Sprouty1 controls genitourinary development via its N-terminal tyrosine

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) is a group of diseases that include a broad spectrum of developmental defects of the genitourinary system. Mouse models indicate that perturbations of the GDNF-Ret signaling pathway are a major genetic cause of CAKUT. Sprouty1 is an intracellular Ret inhibitor whose mutation results in supernumerary kidneys, megaureters, and hydronephrosis in mice. Both the molecular mechanisms and the structural domains critical for Sprouty function are a matter of controversy, partly because studies pursuing this objective rely on ectopic overexpression in cell lines. A conserved N-terminal tyrosine has been frequently, but not always, identified as critical for their function in vitro. Methods: We have generated Sprouty1 knockin mice bearing a tyrosine-to-alanine substitution in position 53, corresponding to the conserved N-terminal tyrosine of Sprouty1. We have characterized development of the genitourinary systems of these mice via different methods, including the use of reporter mice expressing EGFP form the Ret locus, and whole mount cytokeratin staining. Results: Mice lacking this tyrosine grow ectopic ureteric buds that ultimately will form supernumerary kidneys, a phenotype indistinguishable to that of Sprouty1 knockout mice. Sprouty1 knockin mice also present megaureters and vesicoureteral reflux, caused by failure of ureters to separate from Wolffian ducts and migrate to their definitive position. Conclusions: Tyrosine 53 is absolutely necessary to convey Sprouty1 function during genitourinary development.This work was supported by grants BFU2010-47175-P and BFU2017-83646-P (AEI/FEDER, UE) from MINECO to ME. MV was supported by a predoctoral fellowship from AGAUR. CA was supported by a predoctoral fellowship from Universitat de Lleida. SC was supported by a Cofund action from the Marie Curie program of the EU. We are grateful to Dr. Sanjay Jain (Washington University, St Louis) for sharing RetEGFP mice, and to Dr. Tung-Tien Sun (New York University) for Uroplakin antibody. We thank Anna Macià (IRB Lleida) for her contribution to the initial development of this manuscript, as well as Marta Hereu, Maria Santacana, Mónica Domingo and Maria Carrele for their excellent technical assistance

Sprouty1 is a broad mediator of cellular senescence

Genes of the Sprouty family (Spry1-4) restrain signaling by certain receptor tyrosine kinases. Consequently, these genes participate in several developmental processes and function as tumor suppressors in adult life. Despite these important roles, the biology of this family of genes still remains obscure. Here we show that Sprouty proteins are general mediators of cellular senescence. Induction of cellular senescence by several triggers in vitro correlates with upregulation of Sprouty protein levels. More importantly, overexpression of Sprouty genes is sufficient to cause premature cellular senescence, via a conserved N-terminal tyrosine (Tyrosine 53 of Sprouty1). Accordingly, fibroblasts from knockin animals lacking that tyrosine escape replicative senescence. In vivo, heterozygous knockin mice display delayed induction of cellular senescence during cutaneous wound healing and upon chemotherapy-induced cellular senescence. Unlike other functions of this family of genes, induction of cellular senescence appears to be independent of activation of the ERK1/2 pathway. Instead, we show that Sprouty proteins induce cellular senescence upstream of the p38 pathway in these in vitro and in vivo paradigms

A dominant negative mutation uncovers cooperative control of caudal Wolffian duct development by Sprouty genes

The Wolffian ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here, we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting of ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grants BFU2017-83646-P (MINECO) and PID2020-114947 GB-I00 (MCIU) (both supported by funds from AEI/FEDER, UE) to ME. MV was supported by a predoctoral fellowship from AGAUR. GA and CA and GA are supported by a fellowship from Universitat de Lleida. SC was supported by a Cofund action from the Marie Curie program of the E

Noves funcions de Sprouty en el desenvolupament i l’homeòstasi del sistema genitourinari

