Molecular Genetics of Bartter Syndrome

Bartter syndrome (BS) is a heterogeneous disorder, caused by mutations in several genes which mostly encode proteins involved in ions transportation across renal cells in the thick ascending limb of the nephron. It is characterized by deficient renal reabsorption of sodium and chloride, which results in a group of certain symptoms. Different types of BS can be distinguished from different clinical manifestations, and most importantly, via analyzing possible affected gene(s) for its confirmation. A close associated syndrome which was primarily considered as a mild variant of BS, Gitelman syndrome (GS), is characterized by hypokalemic metabolic alkalosis with hypocalciuria, and hypomagnesemia. In this review, we discuss different features of BS and also GS, including clinical and genetic alterations which correspond to each type.  Keywords: Bartter Syndrome; Molecular Genetics; Child

Terminal differentiation of villus tip enterocytes is governed by distinct Tgfβ superfamily members

The protective and absorptive functions of the intestinal epithelium rely on differentiated enterocytes in the villi. The differentiation of enterocytes is orchestrated by sub-epithelial mesenchymal cells producing distinct ligands along the villus axis, in particular Bmps and Tgfβ. Here, we show that individual Bmp ligands and Tgfβ drive distinct enterocytic programs specific to villus zonation. Bmp4 is expressed from the centre to the upper part of the villus and activates preferentially genes connected to lipid uptake and metabolism. In contrast, Bmp2 is produced by villus tip mesenchymal cells and it influences the adhesive properties of villus tip epithelial cells and the expression of immunomodulators. Additionally, Tgfβ induces epithelial gene expression programs similar to those triggered by Bmp2. Bmp2-driven villus tip program is activated by a canonical Bmp receptor type I/Smad-dependent mechanism. Finally, we establish an organoid cultivation system that enriches villus tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Our data suggest that not only a Bmp gradient but also the activity of individual Bmp drives specific enterocytic programs

Epithelial to mesenchymal transition trajectories in developmental and disease

Resumen del trabajo presentado al 19th International Congress of Developmental Biology, celebrado en El Algarve (Portugal) del 16 al 20 de octubre de 2022.The Epithelial to Mesenchymal transition (EMT) triggers cell plasticity during embryonic development and tissue repair, but it can also promote tumor progression and organ degeneration. The reactivation of EMT in the adult promotes cell dedifferentiation and profound remodeling of the epithelial program, leading to multiple phenotypes, observed in response to injury, during organ fibrosis and cancer cell dissemination. Despite recent advances, identifying universal EMT molecular signatures and understanding how EMT can instructs different outcomes have remained elusive due to the intrinsic complexity and heterogeneity of the process. We have dissected how EMT transcription factors (EMT-TFs) orchestrate TGFBinduced EMT including phenotypic and behavioral states. Further, we have combined lineage tracing and single-cell transcriptomics in three EMT contexts, namely the neural crest, renal fibrosis, and breast cancer to reveal conserved EMT transcription factor codes and signaling pathways that discriminate different EMT states. After inferring cellular trajectories, we have reconstructed the evolution of EMT phenotypic and functional states in all these contexts. Finally, multiplex labeling allowed to spatially allocate distinct EMT programs in mouse and human tumor samples. Altogether, this work unveils distinct EMT trajectories in development and disease, which should also help propase improved therapeutic strategies for organ fibrosis and cancer.Peer reviewe

Two distinct epithelial to mesenchymal transition programmes. Control invasion and inflammation in segregated tumour cell populations

Resumen del trabajo presentado al 19th Christmas Meeting del Instituto de Neurociencias (CSIC-UMH) celebrado el 21 de diciembre de 2022.Epithelial plasticity is at the core of crucial processes including embryonic cell migration, cancer progression, organ tibrosis and tissue repair. The epithelial to mesenchymal transition (EMT) triggers cell plasticity in all these contexts, highlighting its pleiotropy and intrinsic complcxity. Seminal studies have classified EMT states in cancer celllines and animal modcls. This varicty ofEMT phenotypes necds further investigation, particularly those relevant to the progression ofprevalent and dcvastating diseases such as cancer. Our objcctive is to analyse at single-cell level how different EMT states are established in tumours and if different EMT states pcrform different functions during tumour progression.Peer reviewe

