RESUMEN
Cochlear melanocytes are intermediate cells in the stria vascularis that generate endocochlear potentials required for auditory function. Human PAX3 mutations cause Waardenburg syndrome and abnormalities of skin and retinal melanocytes, manifested as congenital hearing loss (~ 70%) and hypopigmentation of skin, hair and eyes. However, the underlying mechanism of hearing loss remains unclear. Cochlear melanocytes in the stria vascularis originated from Pax3-traced melanoblasts and Plp1-traced Schwann cell precursors, both of which derive from neural crest cells. Here, using a Pax3-Cre knock-in mouse that allows lineage tracing of Pax3-expressing cells and disruption of Pax3, we found that Pax3 deficiency causes foreshortened cochlea, malformed vestibular apparatus, and neural tube defects. Lineage tracing and in situ hybridization show that Pax3+ derivatives contribute to S100+, Kir4.1+ and Dct+ melanocytes (intermediate cells) in the developing stria vascularis, all of which are significantly diminished in Pax3 mutant animals. Taken together, these results suggest that Pax3 is required for the development of neural crest cell-derived cochlear melanocytes, whose absence may contribute to congenital hearing loss of Waardenburg syndrome in humans.
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Sordera , Pérdida Auditiva Sensorineural , Pérdida Auditiva , Síndrome de Waardenburg , Ratones , Animales , Humanos , Síndrome de Waardenburg/genética , Cóclea , Estría Vascular , Pérdida Auditiva Sensorineural/genética , Melanocitos , Factor de Transcripción PAX3/genéticaRESUMEN
Cochlear melanocytes are intermediate cells in the stria vascularis that generate endocochlear potentials required for auditory function. Human PAX3 mutations cause Waardenburg syndrome and abnormalities of melanocytes, manifested as congenital hearing loss and hypopigmentation of skin, hair and eyes. However, the underlying mechanism of hearing loss remains unclear. During development, cochlear melanocytes in the stria vascularis are dually derived from Pax3-Cre+ melanoblasts migrating from neuroepithelial cells including neural crest cells and Plp1+ Schwann cell precursors originated from also neural crest cells, differentiating in a basal-apical manner. Here, using a Pax3-Cre mouse line, we found that Pax3 deficiency causes foreshortened cochlea, malformed vestibular apparatus, and neural tube defects. Lineage tracing and in situ hybridization show that Pax3-Cre derivatives contribute to S100+ , Kir4.1+ and Dct+ melanocytes (intermediate cells) in the developing stria vascularis, all significantly diminished in Pax3 mutant animals. Taken together, these results suggest that Pax3 is required for the development of neural crest cell-derived cochlear melanocytes, whose absence may contribute to congenital hearing loss of Waardenburg syndrome in human.
RESUMEN
During development, cranial neural crest (NC) cells display a striking transition from collective to single-cell migration and undergo a mesenchymal-to-epithelial transformation to form a part of the middle ear epithelial cells (MEECs). While MEECs derived from NC are known to control homeostasis of the epithelium and repair from otitis media, paracrine action of keratinocyte growth factor (KGF) promotes the growth of MEECs and induces middle ear cholesteatoma (cholesteatoma). The animal model of cholesteatoma was previously established by transfecting a human KGF-expression vector. Herein, KGF-inducing cholesteatoma was studied in Wnt1-Cre/Floxed-enhanced green fluorescent protein (EGFP) mice that conditionally express EGFP in the NC lineages. The cytokeratin 14-positive NC lineage expanded into the middle ear and formed cholesteatoma. Moreover, the green fluorescent protein-positive NC lineages comprising the cholesteatoma tissue expressed p75, an NC marker, with high proliferative activity. Similarly, a large number of p75-positive cells were observed in human cholesteatoma tissues. Injections of the immunotoxin murine p75-saporin induced depletion of the p75-positive NC lineages, resulting in the reduction of cholesteatoma in vivo. The p75 knockout in the MEECs had low proliferative activity with or without KGF protein in vitro. Controlling p75 signaling may reduce the proliferation of NC lineages and may represent a new therapeutic target for cholesteatoma.