La família de gens Sprouty està formada per quatre membres en mamífers (Spry1-4), ortòlegs al gen dSpry de Drosophila Melanogaster. Durant el desenvolupament, les proteïnes Sprouty funcionen principalment com a inhibidors de la senyalització mitjançada per receptors tirosina-cinasa (RTK). Resultats previs del nostre laboratori mostren que a nivell molecular, la tirosina 53 de SPRY1, un residu conservat entre els diferents membres de la família, és essencial per a la funció d’aquesta proteïna durant el desenvolupament del sistema genitourinari, ja que els ratolins Spry1Y53A/Y53A fenocopien les anomalies observades en els ratolins knockout de Spry1. A la vegada, l’expressió de la mutació Y53A en heterozigosi suposa l’aparició d’una sèrie d'anomalies de l'aparell genital intern. Per un costat, les femelles Spry1Y53A/+ manquen d'obertura vaginal i presenten cavitats vaginals septades flanquejades per restes de conductes de Wolff (WD) al llarg de les parets vaginals. D’altra banda, els mascles Spry1Y53A/+ mostren ramificacions anòmales de la vesícula seminal. Tot i que s’ha descartat una hiperactivació de la senyalització per RET com a causa d’aquest fenotip, els mecanismes moleculars responsables son desconeguts. En primer lloc, establim l’increment en els nivells de proteïna Spry1 com la causa de l’efecte dominant negatiu de la mutació Y53A i descartem problemes de localització d’aquesta proteïna. En segon lloc, es demostra que, a diferència de les malformacions renals, aquestes anomalies del WD es rescaten parcialment disminuint la dosi gènica d’Axin2, i totalment disminuint la dosi gènica de Fgf10. Finalment, en aquest treball es confirma una nova funció de les proteïnes Sprouty en l’homeòstasi del fòsfor a través de la inhibició de la senyalització de Fgf23 al ronyó, gràcies a l’estudi fenotípic d’animals triple knockouts per Spry1, Spry2 i Spry4. A més, la deleció d’aquests gens ja en adults també suposa l’aparició d’altres trets com són la pèrdua de pes, el descens en els nivells de glucosa, l’increment en l’activitat de la tiroides i la pèrdua de força.La familia de genes Sprouty consta de cuatro miembros en mamíferos (Spry1-4), que son ortólogos del gen dSpry de Drosophila Melanogaster. Durante el desarrollo, las proteínas Sprouty desempeñan principalmente el papel de inhibidores de la señalización mediada por receptores tirosina quinasa. Sin embargo, gran parte de la función de esta familia de genes en la edad adulta permanece desconocida. Resultados previos de nuestro laboratorio indican que, a nivel molecular, la tirosina 53 de SPRY1, un residuo conservado en todos los miembros de la familia, es esencial para la función de esta proteína durante el desarrollo del sistema genitourinario, ya que los ratones Spry1Y53A/Y53A fenocopian los defectos encontrados en los ratones knockout de Spry1. Además, la expresión en heterocigosis de la mutación Y53A conlleva la aparición de una serie de anomalías en el aparato genital interno. Por un lado, las hembras Spry1Y53A/+ carecen de apertura vaginal y presentan cavidades vaginales septadas con restos de conductos de Wolff (WD) a lo largo de las paredes vaginales. Por otro lado, los machos Spry1Y53A/+ muestran ramificaciones anómalas de la vesícula seminal. Aunque se ha descartado una hiperactivación de la señalización por RET como causa de este fenotipo, los mecanismos moleculares responsables aún son desconocidos. En primer lugar, establecemos que el aumento en los niveles de proteína SPRY1 es la causa del efecto dominante negativo de la mutación Y53A, y descartamos problemas de localización de esta proteína. En segundo lugar, demostramos que, a diferencia de las malformaciones renales, estas anomalías del WD se rescatan parcialmente reduciendo la dosis génica de Axin2 y completamente reduciendo la dosis génica de Fgf10. Finalmente, en este trabajo confirmamos una nueva función de las proteínas Sprouty en la homeostasis del fósforo mediante la inhibición de la señalización de FGF23 en el riñón, gracias al estudio fenotípico de animales triple knockout para Spry1, Spry2 y Spry4. Además, la eliminación de estos genes en adultos también conlleva la aparición de otros rasgos, como són la pérdida de peso, la disminución de los niveles de glucosa, el aumento de la actividad tiroidea y la pérdida de fuerza.Sprouty family of genes in mammals is composed of four members (Spry1-4), orthologous to a single gene in Drosophila Melanogaster (dSpry). During development, Sprouty proteins primarily function as inhibitors of Receptor Tyrosine Kinase (RTK) signaling, especially RET and FGFR. However, much of this gene family's function in adulthood remains largely unknown. Previous data from our group demonstrate that, at a molecular level, Tyrosine 53 of SPRY1, a highly conserved residue among Sprouty family, is essential for its function during genitourinary system development, as Spry1Y53A/Y53A mice phenocopy the renal defects found in Spry1 knockout animals. Moreover, heterozygous expression of the Y53A mutation gives rise to a range of internal genital anomalies not reported previously. Female Spry1Y53A/+ mice lack vaginal opening and show vaginal septum flanked by remnants of Wolffian ducts along the vaginal walls. In male Spry1Y53A/+ mice anomalous branching of the seminal vesicle is observed. Additionally, Spry1 and Spry2 double heterozygous mice also exhibit these anomalies. Although hyperactivation of RET signaling has been rejected as the cause of this phenotype, the molecular mechanisms remain unknown. Firstly, we identify the increase in SPRY1 protein levels as the cause of the dominant-negative effect of Y53A mutation rather than anomalies in protein localization. Secondly, we demonstrate that, unlike renal malformations, these WD anomalies are independent of Ret signaling and are partially rescued by Axin2 gene dosage reduction and fully rescued by Fgf10 gene dosage reduction. Finally, we validate a novel role of Sprouty proteins in phosphorus homeostasis by inhibiting FGF23 signaling in the kidney, as evidenced through phenotypic analysis of triple knockout animals for Spry1, Spry2, and Spry4. Moreover, the deletion of Sprouty genes during adulthood leads physiological impairments such as weight loss, reduced glucose levels, heightened thyroid activity, and decreased grip strength