A gene regulatory network to control EMT programs in development and disease

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 23-04-2019Esta tesis tiene embargado el acceso al texto completo hasta el 23-10-2020Epithelial to Mesenchymal Transition (EMT) plays pivotal roles during development and diseases like cancer and fibrosis, through the activation of several EMT transcription factor (EMT-TF) families, including Snail, Zeb, Twist and Prrx. Prior data from the laboratory showed that the patterns of PRRX1 and SNAIL1 expression were complementary in chicken embryos and cancer cells, and that their functions in EMT subprograms, such as the regulation of stemness, also seemed to be distinct. Here, after examining zebrafish, chicken and mouse embryos, we find that this complementary expression of Snail1 and Prrx1 is conserved during vertebrate development. Moreover, analyzing public single-cell RNA sequencing databases of breast, head-and-neck cancer or melanoma patients and from mouse pulmonary fibrosis, we confirmed that this complementary expression is also present in pathological EMTs. By studying the transcriptome of cancer cells, gain and loss of function experiments for the two EMT-TFs, and the use of animal models, we describe a novel gene regulatory network (GRN) where Snail1 and Prrx1 form a double-negative feedback loop, involving miR-15 family. We have found that this GRN triggers an expression switch from Snail1 to Prrx1, with Snail1 being an early-response gene to EMT-inducing signals, which is followed by the activation of Prrx1 that in turn attenuates Snail1 expression through miR-15 family. We have also validated this GRN in vitro and in vivo highlighting its relevance in development and diseaseLa transición epitelio-mesenquimática (EMT) desempeña un papel fundamental durante la embriogénesis y la progresión de enfermedades como el cáncer y la fibrosis, mediante la activación de varias familias de factores de transcripción inductores de la EMT (EMT-TF), incluyendo Snail, Zeb, Twist y Prrx. Resultados previos obtenidos en el laboratorio mostraron que los patrones de expresión de PRRX1 y SNAIL1 eran complementarios en embriones de pollo y células cancerosas, y que sus funciones en subprogramas de la EMT, como la regulación de la pluripotencia, también parecían ser distintas. En este estudio, tras examinar el patrón de expresión de estos EMT-TFs en embriones de pez cebra, de pollo y de ratón hemos encontrado que la expresión complementaria de Snail1 y Prrx1 está conservada en los distintos vertebrados. Además, el análisis de bases de datos públicas obtenidos tras la secuenciación del transcriptoma de células individuales (scRNA seq) de pacientes con cáncer de mama, cuello y cabeza, melanoma, y de ratones con fibrosis pulmonar, confirmamos que esta complementariedad también existe en los procesos de EMT patológicos. Tras el estudio del transcriptoma de células cancerosas y experimentos de ganancia y pérdida de función para los dos EMT-TF, junto con el uso de modelos animales, hemos encontrado una red de regulación génica (GRN) donde Snail1 y Prrx1 forman un bucle de retroalimentación negativo, involucrando a la familia miR-15. Esta red promueve un cambio de expresión de Snail1 a Prrx1, siendo Snail1 un gen de respuesta temprana a señales inductoras de EMT, que es seguida por la activación de Prrx1 que, a su vez, atenúa la expresión de Snail1 mediante la familia miR-15. Hemos validado este GRN in vitro e in vivo, revelando sus implicaciones en el desarrollo embrionario y la enfermedad

Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis—placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity

Variants in GNPTAB, GNPTG and NAGPA genes are associated with stutterers

Non-syndromic stuttering is a neurodevelopmental disorder characterized by disruptions in normal flow of speech in the form of repetition, prolongation and involuntary halts. Previously, mutations with more severe effects on GNPTAB and GNPTG have been reported to cause Mucolipidosisll (ML-ll) and Mucolipidosislll (ML-lll), two lysosomal storage disorders with multiple pathologies. We used homozygosity mapping and Sanger sequencing to investigate variants of the three genes in 25 Iranian families with at least two first degree related non-syndromic stutterers. Bioinformatic evaluation and Segregation analysis of the found variants helped us define probable consequences. We also compared our findings with those related to Mucolipidosis. 14 variations were found in the three genes 3 of which, including a novel variant within intronic region of GNPTG and a heterozygous 2-bp deletion in coding region of GNPTAB, co-segregated with stuttering in the families they were found. Bioinformatics analysis predicted all three variants causing deleterious effects on gene functioning. Our findings support the role of these three variants in non-syndromic stuttering. This finding may challenge the current belief that variations causing stuttering are at different sites and have less severe consequences than genetic changes that cause ML-ll and ML-lll.Peer reviewe

Ciliated, Mitochondria-Rich Postmitotic Cells are Immune-privileged, and Mimic Immunosuppressive Microenvironment of Tumor-Initiating Stem Cells: From Molecular Anatomy to Molecular Pathway

Cancer whose major problems are metastasis, treatment resistance, and recurrence is the leading cause of death worldwide. Tumor-initiating stem cells (TiSCs) are a subset of the tumor population responsible for tumor resistance and relapse. Understanding the characteristics and shared features between tumor-initiating stem cells (TiSCs) and long-lived postmitotic cells may hold a key to better understanding the biology of cancer. Postmitotic cells have exited the cell cycle and are transitioned into a non-dividing and terminally differentiated state with a specialized function within a tissue. Conversely, a cancer cell with TiSC feature can divide and produce a variety of progenies, and is responsible for disease progression, tumor resistance to therapy and immune system and disease relapse. Surprisingly, our comprehensive evaluation of TiSCs suggests common features with long-lived post-mitotic cells. They are similar in structure (primary cilia, high mitochondrial content, and being protected by a barrier), metabolism (autophagy and senescence), and function (immunoescape and/or immune-privileged by a blood barrier). In-depth exploration showed how mitochondrial metabolism contributes to these shared features, including high energy demands arising from ciliary and microtubular functionality, increased metabolic activity, and movement. These findings can assist in decoding the remaining properties which offer insights into the biology of TiSCs, with potential implications for enhancing cancer treatment strategies and patient prognosis

Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis.

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity