RESUMEN
Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 âkb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 âkb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.
Asunto(s)
Pronefro , Tretinoina , Animales , Xenopus laevis/genética , Xenopus laevis/metabolismo , Tretinoina/farmacología , Tretinoina/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Regulación del Desarrollo de la Expresión Génica , Pronefro/metabolismo , Riñón/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Retinal-Deshidrogenasa/metabolismoRESUMEN
Upon infection, B lymphocytes develop clonal responses. In teleost fish, which lack lymph nodes, the kinetics and location of B cell responses remain poorly characterized. Fish pronephros is the site of B cell differentiation and the main niche for persistence of plasma cells. In this study, we undertook the analysis of the rainbow trout IgHµ repertoire in this critical tissue for humoral adaptive immunity after primary immunization and boost with a rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We used a barcoded 5' RACE-cDNA sequencing approach to characterize modifications of the IgHµ repertoire, including VH usage in expressed V(D)J rearrangements, clonal diversity, and clonotype sharing between individual fish and treatments. In the pronephros, our approach quantified the clonotype frequency across the whole IgH repertoire (i.e., with all VH), measuring the frequency of Ag-responding clonotypes. Viral infection led to extensive modifications of the pronephros B cell repertoire, implicating several VH subgroups after primary infection. In contrast, only modest changes in repertoire persisted 5 mo later, including VHSV-specific public expansions. The IgM public response implicating IgHV1-18 and JH5, previously described in spleen, was confirmed in pronephros in all infected fish, strongly correlated to the response. However, the distribution of top clonotypes showed that pronephros and spleen B cells constitute distinct compartments with different IgH repertoires. Unexpectedly, after boost, the frequency of anti-VHSV clonotypes decreased both in pronephros and spleen, raising questions about B cell circulation. A better monitoring of B cell response kinetics in lymphoid tissues will be an essential step to understand B memory and plasmocyte formation mechanisms in fish.
Asunto(s)
Enfermedades de los Peces , Septicemia Hemorrágica Viral , Novirhabdovirus , Oncorhynchus mykiss , Pronefro , Virosis , Animales , Septicemia Hemorrágica Viral/prevención & control , Oncorhynchus mykiss/genética , BazoRESUMEN
The corpuscles of Stannius (CS) represent a unique endocrine organ of teleostean fish that secrets stanniocalcin-1 (Stc1) to maintain calcium homeostasis. Appearing at 20-25 somite stage in the distal zebrafish pronephros, stc1-expressing cells undergo apical constriction, and are subsequently extruded to form a distinct gland on top of the distal pronephric tubules at 50 âh post fertilization (hpf). Several transcription factors (e.g. Hnf1b, Irx3b, Tbx2a/b) and signaling pathways (e.g. Notch) control CS development. We report now that Fgf signaling is required to commit tubular epithelial cells to differentiate into stc1-expressing CS cells. Inhibition of Fgf signaling by SU5402, dominant-negative Fgfr1, or depletion of fgf8a prevented CS formation and stc1 expression. Ablation experiments revealed that CS have the ability to partially regenerate via active cell migration involving extensive filopodia and lamellipodia formation. Activation of Wnt signaling curtailed stc1 expression, but had no effect on CS formation. Thus, our observations identify Fgf signaling as a crucial component of CS cell fate commitment.
Asunto(s)
Diferenciación Celular , Glándulas Endocrinas/embriología , Factores de Crecimiento de Fibroblastos , Pronefro/embriología , Vía de Señalización Wnt , Proteínas de Pez Cebra , Pez Cebra , Animales , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Pronephric kidneys have a single large nephron that provides essential osmoregulation in amphibians and fish until the adult kidney forms. As mammalian kidneys evolved from the simple pronephric kidneys of the early vertebrates, understanding the structure and function of pronephroi gives insight into the blueprints underlying all nephrons. The article in this issue by Corkins et al. uses single-cell sequencing to demonstrate an extraordinary segmental complexity and the organizational roadmap that mammalian nephrons are based upon.
