard tissue: role of osteoclasts in osteolytic lesion.

Histochem Cell Biol 2018 Apr 22;149(4):405-415. Epub 2018 Jan 22.

Department of Molecular Pathology, Graduate School of Medicine, Ehime University Graduate School of Medicine, 454 Shitsukawa, Toon, Ehime, 791-0295, Japan.

Hard tissue homeostasis is regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. This physiologic process allows adaptation to mechanical loading and calcium homeostasis. Under pathologic conditions, however, this process is ill-balanced resulting in either over-resorption or over-formation of hard tissue. Local over-resorption by osteoclasts is typically observed in osteolytic metastases of malignancies, autoimmune arthritis, and giant cell tumor of bone (GCTB). In tumor-related local osteolysis, tumor-derived osteoclast-activating factors induce bone resorption not by directly acting on osteoclasts but by indirectly upregulating receptor activator of NFκB ligand (RANKL) on osteoblastic cells. Similarly, synovial tissue in the autoimmune arthritis model does overexpress RANKL and contains numerous osteoclast precursors, and like a landing craft, when it comes in contact with eroded bone surfaces, osteoclast precursors are immediately polarized to become mature osteoclasts, inducing rapidly progressive bone destruction at a late stage of the disease. GCTB, on the other hand, is a common primary bone tumor, usually arising at the metaphysis of the long bone in young adults. After the discovery of RANKL, the concept of GCTB as a tumor of RANKL-expressing stromal cells was established, and comprehensive exosome studies finally disclosed the causative single-point mutation at histone H3.3 (H3F3A) in stromal cells. Thus, osteolytic lesions under various pathological conditions are ultimately attributable to the overexpression of RANKL, which opens up a common, practical and useful therapeutic target for diverse osteolytic conditions.
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http://dx.doi.org/10.1007/s00418-018-1639-zDOI Listing
April 2018

Growth plate-derived hedgehog-signal-responsive cells provide skeletal tissue components in growing bone.

Histochem Cell Biol 2018 Apr 22;149(4):365-373. Epub 2018 Jan 22.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Longitudinal bone growth progresses by continuous bone replacement of epiphyseal cartilaginous tissue, known as "growth plate", produced by columnar proliferated- and differentiated-epiphyseal chondrocytes. The endochondral ossification process at the growth plate is governed by paracrine signals secreted from terminally differentiated chondrocytes (hypertrophic chondrocytes), and hedgehog signaling is one of the best known regulatory signaling pathways in this process. Here, to investigate the developmental relationship between longitudinal endochondral bone formation and osteogenic progenitors under the influence of hedgehog signaling at the growth plate, genetic lineage tracing was carried out with the use of Gli1 mice line to follow the fate of hedgehog-signal-responsive cells during endochondral bone formation. Gli1 genetically labeled cells are detected in hypertrophic chondrocytes and osteo-progenitors at the chondro-osseous junction (COJ); these progeny then commit to the osteogenic lineage in periosteum, trabecular and cortical bone along the developing longitudinal axis. Furthermore, in ageing bone, where longitudinal bone growth ceases, hedgehog-signal responsiveness and its implication in osteogenic lineage commitment is significantly weakened. These results show, for the first time, evidence of the developmental contribution of endochondral progenitors under the influence of epiphyseal chondrocyte-derived secretory signals in longitudinally growing bone. This study provides a precise outline for assessing the skeletal lineage commitment of osteo-progenitors in response to growth-plate-derived regulatory signals during endochondral bone formation.
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http://dx.doi.org/10.1007/s00418-018-1641-5DOI Listing
April 2018

Developmental Contribution of Wnt-signal-responsive Cells to Mouse Reproductive Tract Formation.