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A crucial evolutionary change in vertebrate history was the Palaeozoic (Devonian 419-359 million years ago) water-to-land transition, allowed by key morphological and physiological modifications including the acquisition of lungs. Nonetheless, the origin and early evolution of vertebrate lungs remain highly controversial, particularly whether the ancestral state was paired or unpaired. Due to the rarity of fossil soft tissue preservation, lung evolution can only be traced based on the extant phylogenetic bracket. Here we investigate, for the first time, lung morphology in extensive developmental series of key living lunged osteichthyans using synchrotron x-ray microtomography and histology. Our results shed light on the primitive state of vertebrate lungs as unpaired, evolving to be truly paired in the lineage towards the tetrapods. The water-to-land transition confronted profound physiological challenges and paired lungs were decisive for increasing the surface area and the pulmonary compliance and volume, especially during the air-breathing on land.
All life on Earth started out under water. However, around 400 million years ago some vertebrates, such as fish, started developing limbs and other characteristics that allowed them to explore life on land. One of the most pivotal features to evolve was the lungs, which gave vertebrates the ability to breathe above water. Most land-living vertebrates, including humans, have two lungs which sit on either side of their chest. The lungs extract oxygen from the atmosphere and transfer it to the bloodstream in exchange for carbon dioxide which then gets exhaled out in to the atmosphere. How this important organ first evolved is a hotly debated topic. This is largely because lung tissue does not preserve well in fossils, making it difficult to trace how the lungs of vertebrates changed over the course of evolution. To overcome this barrier, Cupello et al. compared the lungs of living species which are crucial to understand the early stages of the water-to-land transition. This included four species of lunged bony fish which breathe air at the water surface, and a four-legged salamander that lives on land. Cupello et al. used a range of techniques to examine how the lungs of the bony fish and salamander changed shape during development. The results suggested that the lungs of vertebrates started out as a single organ, which became truly paired later in evolution once vertebrates started developing limbs. This anatomical shift increased the surface area available for exchanging oxygen and carbon dioxide so that vertebrates could breathe more easily on land. These findings provide new insights in to how the lung evolved into the paired structure found in most vertebrates alive today. It likely that this transition allowed vertebrates to fully adapt to breathing above water, which may explain why this event only happened once over the course of evolution.
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Evolución Biológica , Agua , Animales , Peces/fisiología , Fósiles , Pulmón , Filogenia , VertebradosRESUMEN
OBJECTIVE: Cholesteatoma is an epithelial lesion that expands into the middle ear, resulting in bone destruction. The acceleration of the proliferative activity of epithelial stem/progenitor cells is involved in the pathogenesis of cholesteatoma. Recently, the use of a menin-mixed lineage leukemia 1 (MLL1) inhibitor, MI503, in experiments has resulted in inhibition of the growth of tumors under histone modification. In this study, we investigated the effects of the menin-MLL inhibitor against cholesteatoma growth in an in vivo model. METHODS: We first correlated the expression level of histone H3 trimethylation at lysine 4 (H3K4me3) among cholesteatoma cases, chronic otitis media cases and normal skin tissues. Based on the role of keratinocyte growth factor (KGF) in the development of cholesteatoma, KGF-expression vector was transfected into the ear and we analyzed the expression level of H3K4me3. After cholesteatoma was induced, MI503 was administered daily into the ear for 14 days. RESULTS: We detected the highest labeling index of H3K4me3 in the cholesteatoma specimens. After KGF-expression vector transfection in the mouse ear, a high expression level of H3K4me3 was observed in the epithelial layers. The use of MI503 reduced cholesteatoma in the in vivo model and decreased the proliferation of epithelial stem/progenitor cells in a dose-dependent manner. CONCLUSION: We demonstrated that inhibition of the menin-MLL interaction may be a potentially useful strategy in the conservative treatment of cholesteatoma.
