Satu Kuure - Academia.edu (original) (raw)

Papers by Satu Kuure

Helsinki 2007 3The developmental biology of my life became true in my sons Otto and Oula.

Glial cell line-derived neurotrophic factor signaling through the Ret receptor tyrosine kinase is... more Glial cell line-derived neurotrophic factor signaling through the Ret receptor tyrosine kinase is crucial for ureteric bud branching morphogenesis during kidney development, yet few of the downstream genes are known. Here we show that the ETS transcription factors Etv4 and Etv5 are positively regulated by Ret signaling in the ureteric bud tips. Mice lacking both Etv4 alleles and one Etv5 allele show either renal agenesis or severe hypodysplasia, whereas kidney development fails completely in double homozygotes. We identified several genes whose expression in the ureteric bud depends on Etv4 and Etv5, including Cxcr4, Myb, Met and Mmp14. Thus, Etv4 and Etv5 are key components of a gene network downstream of Ret that promotes and controls renal branching morphogenesis.

Transgenic Research

The 16th transgenic technology (TT) meeting of the International Society of Transgenic technology... more The 16th transgenic technology (TT) meeting of the International Society of Transgenic technology (ISTT) took place on October 26–29th 2020 and was quite unique as it was the first-ever virtual meeting in the history of ISTT events. Dr. Rebecca Haffner-Krausz at Weizmann Institute of Science, Israel, was the local organizer of the meeting, which attracted 756 registered participants from 32 different countries.

The 16th transgenic technology (TT) meeting of the International Society of Transgenic technology... more The 16th transgenic technology (TT) meeting of the International Society of Transgenic technology (ISTT) took place on October 26–29th 2020 and was quite unique as it was the first-ever virtual meeting in the history of ISTT events. Dr. Rebecca Haffner-Krausz at Weizmann Institute of Science, Israel, was the local organizer of the meeting, which attracted 756 registered participants from 32 different countries.

GSK3 inactivation and stabilisation of-catenin induce nephron differentiation in isolated mouse a... more GSK3 inactivation and stabilisation of-catenin induce nephron differentiation in isolated mouse and rat kidney mesenchymes.

Cells

The modification of genes in animal models has evidently and comprehensively improved our knowled... more The modification of genes in animal models has evidently and comprehensively improved our knowledge on proteins and signaling pathways in human physiology and pathology. In this review, we discuss almost 40 monogenic rare diseases that are enriched in the Finnish population and defined as the Finnish disease heritage (FDH). We will highlight how gene-modified mouse models have greatly facilitated the understanding of the pathological manifestations of these diseases and how some of the diseases still lack proper models. We urge the establishment of subsequent international consortiums to cooperatively plan and carry out future human disease modeling strategies. Detailed information on disease mechanisms brings along broader understanding of the molecular pathways they act along both parallel and transverse to the proteins affected in rare diseases, therefore also aiding understanding of common disease pathologies.

Advances in Experimental Medicine and Biology

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects, which caus... more Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects, which cause the majority of chronic kidney diseases in children. CAKUT covers a wide range of malformations that derive from deficiencies in embryonic kidney and lower urinary tract development, including renal aplasia, hypodysplasia, hypoplasia, ectopia, and different forms of ureter abnormalities. The majority of the genetic causes of CAKUT remain unknown. Research on mutant mice has identified multiple genes that critically regulate renal differentiation. The data generated from this research have served as an excellent resource to identify the genetic bases of human kidney defects and have led to significantly improved diagnostics. Furthermore, genetic data from human CAKUT studies have also revealed novel genes regulating kidney differentiation.

Development

Nephron endowment, defined during the fetal period, dictates renal and related cardiovascular hea... more Nephron endowment, defined during the fetal period, dictates renal and related cardiovascular health throughout life. We show here that, despite its negative effects on kidney growth, genetic increase of GDNF prolongs the nephrogenic program beyond its normal cessation. Multi-stage mechanistic analysis revealed that excess GDNF maintains nephron progenitors and nephrogenesis through increased expression of its secreted targets and augmented WNT signaling, leading to a two-part effect on nephron progenitor maintenance. Abnormally high GDNF in embryonic kidneys upregulates its known targets but also Wnt9b and Axin2, with concomitant deceleration of nephron progenitor proliferation. Decline of GDNF levels in postnatal kidneys normalizes the ureteric bud and creates a permissive environment for continuation of the nephrogenic program, as demonstrated by morphologically and molecularly normal postnatal nephron progenitor self-renewal and differentiation. These results establish that exce...