Acta Histochem Cytochem 2017 Aug 10;50(4):127-133. Epub 2017 Aug 10.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

In mammals, the müllerian duct (MD) is an embryonic tubular structure that gives rise to the female reproductive tract (FRT). The MD originates from the coelomic epithelium (CoE) and takes on a rostral to caudal shape to establish the primary structure of the FRT under the regulation of morphogenetic signals. During these developmental processes, the MD and its derivatives require proper regulation of the Wnt-signaling-pathway. Here, to investigate the developmental contribution of FRT primordia under the influence of the Wnt-signaling, genetic lineage tracing was carried out using TopCreER/Rosa-LacZ mice to follow the fate of Wnt-signal-responsive cells during reproductive tract formation. TopCreER-marked-LacZ+ cells, arising from the Wnt-signal-responsive progenitors in CoE, give rise to spatially restricted MD and the uterine luminal epithelium. Similarly, the progeny from LacZ+ mesenchymal cells surrounding the MD contribute to both the uterine smooth muscle and stroma. Furthermore, in males, the Wnt-signal-responsive MD mesenchyme develops into the epididymis. These results show, for the first time, evidence of the sequential involvement of reproductive tract progenitors under the influence of Wnt-signal throughout the developmental term. This study provides a precise outline for assessing the lineage relation between the reproductive tract and the cell fate of its primordia in a temporally regulated manner.
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http://dx.doi.org/10.1267/ahc.17017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593815PMC
August 2017

Primary Type3 (Non-ABC, Non-GCB) Subtype of Extranodal Diffuse Large B-Cell Lymphoma of the Thyroid Bearing No Mutation by Padlock Probe Hybridization.

Case Rep Oncol 2017 May-Aug;10(2):508-514. Epub 2017 Jun 14.

Department of Molecular Pathology, Ehime University, Graduate School of Medicine, Toon City, Japan.

Primary extranodal malignant lymphoma of the thyroid is a rare entity composed of mostly neoplastic transformation of germinal center-like B cells (GCB) or memory B cells. Other B-cell-type malignancies arising primarily in the thyroid have rarely been described. Immunohistochemical examination of autopsied primary malignant lymphoma of the thyroid in an 83-year-old Japanese female revealed the presence of a non-GCB subtype of diffuse large B-cell lymphoma (DLBCL) without the typical codon 206 or 265 missense mutation of . The lack of the highly oncogenic gene mutation, frequently observed in DLBCL of the activated B-cell (ABC) subtype, and the detection of an extremely aggressive yet local clinical phenotype demonstrated that the present case was an exceptional entity of the type3 (non-GCB and non-ABC) subtype.
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http://dx.doi.org/10.1159/000477489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498938PMC
June 2017

sFRP4-dependent Wnt signal modulation is critical for bone remodeling during postnatal development and age-related bone loss.

Sci Rep 2016 04 27;6:25198. Epub 2016 Apr 27.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

sFRP4 is an extracellular Wnt antagonist that fine-tunes its signal activity by direct binding to Wnts. Bone fragility under oxidative stress by diabetes and aging is partly related to the suppression of the Wnt signal through upregulated sFRP4. Here, to explore the functions of sFRP4 as a balancer molecule in bone development and remodeling, we analyzed the sFRP4 knock-in mouse strain. X-gal and immunohistochemically stained signals in sFRP4-LacZ heterozygous mice were detectable in restricted areas, mostly in osteoblasts and osteoclasts, of the femoral diaphysis after neonatal and postnatal stages. Histological and μCT analyses showed increased trabecular bone mass with alteration of the Wnt signal and osteogenic activity in sFRP4 mutants; this augmented the effect of the buildup of trabecular bone during the ageing period. Our results indicate that sFRP4 plays a critical role in bone development and remodeling by regulating osteoblasts and osteoclasts, and that its functional loss prevents age-related bone loss in the trabecular bone area. These findings imply that sFRP4 functions as a key potential endogenous balancer of the Wnt signaling pathway by efficiently having direct influence on both bone formation and bone absorption during skeletal bone development and maintenance through remodeling.
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http://dx.doi.org/10.1038/srep25198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846872PMC
April 2016

Reactivation of CDX2 in Gastric Cancer as Mark for Gene Silencing Memory.

Acta Histochem Cytochem 2015 Aug 20;48(4):115-24. Epub 2015 Aug 20.

Department of Molecular Pathology, Ehime University Graduate School of Medicine.