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Colesteatoma del Oído Medio , Animales , Oído Medio , Ratones , Otitis Media , Células Madre , TransfecciónRESUMEN
In acute kidney injury (AKI), the S3 segment of the proximal tubule is particularly damaged, as it is most vulnerable to ischemia. However, this region is also involved in renal tubular regeneration. To deeply understand the mechanism of the repair process after ischemic injury in AKI, we focused on glial cells missing 1 (Gcm1), which is one of the genes expressed in the S3 segment. Gcm1 is essential for the development of the placenta, and Gcm1 knockout (KO) is embryonically lethal. Thus, the function of Gcm1 in the kidney has not been analyzed yet. We analyzed the function of Gcm1 in the kidney by specifically knocking out Gcm1 in the kidney. We created an ischemia-reperfusion injury (IRI) model to observe the repair process after AKI. We found that Gcm1 expression was transiently increased during the recovery phase of IRI. In Gcm1 conditional KO mice, during the recovery phase of IRI, tubular cell proliferation reduced and transforming growth factor-ß1 expression was downregulated resulting in a reduction in fibrosis. In vitro, Gcm1 overexpression promoted cell proliferation and upregulated TGF-ß1 expression. These findings indicate that Gcm1 is involved in the mechanisms of fibrosis and cell proliferation after ischemic injury of the kidney.
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Lesión Renal Aguda/patología , Proteínas de Unión al ADN/metabolismo , Túbulos Renales Proximales/patología , Regeneración/fisiología , Daño por Reperfusión/patología , Factores de Transcripción/metabolismo , Lesión Renal Aguda/etiología , Animales , Proliferación Celular/fisiología , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Fibrosis , Humanos , Túbulos Renales Proximales/irrigación sanguínea , Masculino , Ratones , Ratones Noqueados , Daño por Reperfusión/complicaciones , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Glial cells missing homolog 2 (GCM2), a zinc finger-type transcription factor, is essential for the development of parathyroid glands. It is considered to be a master regulator because the glands do not form when Gcm2 is deficient. Remarkably, Gcm2 expression is maintained throughout the fetal stage and after birth. Considering the Gcm2 function in embryonic stages, it is predicted that Gcm2 maintains parathyroid cell differentiation and survival in adults. However, there is a lack of research regarding the function of Gcm2 in adulthood. Therefore, we analyzed Gcm2 function in adult tamoxifen-inducible Gcm2 conditional knockout mice. One month after tamoxifen injection, Gcm2-knockout mice showed no significant difference in serum calcium, phosphate, and PTH levels and in the expressions of calcium-sensing receptor (Casr) and parathyroid hormone (Pth), whereas Ki-67 positive cells were decreased and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) positive cell number did not change, as compared with those of controls. Seven months after tamoxifen injection, Gcm2-knockout mice showed shrinkage of the parathyroid glands and fewer parathyroid cells. A significant decrease was noted in Casr- and Pth-expressing cells and serum PTH and Ca levels, whereas serum phosphate levels increased, as compared with those of controls. All our results concluded that a reduction of Gcm2 expression leads to a reduction of parathyroid cell proliferation, an increase in cell death, and an attenuation of parathyroid function. Therefore, we indicate that Gcm2 plays a prominent role in adult parathyroid cell proliferation and maintenance.
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Proteínas Nucleares/metabolismo , Glándulas Paratiroides/citología , Glándulas Paratiroides/metabolismo , Factores de Transcripción/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Femenino , Técnicas de Genotipaje , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Glándulas Paratiroides/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-ß depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-ß signaling, thus regulating normal lung development.