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Nefronas , Pronefro , Animales , Riñón , MamíferosRESUMEN
Ciliopathies affect a variety of tissues during development including the heart, kidneys, respiratory tract, and retina. Though an increasing number of monogenic causes of ciliopathies have been described, many remain unexplained. Recently, recessive variants in NUP93 and NUP205 encoding two proteins of the inner ring of the nuclear pore complex were implicated as causes of steroid resistant nephrotic syndrome. In addition, we previously found that the inner ring nucleoporins NUP93 and NUP188 function in proper left-right patterning in developing embryos via a role at the cilium. Here, we describe the role of an additional inner ring nucleoporin NUP205 in cilia biology and establishment of normal organ situs. Using knockdown in Xenopus, we show that Nup205 depletion results in loss of cilia and abnormal cardiac morphology. Furthermore, by transmission electron microscopy, we observe a loss of cilia and mispositioning of intracellular ciliary structures such as basal bodies and rootlets upon depleting inner ring nucleoporins. We describe a model wherein NUP93 interacting with either NUP188 or NUP205 is necessary for cilia. We thus provide evidence that dysregulation of inner ring nucleoporin genes that have been identified in patients may contribute to pathogenesis through cilia dysfunction.
Asunto(s)
Cilios/fisiología , Proteínas de Complejo Poro Nuclear/fisiología , Proteínas de Xenopus/fisiología , Animales , Tipificación del Cuerpo , Cilios/ultraestructura , Epidermis/embriología , Epidermis/ultraestructura , Técnicas de Silenciamiento del Gen , Cardiopatías Congénitas/genética , Humanos , Proteínas de Complejo Poro Nuclear/genética , Pronefro/ultraestructura , Xenopus/embriología , Proteínas de Xenopus/genéticaRESUMEN
Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular compartments with discrete physiological identities. Here, from a forward genetic screen using zebrafish, we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that activates the terminal differentiation program of distal segments in the pronephros. We found that tfap2a acts downstream of Iroquois homeobox 3b (irx3b), a distal lineage transcription factor, to operate a circuit consisting of tfap2b, irx1a and genes encoding solute transporters that dictate the specialized metabolic functions of distal nephron segments. Interestingly, this regulatory node is distinct from other checkpoints of differentiation, such as polarity establishment and ciliogenesis. Thus, our studies reveal insights into the genetic control of differentiation, where tfap2a is essential for regulating a suite of segment transporter traits at the final tier of zebrafish pronephros ontogeny. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to facilitate drug discovery and regenerative therapies.
Asunto(s)
Riñón/embriología , Nefronas/embriología , Organogénesis/genética , Factor de Transcripción AP-2/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Genes de Cambio/fisiología , Riñón/metabolismo , Nefronas/metabolismo , Pronefro/embriología , Pronefro/crecimiento & desarrollo , Pronefro/metabolismo , Factor de Transcripción AP-2/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease. METHODS: Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified. RESULTS: Three biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes. CONCLUSIONS: Variants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.
Asunto(s)
Proteínas de Unión al ADN/genética , Hernia Hiatal/genética , Microcefalia/genética , Mutación Missense , Nefrosis/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Preescolar , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/deficiencia , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Masculino , Modelos Moleculares , Síndrome Nefrótico/genética , Podocitos/metabolismo , Polimorfismo de Nucleótido Simple , Pronefro/embriología , Pronefro/metabolismo , Estabilidad Proteica , Factores de Transcripción/química , Factores de Transcripción/deficiencia , Xenopus laevis/embriología , Xenopus laevis/genética , Dedos de Zinc/genéticaRESUMEN
Acute kidney injury (AKI) is commonly associated with severe human diseases, and often worsens the outcome in hospitalized patients. The mammalian kidney has the ability to recover spontaneously from AKI; however, little progress has been made in the development of supportive treatments. Increasing evidence suggest that histone deacetylases (HDAC) and NF-κB promote the pathogenesis of AKI, and inhibition of Hdac activity has a protective effect in murine models of AKI. However, the role of HDAC at the early stages of recovery is unknown. We used the zebrafish pronephros model to study the role of epigenetic modifiers in the immediate repair response after injury to the tubular epithelium. Using specific inhibitors, we found that the histone deacetylase Hdac2, Hdac6, and Hdac8 activities are required for the repair via collective cell migration. We found that hdac6, hdac8, and nfkbiaa expression levels were upregulated in the repairing epithelial cells shortly after injury. Depletion of hdac6, hdac8, or nfkbiaa with morpholino oligonucleotides impaired the repair process, whereas the combined depletion of all three genes synergistically suppressed the recovery process. Furthermore, time-lapse video microscopy revealed that the lamellipodia and filopodia formation in the flanking cells was strongly reduced in hdac6-depleted embryos. Our findings suggest that Hdac activity and NF-κB are synergistically required for the immediate repair response in the zebrafish pronephros model of AKI, and the timing of HDAC inhibition might be important in developing supportive protocols in the human disease.