To explore the epigenetic mechanism that reactivates CDX2 (a homeobox transcription factor that serves as a tumor-suppressor gene) in intestinal-type gastric cancer during cancer progression, we examined the methylation status of the CDX2 gene promoter and the expression pattern of methyl-CpG binding protein-2 (MeCP2). From archives of the pathology records of surgically excised advanced stomach cancer cases in the Department of Molecular Pathology, Ehime University in a past decate (n=265), 10 cases of intestinal-type tubular adenocarcinoma, well-differentiated type (wel) with minor poorly-differentiated adenocarcinoma (por) components were selected. The expression pattern of CDX2, MUC2 and MeCP2 in these 10 cases was analyzed by immunohistochemistry. The cancerous and non-cancerous areas were selectively obtained by microdissection, and the methylation status of the CDX2 promoter of each area was assessed by methylation-specific polymerase chain reaction (MSP). In all 10 cases, CDX2 expression was clearly observed in the nucleus of the non-cancerous background of the intestinal metaplasic area, where the unmethylation pattern of the CDX2 gene promoter prevailed with reduced MeCP2 expression. In this metaplastic area, CDX2 expression was co-localized with its target gene, MUC2. CDX2 expression then disappeared from the deep invasive wel area. Reflecting the reduced CDX2 expression, microdissected samples from all the wel areas showed hypermethylation of the CDX2 gene promoter by MSP, with prominent MeCP2 expression. Interestingly, while hypermethylation of the CDX2 gene promoter was maintained in the por area in 8 of the 10 cases, CDX2 expression was restored in por areas where MeCP2 expression was markedly and selectively reduced. The other two cases, however, showed a constant MeCP2 expression level comparable to the surrounding deep invasive wel area with negative CDX2 expression. Therefore, gene silencing by hypermethylation may be overcome by the reduction of methyl-CpG binding proteins, resulting in apparent but non-functional reactivation of CDX2 as a mere molecular mark for gene silencing memory.
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http://dx.doi.org/10.1267/ahc.15014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564377PMC
August 2015

Epigenetic regulation of Tbx18 gene expression during endochondral bone formation.

Cell Tissue Res 2015 Feb 8;359(2):503-512. Epub 2014 Nov 8.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Endochondral bone formation is tightly regulated by the spatial and sequential expression of a series of transcription factors. To disclose the roles of TBX18, a member of the T-box transcription factor family, during endochondral bone formation, its spatial and temporal expression patterns were characterized in the limb skeletal region of the developing mouse together with those of established osteochondrogenic markers Sox9, Col2a1, and Runx2. TBX18 expression first appeared in condensed mesenchymal cells (chondro-progenitors) in embryonic-day-10.5 (E10.5) limb bud and was co-localized with Sox9 expression, whereas at E11.5 and E12.5, it became undetectable in mesenchymal cells committed to the chondrocyte lineage. From E13.5 to E18.5, TBX18 expression reappeared in chondrocytes, correlating strongly with Col2a1 expression; furthermore, low level TBX18 expression was found in the Runx2-positive perichondral osteoblastic cell lineage. At the postnatal stage, TBX18 expression was observed in epiphyseal chondrocytes and osteocytes within the lacunae of mature trabecular bone. On the assumption that such characteristic Tbx18 gene expression is epigenetically regulated during mouse limb development, we examined the methylation status of the CpG-island in the mouse Tbx18 gene by methylation-specific polymerase chain reaction. Hypermethylation of the Tbx18 gene promoter became evident at an early embryonic stage in TBX18-negative cells and then disappeared at a late embryonic stage in TBX18-positive cells. Therefore, the temporal suppression of Tbx18 gene expression by the hypermethylation of its promoter seems to trigger the differentiation of mesenchymal cells into hypertrophic chondrocytes in the early stages of endochondral ossification.
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http://dx.doi.org/10.1007/s00441-014-2028-0DOI Listing
February 2015

BOB.1-positive Classical Hodgkin's Lymphoma Carries Hypermethylation of Its Promoter as Epigenetic Marker of Gene-silencing Memory.

Acta Histochem Cytochem 2014 Jun 26;47(3):125-31. Epub 2014 Jun 26.

Department of Molecular Pathology, Ehime University Graduate School of Medicine , Shitsukawa, Toon City, Ehime 791-0295, Japan.