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Plaquetas/metabolismo , Diferenciación Celular/fisiología , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Alveolos Pulmonares/embriología , Transducción de Señal/fisiología , Animales , Plaquetas/citología , Células Endoteliales , Células Epiteliales/citología , Células Epiteliales/metabolismo , Lectinas Tipo C/genética , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Miofibroblastos/citología , Miofibroblastos/metabolismo , Alveolos Pulmonares/citología , Mucosa Respiratoria/citología , Mucosa Respiratoria/embriologíaRESUMEN
Chronic kidney disease (CKD) disrupts mineral homeostasis and its representative pathosis is defined as secondary hyperparathyroidism (SHPT). SHPT occurs during the early course of progressive renal insufficiency, and is associated with mortality and cardiovascular events. SHPT results in reduction of calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) in the parathyroid glands during CKD. However, the precise mechanism of CaSR and VDR reduction is largely unknown. CKD was induced through two-step 5/6 nephrectomy, and then CKD rats and sham-operated rats were maintained for 8 weeks on diets containing 0.7 % phosphorus (normal phosphate) or 1.2 % phosphorus (high phosphate). In gene expression analysis, TaqMan probes were used for quantitative real-time polymerase chain reaction. Finally, CaSR and VDR protein expressions were analyzed using immunohistochemistry. DNA methylation analysis was performed using a restriction digestion and quantitative PCR. CaSR and VDR mRNA were reduced only in CKD rats fed the high-phosphorus diets (CKD HP), then CaSR and VDR immunohistochemical expressions were compatible with gene expression assay. SHPT was then confirmed only in CKD HP rats. Furthermore, sole CKD HP rats showed the hypermethylation in CaSR and VDR genes; however, the percentage methylation of both genes was low. Although CaSR and VDR hypermethylation was demonstrated in PTGs of CKD HP rats, the extent of hypermethylation was insufficient to support the relevance between hypermethylation and down-regulation of gene expression because of the low percentage of methylation. Consequently, our data suggest that mechanisms, other than DNA hypermethylation, were responsible for the reduction in mRNA and protein levels of CaSR and VDR in PTGs of CKD HP rats.
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Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Glándulas Paratiroides/metabolismo , Fósforo Dietético/administración & dosificación , Receptores de Calcitriol/metabolismo , Receptores Sensibles al Calcio/metabolismo , Animales , Enfermedad Crónica , Metilación de ADN , Modelos Animales de Enfermedad , Expresión Génica , Hiperparatiroidismo Secundario/etiología , Enfermedades Renales/complicaciones , Masculino , Metilación , Proteínas/análisis , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores de Calcitriol/genética , Receptores Sensibles al Calcio/genéticaRESUMEN
The lung is an important organ for air breathing in tetrapods and originated well before the terrestrialization of vertebrates. Therefore, to better understand lung evolution, we investigated lung development in the extant basal actinopterygian fish Senegal bichir (Polypterus senegalus). First, we histologically confirmed that lung development in this species is very similar to that of tetrapods. We also found that the mesenchymal expression patterns of three genes that are known to play important roles in early lung development in tetrapods (Fgf10, Tbx4, and Tbx5) were quite similar to those of tetrapods. Moreover, we found a Tbx4 core lung mesenchyme-specific enhancer (C-LME) in the genomes of bichir and coelacanth (Latimeria chalumnae) and experimentally confirmed that these were functional in tetrapods. These findings provide the first molecular evidence that the developmental program for lung was already established in the common ancestor of actinopterygians and sarcopterygians.
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Proteínas de Peces/genética , Peces/crecimiento & desarrollo , Pulmón/crecimiento & desarrollo , Animales , Evolución Biológica , Embrión de Pollo , Pollos/genética , Embrión no Mamífero , Elementos de Facilitación Genéticos , Femenino , Peces/embriología , Peces/genética , Peces/fisiología , Regulación del Desarrollo de la Expresión Génica , Larva , Pulmón/fisiología , Masculino , MesodermoRESUMEN
Ulcerative colitis (UC) is a representative clinical manifestation of inflammatory bowel disease that causes chronic gastrointestinal tract inflammation. Dextran sulfate sodium (DSS)-induced colitis mice have been used to investigate UC pathogenesis, and in this UC model, disturbance and impairment of the mucosal epithelium have been reported to cause colitis. However, how DSS sporadically breaks down the epithelium remains unclear. In this study, we focused on the colonic microcirculation and myenteric neurons of DSS-induced colitis. Moreover, we examined the potential of myenteric neurons as a target to prevent exacerbation of colitis. Fluorescent angiographic and histopathological studies revealed that DSS administration elicited blood vessel disruption before epithelial disorders appeared. Ischemic conditions in the lamina propria induced inducible nitric oxide synthase (iNOS) expression in myenteric neurons as colitis aggravated. When neuronal activity was inhibited with butylscopolamine, neuronal iNOS expression decreased, and the exacerbation of colitis was prevented. These results suggested that DSS-induced colitis was triggered by microcirculatory disturbance in the mucosa, and that excessive neuronal excitation aggravated colitis. During remission periods of human UC, endoscopic inspection of the colonic microcirculation may enable the early detection of disease recurrence, and inhibition of neuronal iNOS expression may prevent the disease from worsening.