Asunto(s)
Lesión Renal Aguda , Histona Desacetilasa 6/metabolismo , Histona Desacetilasas/metabolismo , Pronefro , Proteínas de Pez Cebra/metabolismo , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Animales , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ratones , FN-kappa B , Pronefro/metabolismo , Pronefro/patología , Proteínas Represoras , Pez Cebra/metabolismoRESUMEN
Developing organisms need to adapt to environmental variations as well as to rapid changes in substrate availability and energy demands imposed by fast-growing tissues and organs. Little is known about the adjustments that kidneys undergo in response to these challenges. We performed single-cell RNA sequencing of zebrafish pronephric duct cells to understand how the developing kidney responds to changes in filtered substrates and intrinsic energy requirements. We found high levels of glucose transporters early in development and increased expression of monocarboxylate transporters at later times. This indicates that the zebrafish embryonic kidney displays a high glucose transporting capacity during early development, which is replaced by the ability to absorb monocarboxylates and amino acids at later stages. This change in transport capacity was accompanied by the upregulation of mitochondrial carriers, indicating a switch to increased oxidative phosphorylation to meet the increasing energy demand of a developing kidney.NEW & NOTEWORTHY The zebrafish embryonic kidney has high levels of glucose transporters during early development, which are replaced by monocarboxylate and amino acid transporters later on. Inhibition of Na+-glucose cotransporter-dependent glucose transport by sotagliflozin also increased slc2a1a expression, supporting the idea that the glucose transport capacity is dynamically adjusted during zebrafish pronephros development. Concurrent upregulation of mitochondrial SCL25 transporters at later stages supports the idea that the pronephros adjusts to changing substrate supplies and/or energy demands during embryonic development.
Asunto(s)
Metabolismo Energético/genética , Perfilación de la Expresión Génica , Pronefro/metabolismo , ARN Mensajero/genética , Análisis de la Célula Individual , Proteínas Transportadoras de Solutos/genética , Transcriptoma , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Pronefro/embriología , ARN Mensajero/metabolismo , RNA-Seq , Proteínas Transportadoras de Solutos/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Inherited and acquired disorders that affect proximal tubule endocytosis and lysosomal processing manifest with improper loss of solutes and proteins. The zebrafish pronephros is conserved with humans and is used to model numerous renal conditions, but has few quantitative measures for proximal tubule function. Here, Chen et al. developed a high-throughput assay to quantify proteinuria and lysosomal processing in transgenic zebrafish by labeling vitamin D protein, allowing for precise reporting of proximal tubule function.
Asunto(s)
Síndrome de Fanconi , Pronefro , Animales , Síndrome de Fanconi/genética , Humanos , Riñón , Túbulos Renales Proximales , Pez Cebra/genéticaRESUMEN
Congenital Anomalies of the Kidney and of the Urinary Tract (CAKUT) cover a broad range of disorders including abnormal kidney development caused by defective nephrogenesis. Here we explored the possible involvement of the low affinity p75 neurotrophin receptor (p75NTR) in CAKUT and nephrogenesis. In mouse, p75NTR was highly expressed in fetal kidney, located within cortical early nephrogenic bodies, and decreased rapidly after birth. In human control fetal kidney, p75NTR was also located within the early nephrogenic bodies as well as in the mature glomeruli, presumably in the mesangium. In CAKUT fetal kidneys, the kidney cortical structure and the localization of p75NTR were often disorganized, and quantification of p75NTR in amniotic fluid revealed a significant reduction in CAKUT compared to control. Finally, invalidation of p75NTR in zebrafish embryo with an antisense morpholino significantly altered pronephros development. Our results indicate that renal p75NTR is altered in CAKUT fetuses, and could participate to early nephrogenesis.