Analysis of archival formalin-fixed, paraffin-embedded (FFPE) pathological specimens of three case of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) and three cases of classical Hodgkin lymphoma (CHL) revealed that hypermethylation of the BOB.1 gene promoter was exclusively observed in CHL. A discrepancy was observed, however, between the methylation status of the BOB.1 gene promoter and its expression in the EBV-positive mixed cellular CHL (MCCHL). Since MCCHL lacks the typical B-cell phenotype even in the presence of abundant BOB.1 transcription factors, functional activity of BOB.1 may be lost or reduced by a mechanism other than epigenetic gene silencing. When some tumor-suppressor gene products have lost their biological function, impact or significance of derepression of such genes may be little. Therefore, when interpreting immunohistochemical results for diagnostic or research purposes, it must be borne in mind that apparent positive immunostaining can merely be the result of chromatin remodeling and that such transient expression often has little functional significance. Any apparent positive immunohistochemical result needs to be interpreted carefully with the help of the hypermethylation status as a molecular marker of gene silencing memory.
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http://dx.doi.org/10.1267/ahc.14012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164698PMC
June 2014

Diabetic osteopenia by decreased β-catenin signaling is partly induced by epigenetic derepression of sFRP-4 gene.

PLoS One 2014 18;9(7):e102797. Epub 2014 Jul 18.

Department of Pathology, Division of Diagnostic Molecular Pathology, Kobe University Graduate School of Medicine, Kobe City, Japan; Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, Japan; Department of Diagnostic Pathology, Ehime University Hospital, Shitsukawa, Toon City, Ehime, Japan.

In diabetics, methylglyoxal (MG), a glucose-derived metabolite, plays a noxious role by inducing oxidative stress, which causes and exacerbates a series of complications including low-turnover osteoporosis. In the present study, while MG treatment of mouse bone marrow stroma-derived ST2 cells rapidly suppressed the expression of osteotrophic Wnt-targeted genes, including that of osteoprotegerin (OPG, a decoy receptor of the receptor activator of NF-kappaB ligand (RANKL)), it significantly enhanced that of secreted Frizzled-related protein 4 (sFRP-4, a soluble inhibitor of Wnts). On the assumption that upregulated sFRP-4 is a trigger that downregulates Wnt-related genes, we sought out the molecular mechanism whereby oxidative stress enhanced the sFRP-4 gene. Sodium bisulfite sequencing revealed that the sFRP-4 gene was highly methylated around the sFRP-4 gene basic promoter region, but was not altered by MG treatment. Electrophoretic gel motility shift assay showed that two continuous CpG loci located five bases upstream of the TATA-box were, when methylated, a target of methyl CpG binding protein 2 (MeCP2) that was sequestered upon induction of 8-hydroxy-2-deoxyguanosine, a biomarker of oxidative damage to DNA. These in vitro data suggest that MG-derived oxidative stress (not CpG demethylation) epigenetically and rapidly derepress sFRP-4 gene expression. We speculate that under persistent oxidative stress, as in diabetes and during aging, osteopenia and ultimately low-turnover osteoporosis become evident partly due to osteoblastic inactivation by suppressed Wnt signaling of mainly canonical pathways through the derepression of sFRP-4 gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102797PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103869PMC
November 2015

Duodenal gastric heterotopia, sporadic or fundic gland polyp-associated, frequently carries β-catenin mutation.

Virchows Arch 2014 Sep 27;465(3):253-6. Epub 2014 Jun 27.

Department of Molecular Pathology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Duodenal gastric heterotopia (DGH) is a benign asymptomatic condition assumed to be of congenital origin. Since DGH is often associated with fundic gland polyps (FGPs) that frequently carry a somatic β-catenin gene mutation, we examined whether DGH, either sporadic or FGP-associated, is attributable to alterations of the Wnt/β-catenin pathway. Genetic analysis revealed frequent somatic β-catenin gene mutations in DGH; some of which showed the same mutation pattern as coexisting FGPs. All missense mutations were confined to codons 32, 33, and 37. No such mutations were observed, however, in any of the specimens from focal gastric foveolar metaplasia (GFM). Therefore, DGH is not a mere congenital lesion due to aberrant migration of normal gastric mucosa or a simple reactive metaplasia after regenerative stimuli of the duodenal mucosa, but a distinct condition based upon molecular genetic changes in the Wnt/β-catenin pathway.
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http://dx.doi.org/10.1007/s00428-014-1612-8DOI Listing
September 2014

Acquisition of MYD88 L265P mutation during treatment of diffuse large B cell lymphoma of the parotid gland.