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Colitis/inducido químicamente , Colon/irrigación sanguínea , Sistema Nervioso Entérico/metabolismo , Microcirculación , Óxido Nítrico Sintasa de Tipo II/metabolismo , Actinas/metabolismo , Animales , Anticuerpos Monoclonales Humanizados , Bevacizumab , Bromuro de Butilescopolamonio , Capilares/patología , Permeabilidad Capilar , Colitis/enzimología , Colitis/patología , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos C57BL , Periferinas/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Embryonic stem cell research has facilitated the generation of many cell types for the production of tissues and organs for both humans and companion animals. Because ≥30% of pet cats suffer from chronic kidney disease (CKD), xenotransplantation between pigs and cats has been studied. For a successful pig to cat xenotransplant, the immune reaction must be overcome, especially hyperacute rejection. In this study, we isolated the gene for feline decay-accelerating factor (fDAF), an inhibitor of complement proteins, and transfected a swine endothelial cell line with fDAF to "felinize" the pig cells. These fDAF-expressing cells were resistant to feline serum containing anti-pig antibodies, suggesting that felinized pig cells were resistant to hyperacute rejection. Our results suggest that a "felinized" pig kidney can be generated for the treatment of CKD in cats in the future.
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Antígenos CD55/genética , Células Endoteliales/metabolismo , Trasplante Heterólogo , Secuencia de Aminoácidos , Animales , Antígenos CD55/química , Enfermedades de los Gatos/cirugía , Gatos , Línea Celular , Células Clonales/metabolismo , Clonación Molecular , Expresión Génica , Rechazo de Injerto , Humanos , L-Lactato Deshidrogenasa/metabolismo , Ratones , Datos de Secuencia Molecular , Insuficiencia Renal Crónica/veterinaria , Porcinos , Transfección , Trasplante Heterólogo/efectos adversosRESUMEN
OBJECTIVE: Vascular endothelial growth factor (VEGF)-C overexpression in extrahepatic cholangiocarcinoma (ECC) has been shown to be correlated with lymph node metastasis. The intensity of immunohistochemical staining of VEGF-C protein in surgical samples has been used as index of VEGF-C overexpression in previous studies. The aim of the study was to examine if VEGF-C overexpression in ECC could be preoperatively detected by using samples obtained during ERCP. METHODS: Consecutive patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) for biliary stricture during the study period were prospectively analyzed. VEGF-C mRNA was quantified by real-time PCR methods using endoscopic samples obtained during ERCP. The high intensity of immunohistochemical staining of VEGF-C protein in surgical samples was used for the reference standard of VEGF-C overexpression. The level of S100P mRNA which was a novel diagnostic marker of ECC was also quantified to evaluate whether the endoscopic samples contained ECC cells. RESULTS: Twenty-five patients were enrolled in this study. Eighteen patients were diagnosed as ECC and seven patients were diagnosed as benign biliary structure. Nine of eighteen patients with ECC, who showed positive S100P mRNA in endoscopic samples and received surgical resection, were finally analyzed. Receiver operating characteristics analysis yielded VEGF-C mRNA cut-off value of 3.85 for detection of VEGF-C overexpression, and the diagnostic performance of VEGF-C mRNA measurement in the endoscopic sample for VEGF-C overexpression reached sensitivity of 75.0%, specificity of 100%, and accuracy of 88.9%. CONCLUSION: The quantification of VEGF-C mRNA of ECC with real-time PCR using endoscopic samples was useful for preoperative detection of VEGF-C overexpression.