Asunto(s)
Riñón/anomalías , Riñón/embriología , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Sistema Urinario/anomalías , Animales , Regulación hacia Abajo , Humanos , Riñón/metabolismo , Ratones , Pronefro , ARN Mensajero/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Pez Cebra/embriologíaRESUMEN
Cadmium (Cd) is a type of toxic metal, in most cases, coming from fuel burning and aquatic plants. The cells of organisms can be caused serious damage, including pyroptosis, exposure to low concentrations of Cd in long-term. Pyroptosis is a recently discovered Caspase-1-mediated cell death. In this study, lymphocytes were extracted from the pronephros and spleens in carps, respectively. After treating cells with low concentration of Cd, the mRNA and protein expression levels of pyroptosis-related genes, NLRP3, Caspase-1, and pro-inflammatory cytokines, increased obviously. And the content of reactive oxygen species (ROS) and mitochondria reactive oxygen species (mtROS) increased significantly, we also found the activities of CAT, GSH-px and T-SOD reduce significantly, and the content of MDA have a clear upward trend. We then added NLRP3 inhibitor, Glyburide, to the Cd-treated group, further confirming that NLRP3 is a key gene in pyroptosis pathways by detecting the mRNA and protein expression levels. Besides, the rupture of the cell membrane was also confirmed by Hoechst/PI double staining, red fluorescence increased obviously in the Cd treatment group. The experiment revealed that Cd exposure induces pyroptosis of lymphocytes in carp pronephros and spleens by activating NLRP3. Inhibition of NLRP3 activity can slow down the degree of lymphocytes pyroptosis. Thus, the above information provides a new avenue toward understanding the partial mechanism of Cd exposure-induced pyroptosis.
Asunto(s)
Cadmio/toxicidad , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Pronefro/metabolismo , Contaminantes Químicos del Agua/toxicidad , Animales , Cadmio/metabolismo , Carpas/metabolismo , Carpas/fisiología , Caspasa 1 , Inflamasomas/metabolismo , Linfocitos , Mitocondrias/metabolismo , Piroptosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Bazo/metabolismoRESUMEN
The zebrafish kidney is conserved with other vertebrates, making it an excellent genetic model to study renal development. The kidney collects metabolic waste using a blood filter with specialized epithelial cells known as podocytes. Podocyte formation is poorly understood but relevant to many kidney diseases, as podocyte injury leads to progressive scarring and organ failure. zeppelin (zep) was isolated in a forward screen for kidney mutants and identified as a homozygous recessive lethal allele that causes reduced podocyte numbers, deficient filtration, and fluid imbalance. Interestingly, zep mutants had a larger interrenal gland, the teleostean counterpart of the mammalian adrenal gland, which suggested a fate switch with the related podocyte lineage since cell proliferation and cell death were unchanged within the shared progenitor field from which these two identities arise. Cloning of zep by whole genome sequencing (WGS) identified a splicing mutation in breast cancer 2, early onset (brca2)/fancd1, which was confirmed by sequencing of individual fish. Several independent brca2 morpholinos (MOs) phenocopied zep, causing edema, reduced podocyte number, and increased interrenal cell number. Complementation analysis between zep and brca2ZM_00057434 -/- zebrafish, which have an insertional mutation, revealed that the interrenal lineage was expanded. Importantly, overexpression of brca2 rescued podocyte formation in zep mutants, providing critical evidence that the brca2 lesion encoded by zep specifically disrupts the balance of nephrogenesis. Taken together, these data suggest for the first time that brca2/fancd1 is essential for vertebrate kidney ontogeny. Thus, our findings impart novel insights into the genetic components that impact renal development, and because BRCA2/FANCD1 mutations in humans cause Fanconi anemia and several common cancers, this work has identified a new zebrafish model to further study brca2/fancd1 in disease.
Asunto(s)
Proteína BRCA2/genética , Regulación del Desarrollo de la Expresión Génica , Organogénesis/genética , Podocitos/citología , Pronefro/embriología , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Diferenciación Celular , Proliferación Celular , Clonación Molecular , Modelos Animales de Enfermedad , Hibridación Fluorescente in Situ , Morfolinos/genética , Pronefro/citología , Pez Cebra/genéticaRESUMEN
The simplified and genetically conserved zebrafish pronephros is an excellent model to examine the cryptic processes of cell fate decisions during the development of nephron segments as well as the origins of associated endocrine cells that comprise the corpuscles of Stannius (CS). Using whole mount in situ hybridization, we found that transcripts of the zebrafish genes t-box 2a (tbx2a) and t-box 2b (tbx2b), which belong to the T-box family of transcription factors, were expressed in the caudal intermediate mesoderm progenitors that give rise to the distal pronephros and CS. Deficiency of tbx2a, tbx2b or both tbx2a/b reduced the size of the distal late (DL) segment, which was accompanied by a proximal convoluted segment (PCT) expansion. Further, tbx2a/b deficiency led to significantly larger CS clusters. These phenotypes were also observed in embryos with the from beyond (fby)c144 mutation, which encodes a premature stop codon in the tbx2b T-box sequence. Conversely, overexpression of tbx2a and tbx2b in wild-type embryos expanded the DL segment where cells were comingled with the adjacent DE, and also decreased CS cell number, but notably did not alter PCT development-providing independent evidence that tbx2a and tbx2b are each necessary and sufficient to promote DL fate and suppress CS genesis. Epistasis studies indicated that tbx2a acts upstream of tbx2b to regulate the DL and CS fates, and likely has other targets as well. Retinoic acid (RA) addition and inhibition studies revealed that tbx2a and tbx2b are negatively regulated by RA signaling. Interestingly, the CS cell expansion that typifies tbx2a/b deficiency also occurred when blocking Notch signaling with the chemical DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). Ectopic activation of Notch in Tg(hsp70::Gal4; UAS::NICD)(NICD) embryos led to a reduced CS post heat-shock induction. To further examine the link between the tbx2a/b genes and Notch during CS formation, DAPT treatment was used to block Notch activity in tbx2a/b deficient embryos, and tbx2a/b knockdown was performed in NICD transgenic embryos. Both manipulations caused similar CS expansions, indicating that Notch functions upstream of the tbx2a/b genes to suppress CS ontogeny. Taken together, these data reveal for the first time that tbx2a/b mitigate pronephros segmentation downstream of RA, and that interplay between Notch signaling and tbx2a/b regulate CS formation, thus providing several novel insights into the genetic regulatory networks that influence these lineages.