Virchows Arch 2014 Jan 22;464(1):121-4. Epub 2013 Nov 22.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

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http://dx.doi.org/10.1007/s00428-013-1514-1DOI Listing
January 2014

Genetic analysis of the role of Alx4 in the coordination of lower body and external genitalia formation.

Eur J Hum Genet 2014 Mar 14;22(3):350-7. Epub 2013 Aug 14.

1] Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan [2] Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan.

Although several syndromes include abnormalities of both the ventral body wall and external genitalia, the developmental bases of this correlation are largely unknown. Naturally occurring mutations in Aristaless-like 4 (Alx4, Strong's luxoid: Alx4Lst) have ventral body wall and pelvic girdle abnormalities. We sought to determine whether the development of the genital tubercle (GT) and its derivatives, the external genitalia, is affected by this mutation. We thus performed genetic and tissue labeling analyses in mutant mice. Alx4Lst/Lst mutants displayed hypoplasia of the dorsal GT and reduced expression of Fibronectin. We analyzed cell migration during GT formation by tissue labeling experiments and discovered that the cells located in the proximal segment of the umbilical cord (infra-umbilical mesenchyme) migrate toward the dorsal part of the GT. The Alx4Lst/Lst mutants also displayed augmented expression of Hh signal-related genes. Hence, we analyzed a series of combinatorial mutants for Alx4, Sonic hedgehog (Shh) and GLI-Kruppel family member 3 (Gli3). These phenotype-genotype analyses suggested a genetic interaction between Alx4 and Hh signaling during GT formation. Moreover, Hh gain-of-function mutants phenocopied some of these phenotypes. These observations reveal novel information regarding the pathogenic mechanisms of syndromic lower ventral body malformations, which are largely unknown.
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http://dx.doi.org/10.1038/ejhg.2013.160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3925283PMC
March 2014

Efficient Genetic Analysis of Microdissected Samples by Agarose-Bead Method: Alterations of β-Catenin Gene in Fundic Gland Polyp and Heterotopic Gastric Mucosa of Duodenum.

Acta Histochem Cytochem 2013 Feb 1;46(1):19-24. Epub 2013 Feb 1.

Division of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

Molecular genetic analyses of archival formalin-fixed, paraffin-embedded (FFPE) pathological specimens taken at biopsy or autopsy, are occasionally compromised because the DNA molecules therein are inevitably degraded. Furthermore, since these tissue samples comprise various cell types, the analyses based on mixtures of such heterogeneous populations often fail to reflect the nature of the affected cells. In the present study, to elucidate the contribution of β-catenin gene mutation to the fundic gland polyp and the heterotopic gastric mucosa in the duodenum, we successfully introduced an agarose-bead mediated technique as an effectual tool for retrospective morphology-oriented genetic analyses. Microdissected samples were embedded in low-melting agarose, and directly treated with proteinase K. A fragment of the agarose-bead was used as a template for polymerase chain reaction to analyze β-catenin mutation. Of the six cases of heterotopic gastric mucosa in the duodenum associated with fundic gland polyps, one showed a common 1-bp missense mutation at codon 37 shared by both the fundic gland polyp and the heterotopic gastric mucosa. Alternatively, a 1-bp silent mutation at codon 33 and missense mutation at codon 32 were identified only in the heterotopic gastric mucosa. Agarose-bead mediated technique shows superior sensitivity to the previously described techniques and is an effectual tool for retrospective morphology-oriented genetic analyses using a large number of archival pathological samples stored for long periods in the pathology laboratory.
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http://dx.doi.org/10.1267/ahc.12022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596603PMC
February 2013

Gastric adenocarcinoma arising in gastritis cystica profunda presenting with selective loss of KCNE2 expression.