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Neoplasias de los Conductos Biliares/metabolismo , Conductos Biliares Extrahepáticos/metabolismo , Biomarcadores de Tumor/metabolismo , Colangiocarcinoma/metabolismo , Colangiopancreatografia Retrógrada Endoscópica , ARN Mensajero/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de los Conductos Biliares/diagnóstico por imagen , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Extrahepáticos/diagnóstico por imagen , Conductos Biliares Extrahepáticos/patología , Biomarcadores de Tumor/genética , Biopsia , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Colangiocarcinoma/diagnóstico por imagen , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Periodo Preoperatorio , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Regulación hacia Arriba , Factor C de Crecimiento Endotelial Vascular/genéticaRESUMEN
During development of left-right asymmetry in the vertebrate embryo, Nodal plays a central role for determination of left-handedness. Bone morphogenetic protein (BMP) signaling has an important role for regulation of Nodal expression, although there is controversy over whether BMP signaling has a positive or negative effect on Nodal expression in the chick embryo. As BMP is a morphogen, we speculated that different concentrations might induce different responses in the cells of the lateral plate mesoderm (LPM). To test this hypothesis, we analyzed the effects of various concentrations of BMP4 and NOGGIN on Nodal expression in the LPM. We found that the effect on Nodal expression varied in a complex fashion with the concentration of BMP. In agreement with previous reports, we found that a high level of BMP signaling induced Nodal expression in the LPM, whereas a low level inhibited expression. However, a high intermediate level of BMP signaling was found to suppress Nodal expression in the left LPM, whereas a low intermediate level induced Nodal expression in the right LPM. Thus, the high and the low intermediate levels of BMP signaling up-regulated Nodal expression, but the high intermediate and low levels of BMP signaling down-regulated Nodal expression. Next, we sought to identify the mechanisms of this complex regulation of Nodal expression by BMP signaling. At the low intermediate level of BMP signaling, regulation depended on a NODAL positive-feedback loop suggesting the possibility of crosstalk between BMP and NODAL signaling. Overexpression of a constitutively active BMP receptor, a constitutively active ACTIVIN/NODAL receptor and SMAD4 indicated that SMAD1 and SMAD2 competed for binding to SMAD4 in the cells of the LPM. Nodal regulation by the high and low levels of BMP signaling was dependent on Cfc up-regulation or down-regulation, respectively. We propose a model for the variable effects of BMP signaling on Nodal expression in which different levels of BMP signaling regulate Nodal expression by a balance between BMP-pSMAD1/4 signaling and NODAL-pSMAD2/4 signaling.
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Proteínas Morfogenéticas Óseas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteína Nodal/metabolismo , Transducción de Señal , Animales , Unión Competitiva , Tipificación del Cuerpo , Embrión de Pollo , Electroporación , Perfilación de la Expresión Génica , Mesodermo/metabolismo , Modelos Biológicos , Modelos Genéticos , Oligonucleótidos/genética , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-ß (TGF-ß) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node.