Asunto(s)
Tipificación del Cuerpo/genética , Pronefro/embriología , Pronefro/metabolismo , Proteínas de Dominio T Box/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Tipificación del Cuerpo/efectos de los fármacos , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Mesodermo/efectos de los fármacos , Mesodermo/embriología , Mesodermo/metabolismo , Modelos Biológicos , Organogénesis/efectos de los fármacos , Organogénesis/genética , Pronefro/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Dominio T Box/genética , Tretinoina/farmacología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
During zebrafish embryogenesis the pronephric kidney arises from a small population of posterior mesoderm cells that then undergo expansion during early stages of renal organogenesis. While wnt8 is required for posterior mesoderm formation during gastrulation, it is also transiently expressed in the post-gastrula embryo in the intermediate mesoderm, the precursor to the pronephros and some blood/vascular lineages. Here, we show that knockdown of wnt8a, using a low dose of morpholino that does not disrupt early mesoderm patterning, reduces the number of kidney and blood cells. For the kidney, wnt8a deficiency decreases renal progenitor growth during early somitogenesis, as detected by EdU incorporation, but has no effect on apoptosis. The depletion of the renal progenitor pool in wnt8a knockdown embryos leads to cellular deficits in the pronephros at 24 hpf that are characterised by a shortened distal-most segment and stretched proximal tubule cells. A pulse of the canonical Wnt pathway agonist BIO during early somitogenesis is sufficient to rescue the size of the renal progenitor pool while longer treatment expands the number of kidney cells. Taken together, these observations indicate that Wnt8, in addition to its well-established role in posterior mesoderm patterning, also plays a later role as a factor that expands the renal progenitor pool prior to kidney morphogenesis.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Embrión no Mamífero/citología , Riñón/citología , Riñón/embriología , Células Madre/citología , Proteínas Wnt/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células Sanguíneas/citología , Células Sanguíneas/efectos de los fármacos , Tipificación del Cuerpo/efectos de los fármacos , Recuento de Células , Proliferación Celular/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Indoles/farmacología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Mesodermo/efectos de los fármacos , Mesodermo/embriología , Mesodermo/metabolismo , Morfolinos/farmacología , Oximas/farmacología , Pronefro/citología , Pronefro/embriología , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Eph/ephrin molecules are widely expressed during embryonic development, and function in a variety of developmental processes. Here we studied the roles of the Eph receptor EphA7 and its soluble form in Xenopus pronephros development. EphA7 is specifically expressed in pronephric tubules at tadpole stages and knockdown of EphA7 by a translation blocking morpholino led to defects in tubule cell differentiation and morphogenesis. A soluble form of EphA7 (sEphA7) was also identified. Interestingly, the membrane level of claudin6 (CLDN6), a tetraspan transmembrane tight junction protein, was dramatically reduced in the translation blocking morpholino injected embryos, but not when a splicing morpholino was used, which blocks only the full length EphA7. In cultured cells, EphA7 binds and phosphorylates CLDN6, and reduces its distribution at the cell surface. Our work suggests a role of EphA7 in the regulation of cell adhesion during pronephros development, whereas sEphA7 works as an antagonist.