World J Gastroenterol 2013 Feb;19(8):1314-7

Department of Molecular Pathology, Ehime University, Graduate School of Medicine, Ehime 791-0295, Japan.

Gastritis cystica profunda (GCP) is a rare condition caused by ectopic entrapment of gastric glands, probably secondary to the disruption of muscularis mucosae. GCP is often associated with gastric adenocarcinoma, and loss of the KCNE2 subunit from potassium channel complexes is considered a common primary target molecule leads to both GCP and malignancy. In this study, we, for the first time, analyzed the expression of KCNE2 in surgically excised tissue from human gastric cancer associated with GCP and confirmed that reduced KCNE2 expression correlates with disease formation.
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http://dx.doi.org/10.3748/wjg.v19.i8.1314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587490PMC
February 2013

Pulmonary hypertension associated with diffuse deposition of pentosidine in pulmonary arterioles.

Diabetes Res Clin Pract 2013 May 9;100(2):e59-62. Epub 2013 Feb 9.

Division of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa 454, Toon City, Ehime 791-0295, Japan.

Diabetes induces advanced glycation end products (AGEs) that per se are not only a major cause of oxidative stress but also reduce the plasticity of connective tissue by pathological collagen cross-linking. We describe a case of severe pulmonary hypertension manifesting as a major diabetic complication. Impaired pulmonary arteriolar plasticity attributed to pentosidine, together with increased circulation volume by hyperosmotic pressure and reduction in myocardial compliance by multiple patchy fibrosis, may contribute to the clinical manifestation of severe pulmonary hypertension.
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http://dx.doi.org/10.1016/j.diabres.2013.01.019DOI Listing
May 2013

Cdx2 expression and its promoter methylation during metaplasia-dysplasia-carcinoma sequence in Barrett's esophagus.

World J Gastroenterol 2013 Jan;19(4):536-41

Division of Molecular Pathology, Ehime University Graduate School of Medicine, Toon 791-0295, Japan.

Aim: To examine how the expression of caudal type homebox transcription factor 2 (Cdx2) is regulated in the development of malignancy in Barrett's esophagus.

Methods: Cdx2, mucin (MUC) series (MUC2, MUC5AC and MUC6), p53 and E-cadherin expression in Barrett's esophagus and adenocarcinoma specimens were examined by immunostaining. Isolated clusters of cells from (1) MUC2 and Cdx2-positive intestinal metaplastic mucosa; (2) MUC5AC and MUC6-positive, and MUC2 and Cdx2-negative high-grade dysplasia (HD), or intramucosal adenocarcinoma (IMC); and (3) MUC5AC, MUC6 and Cdx2-positive poorly-differentiated invasive adenocarcinoma (PDA) were analyzed by methylation-specific polymerase chain reaction using sets of primers for detecting methylation status of the Cdx2 gene.

Results: Most of the non-neoplastic Barrett's esophageal mucosa showing intestinal-type metaplasia with or without low-grade dysplasia was positive for E-cadherin, MUC series and Cdx2, but negative for p53. A portion of the low-grade to HD was positive for E-cadherin, MUC5AC, MUC6 and p53, but negative for MUC2 and Cdx2. The definite IMC area was strongly positive for MUC5AC, MUC6 and p53, but negative for MUC2 and Cdx2. Methylation of the Cdx2 promoter was not observed in intestinal metaplasia, while hypermethylation of part of its promoter was observed in hot dipped and IMC. Hypermethylation of a large fraction of the Cdx2 promoter was observed in PDA.

Conclusion: Cdx2 expression is restored irrespective of the methylation status of its promoter. Apparent positive immunohistochemical results can be a molecular mark for gene silencing memory.
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http://dx.doi.org/10.3748/wjg.v19.i4.536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558578PMC
January 2013

Reduced BMP signaling results in hindlimb fusion with lethal pelvic/urogenital organ aplasia: a new mouse model of sirenomelia.

PLoS One 2012 17;7(9):e43453. Epub 2012 Sep 17.

Department of Development of Genetics, Institute of Advanced Medicine, Wakayama Medical University, Kimiidera, Wakayama, Japan.

Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4(flox/flox)) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4(flox/flox) conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043453PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444444PMC
March 2013
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