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Proteínas Aviares/genética , Tipificación del Cuerpo/genética , Proteína Morfogenética Ósea 4/genética , Embrión de Pollo/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Organizadores Embrionarios/metabolismo , Amisulprida , Animales , Proteínas Aviares/metabolismo , Benzamidas/farmacología , Proteína Morfogenética Ósea 4/metabolismo , Células COS , Embrión de Pollo/embriología , Chlorocebus aethiops , Dioxoles/farmacología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Determinación Derecha-Izquierda/genética , Factores de Determinación Derecha-Izquierda/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Modelos Genéticos , Proteína Nodal/genética , Proteína Nodal/metabolismo , Notocorda/embriología , Notocorda/metabolismo , Organizadores Embrionarios/embriología , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulpirida/análogos & derivados , XenopusRESUMEN
Hepatic epithelial morphogenesis, including hepatoblast migration and proliferation in the septum transversum, requires the interaction of hepatic epithelium with the embryonic sinusoidal wall. No factors that mediate this interaction have yet been identified. As the beta-catenin pathway is active in hepatoblast proliferation, then Wnt ligands might activate the canonical Wnt pathway during liver development. Here, we investigated the role of Wnts in mediating epithelial vessel interactions in the developing chick liver. We found that Wnt9a was specifically expressed in both endothelial and stellate cells of the embryonic sinusoidal wall. Induced overexpression of Wnt9a resulted in hepatomegaly with hyperplasia of the hepatocellular cords, and in hyperproliferation of hepatocytes. Knockdown of Wnt9a caused a reduction in liver size, with hypoplasia of hepatocellular cord branching, and hypoproliferation of hepatoblasts, and also inhibited glycogen accumulation at later developmental stages. Wnt9a promoted in vivo stabilization of beta-catenin through binding with Frizzled 4, 7, and 9, and activated TOPflash reporter expression in vitro via Frizzled 7 and 9. Our results demonstrate that Wnt9a from the embryonic sinusoidal wall is required for the proper morphogenesis of chick hepatocellular cords, proliferation of hepatoblasts/hepatocytes, and glycogen accumulation in hepatocytes. Wnt9a signaling appears to be mediated by an Fzd7/9-beta-catenin pathway.
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División Celular/fisiología , Receptores Frizzled/genética , Hígado/citología , Hígado/fisiología , Morfogénesis/fisiología , Proteínas Wnt/fisiología , Animales , Pollos , Cartilla de ADN , Células Epiteliales/citología , Células Epiteliales/fisiología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Reacción en Cadena de la PolimerasaRESUMEN
Intrahepatic bile ducts (IHBDs) are indispensable for transporting bile secreted from hepatocytes to the hepatic duct. The biliary epithelial cells (BECs) of the IHBD arise from bipotent hepatoblasts around the portal vein, suggesting the portal mesenchyme is essential for their development. However, except for Notch or Activin/TGF-beta signaling molecules, it is not known which molecules regulate IHBD development. Here, we found that FGF receptors and BMP4 are specifically expressed in the developing IHBD and the hepatic mesenchyme, respectively. Using a mesenchyme-free culture of liver bud, we showed that bFGF and FGF7 induce the hepatoblasts to differentiate into BECs, and that BMP4 enhances bFGF-induced BEC differentiation. The extracellular matrix (ECM) components in the hepatic mesenchyme induced BEC differentiation. Forced expression of a constitutively active form of the FGF receptor partially induced BEC differentiation markers in vivo. These data strongly suggest that bFGF and FGF7 promote BEC differentiation cooperatively with BMP4 and ECMs in vivo.
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Proteínas Morfogenéticas Óseas/metabolismo , Matriz Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Células Madre Hematopoyéticas , Hígado , Transducción de Señal/fisiología , Animales , Conductos Biliares/anatomía & histología , Conductos Biliares/fisiología , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular/fisiología , Linaje de la Célula , Células Cultivadas , Pollos , Células Epiteliales/citología , Células Epiteliales/fisiología , Matriz Extracelular/química , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Factor de Crecimiento de Hepatocito/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Queratina-19/genética , Queratina-19/metabolismo , Hígado/anatomía & histología , Hígado/fisiología , Mesodermo/citología , Mesodermo/fisiología , Morfogénesis , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Hepatoblasts have the potential to differentiate into both hepatocytes and biliary epithelial cells through a differentiation program that has not been fully elucidated. With the aim to better define the mechanism of differentiation of hepatoblasts, we isolated hepatoblasts and established new culture systems. We isolated hepatoblasts from E12.5 fetal mouse liver by using E-cadherin. The E-cadherin+ cells expressed alpha-fetoprotein (AFP) and albumin (Alb) but not cytokeratin 19 (CK19). Transplantation of the E-cadherin+ cells into mice that had been subjected to liver injury or biliary epithelial injury led to differentiation of the cells into hepatocytes or biliary epithelial cells, respectively. In a low-cell-density culture system in the absence of additional growth factors, E-cadherin+ cells formed colonies of various sizes, largely comprising Alb-positive cells. Supplementation of the culture medium with hepatocyte growth factor and epidermal growth factor promoted proliferation of the cells. Thus the low-cell-density culture system should be useful to identify inductive factors that regulate the proliferation and differentiation of hepatoblasts. In a high-cell-density system in the presence of oncostatin M+dexamethasone, E14.5, but not E12.5, E-cadherin+ cells differentiated into mature hepatocytes, suggesting that unidentified factors are involved in hepatic maturation. Culture of E-cadherin+ cells derived from E12.5 or E14.5 liver under high-cell-density conditions should allow elucidation of the mechanism of hepatic differentiation in greater detail. These new culture systems should be of use to identify growth factors that induce hepatoblasts to proliferate or differentiate into hepatocytes and biliary epithelial cells.