Quantitative Operating Principles of Yeast Metabolism during Adaptation to Heat Stress

Summary: Microorganisms evolved adaptive responses to survive stressful challenges in ever-changing environments. Understanding the relationships between the physiological/metabolic adjustments allowing cellular stress adaptation and gene expression changes being used by organisms to achieve such adjustments may significantly impact our ability to understand and/or guide evolution. Here, we studied those relationships during adaptation to various stress challenges in Saccharomyces cerevisiae, focusing on heat stress responses. We combined dozens of independent experiments measuring whole-genome gene expression changes during stress responses with a simplified kinetic model of central metabolism. We identified alternative quantitative ranges for a set of physiological variables in the model (production of ATP, trehalose, NADH, etc.) that are specific for adaptation to either heat stress or desiccation/rehydration. Our approach is scalable to other adaptive responses and could assist in developing biotechnological applications to manipulate cells for medical, biotechnological, or synthetic biology purposes. : Evolution selects coordinated adaptive changes in gene expression and metabolism that ensure survival to stress challenges. Pereira et al. identify quantitative ranges for those changes in a set of genes and physiological variables (production of ATP, trehalose, NADH, etc.) that are specific for adaptation to heat stress, desiccation/rehydration, or pH. Keywords: biological design principles, systems biology, computational biology, multilevel modeling, integrative biology, metabolism, optimizatio

In Vivo Intra‐Uterine Delivery of TAT‐Fused Cre Recombinase and CRISPR/Cas9 Editing System in Mice Unveil Histopathology of Pten/p53‐Deficient Endometrial Cancers

Abstract Phosphatase and TENsin homolog (Pten) and p53 are two of the most frequently mutated tumor suppressor genes in endometrial cancer. However, the functional consequences and histopathological manifestation of concomitant p53 and Pten loss of function alterations in the development of endometrial cancer is still controversial. Here, it is demonstrated that simultaneous Pten and p53 deletion is sufficient to cause epithelial to mesenchymal transition phenotype in endometrial organoids. By a novel intravaginal delivery method using HIV1 trans‐activator of transcription cell penetrating peptide fused with a Cre recombinase protein (TAT‐Cre), local ablation of both p53 and Pten is achieved specifically in the uterus. These mice developed high‐grade endometrial carcinomas and a high percentage of uterine carcinosarcomas resembling those found in humans. To further demonstrate that carcinosarcomas arise from epithelium, double Pten/p53 deficient epithelial cells are mixed with wild type stromal and myometrial cells and subcutaneously transplanted to Scid mice. All xenotransplants resulted in the development of uterine carcinosarcomas displaying high nuclear pleomorphism and metastatic potential. Accordingly, in vivo CRISPR/Cas9 disruption of Pten and p53 also triggered the development of metastatic carcinosarcomas. The results unfadingly demonstrate that simultaneous deletion of p53 and Pten in endometrial epithelial cells is enough to trigger epithelial to mesenchymal transition that is consistently translated to the formation of uterine carcinosarcomas in vivo