Asunto(s)
Claudinas/metabolismo , Pronefro/embriología , Receptor EphA7/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Animales , Membrana Celular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Oligodesoxirribonucleótidos Antisentido/genética , Pronefro/metabolismo , Receptor EphA7/antagonistas & inhibidores , Receptor EphA7/genética , Solubilidad , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
The renin-angiotensin system (RAS) is highly conserved, and components of the RAS are present in all vertebrates to some degree. Although the RAS has been studied since the discovery of renin, its biological role continues to broaden with the identification and characterization of new peptides. The evolutionarily distant zebrafish is a remarkable model for studying the kidney due to its genetic tractability and accessibility for in vivo imaging. The zebrafish pronephros is an especially useful kidney model due to its structural simplicity yet complex functionality, including capacity for glomerular and tubular filtration. Both the pronephros and mesonephros contain renin-expressing perivascular cells, which respond to RAS inhibition, making the zebrafish an excellent model for studying the RAS. This review summarizes the physiological and genetic tools currently available for studying the zebrafish kidney with regards to functionality of the RAS, using novel imaging techniques such as SPIM microscopy coupled with targeted single cell ablation and synthesis of vasoactive RAS peptides.
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Pronefro/metabolismo , Sistema Renina-Angiotensina , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Antagonistas de Receptores de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Animales Modificados Genéticamente , Regulación del Desarrollo de la Expresión Génica , Enfermedades Renales/inducido químicamente , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Pronefro/efectos de los fármacos , Pronefro/patología , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/genética , Transducción de Señal , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease.
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Endocitosis , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Pronefro/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Polaridad Celular , Endosomas/metabolismo , Eliminación de Gen , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a "salt-and-pepper" fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA treatment expanded the etv5a domain within the renal progenitor field and RA inhibition blocked etv5a in this populace, indicating that etv5a acts downstream of RA. Additionally, treatment with exogenous RA partially rescued the reduced MCC phenotype after loss of etv5a. Further, abrogation of Notch with the small molecule inhibitor DAPT increased the renal progenitor etv5a expression domain as well as MCC density in etv5a deficient embryos, suggesting Notch acts upstream to inhibit etv5a. In contrast, etv4 levels in renal progenitors were unaffected by changes in RA or Notch signaling levels, suggesting a possible non-cell autonomous role during pronephros formation. Taken together, these findings have revealed new insights about the genetic mechanisms of epithelial cell development during nephrogenesis.
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Células Epiteliales/citología , Riñón/embriología , Nefronas/embriología , Pronefro/embriología , Proteínas Proto-Oncogénicas c-ets/fisiología , Factores de Transcripción/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Naranja de Acridina/química , Animales , Diferenciación Celular , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Hibridación Fluorescente in Situ , Organogénesis/genética , Receptores Notch/metabolismo , Transducción de Señal , Tretinoina/metabolismo , Pez Cebra/fisiologíaRESUMEN
Mutations in the homeobox transcription factor MNX1 are the major cause of dominantly inherited sacral agenesis. Studies in model organisms revealed conserved mnx gene requirements in neuronal and pancreatic development while Mnx activities that could explain the caudal mesoderm specific agenesis phenotype remain elusive. Here we use the zebrafish pronephros as a simple yet genetically conserved model for kidney formation to uncover a novel role of Mnx factors in nephron morphogenesis. Pronephros formation can formally be divided in four stages, the specification of nephric mesoderm from the intermediate mesoderm (IM), growth and epithelialisation, segmentation and formation of the glomerular capillary tuft. Two of the three mnx genes in zebrafish are dynamically transcribed in caudal IM in a time window that proceeds segmentation. We show that expression of one mnx gene, mnx2b, is restricted to the pronephric lineage and that mnx2b knock-down causes proximal pronephric tubule dilation and impaired pronephric excretion. Using expression profiling of embryos transgenic for conditional activation and repression of Mnx regulated genes, we further identified irx1b as a direct target of Mnx factors. Consistent with a repression of irx1b by Mnx factors, the transcripts of irx1b and mnx genes are found in mutual exclusive regions in the IM, and blocking of Mnx functions results in a caudal expansion of the IM-specific irx1b expression. Finally, we find that knock-down of irx1b is sufficient to rescue proximal pronephric tubule dilation and impaired nephron function in mnx-morpholino injected embryos. Our data revealed a first caudal mesoderm specific requirement of Mnx factors in a non-human system and they demonstrate that Mnx-dependent restriction of IM-specific irx1b activation is required for the morphogenesis and function of the zebrafish pronephros.