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Hepatocitos/fisiología , Hígado/citología , Hígado/embriología , Animales , Conductos Biliares/citología , Cadherinas/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular , Separación Celular , Tamaño de la Célula , Trasplante de Células , Células Cultivadas , Factor de Crecimiento Epidérmico/fisiología , Femenino , Factor de Crecimiento de Hepatocito/fisiología , Inmunohistoquímica , Ratones , Ratones Endogámicos ICR , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
During chick liver development, the liver bud arises from the foregut, invaginates into the septum transversum, and elongates along and envelops the ductus venosus. However, the mechanism of liver bud migration is only poorly understood. Here, we demonstrate that a GDNF family ligand involved in neuronal outgrowth and migration, neurturin (NRTN), and its receptor, GFRalpha2, are essential for liver bud migration. In the chick embryo, we found that GFRalpha2 was expressed in the liver bud and that NRTN was expressed in the endothelial cells of the ductus venosus. Inhibition of GFRalpha2 signaling suppressed liver bud elongation along the ductus venous without affecting cell proliferation and apoptosis. Moreover, ectopic expression of NRTN perturbed the directional migration along the ductus venosus, leading to splitting or ectopic branching of the liver. We showed that liver buds selectively migrated toward an NRTN-soaked bead in vitro. These data represent a new model for liver bud migration: NRTN secreted from endothelial cells functions as a chemoattractant to direct the migration of the GFRalpha2-expressing liver bud in early liver development.
Asunto(s)
Movimiento Celular , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Hígado/embriología , Neurturina/fisiología , Animales , Circulación Sanguínea , Factores Quimiotácticos/fisiología , Embrión de Pollo , Células Endoteliales , Hígado/citología , Hígado/crecimiento & desarrollo , Morfogénesis , Organogénesis , Transducción de SeñalRESUMEN
The specified hepatic endoderm (hepatoblasts), the bipotential progenitor for hepatocytes and bile duct epithelial cells, proliferates during the primordial stages of liver development. Despite extensive studies, the mechanism that regulates proliferation of bipotential hepatoblasts is not fully understood. Here we show that Id3, a negative regulator of helix-loop-helix transcription factors, is an important regulator of hepatoblast proliferation in the developing chick liver. Id3 was expressed in hepatoblasts at early developmental stages (stages 12-29) but not in hepatocytes at later developmental stages (stage 34 onwards). Depletion of Id3 in hepatoblasts by siRNA results in failure of cell proliferation, but is not associated with either cell death or failure of expression of Hhex and Fibrinogen, the earliest hepatoblast markers. These observations suggest that at early developmental stages, Id3 functions as a positive regulator of hepatoblast proliferation, independent of cell death or maintenance of the non-terminally differentiated state. Interestingly at later developmental stages, the expression pattern of Id3 is complementary to that of Albumin, a marker of mature hepatocytes. Overexpression of Id3 in liver explants delayed the initiation of Albumin expression. Taken together, our observations show that Id3 is not only a positive regulator of hepatoblast proliferation, but also an inhibitor of their differentiation into hepatocytes in the developing chick liver.