Methods in Molecular Biology

Kidney organogenesis has been a widely used classical model system to study inductive tissue inte... more Kidney organogenesis has been a widely used classical model system to study inductive tissue interactions that guide differentiation of many organs. The basis for this is in the pioneering work done during the early 1950s when the conditions of how to support ex vivo growth and differentiation of developing kidneys were revealed. Importantly, culturing developing kidneys remains as an essential instrument to advance our understanding of molecular and cellular regulation of morphogenesis even today. Despite the fact that embryonic kidneys have been cultured for decades, it is not a trivial method and requires specific anatomical and developmental biology knowledge. This chapter outlines the general steps in organ culture and details the requirements for successful kidney explant differentiation.

Genes

The adult mammalian kidney is a poorly regenerating organ that lacks the stem cells that could re... more The adult mammalian kidney is a poorly regenerating organ that lacks the stem cells that could replenish functional homeostasis similarly to, e.g., skin or the hematopoietic system. Unlike a mature kidney, the embryonic kidney hosts at least three types of lineage-specific stem cells that give rise to (a) a ureter and collecting duct system, (b) nephrons, and (c) mesangial cells together with connective tissue of the stroma. Extensive interest has been raised towards these embryonic progenitor cells, which are normally lost before birth in humans but remain part of the undifferentiated nephrogenic rests in the pediatric renal cancer Wilms tumor. Here, we discuss the current understanding of kidney-specific embryonic progenitor regulation in the innate environment of the developing kidney and the types of disruptions in their balanced regulation that lead to the formation of Wilms tumor.

ABSTRACTNephron endowment is defined by fetal kidney growth and critically dictates renal health ... more ABSTRACTNephron endowment is defined by fetal kidney growth and critically dictates renal health in adults. Despite the advances in understanding the molecular regulation of nephron progenitors, the causes for low congenital nephron count and contribution of basic metabolism to nephron progenitor biology remain poorly understood. Here we characterized the metabolic consequences of MAPK/ERK-deficiency in nephron progenitors, whose maintenance and propagation in developing kidney critically depends on ERK activation. Our LC/MS-based metabolomics profiling identified 42 reduced metabolites, of which 26 were further supported by in vivo transcriptional characterization of MAPK/ERK-deficient nephron progenitors. This revealed a severe shortage of energy and nucleotide biosynthesis precursors, blockage in glycolysis and diminished pyruvate and proline metabolism. Utilization of in vitro kidney cultures demonstrated a dosage-specific function for glycolytic pyruvate as an energy source tha...

EMBO reports

Transforming growth factor-beta (TGFβ) is a multifunctional cytokine with a well-established role... more Transforming growth factor-beta (TGFβ) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent-TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent-TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.

ABSTRACTDue to poor regenerative capacity of adult kidneys, nephron endowment defined by the neph... more ABSTRACTDue to poor regenerative capacity of adult kidneys, nephron endowment defined by the nephrogenic program during the fetal period dictates renal and related cardiovascular health throughout life. We show that the neurotropic factor GDNF, which is in clinical trials for Parkinson’s disease, is capable of prolonging the nephrogenic program beyond its normal cessation without increasing the risk of kidney tumors. Our data demonstrates that excess GDNF expands the nephrogenic program by maintaining nephron progenitors and nephrogenesis in postnatal mouse kidneys. GDNF, through its transcriptional targets excreted from the adjacent epithelium, has a major effect on nephron progenitor self-renewal and maintenance; abnormally high GDNF inhibits nephron progenitor proliferation, but lowering its level normalizes the nephrogenic program to that permissive for nephron progenitor self-renewal and differentiation. Based on our results, we propose that the lifespan of nephron progenitors ...

Developmental Biology

The demand for single-cell level data is constantly increasing within life sciences. In order to ... more The demand for single-cell level data is constantly increasing within life sciences. In order to meet this demand, robust cell segmentation methods that can tackle challenging in vivo tissues with complex morphology are required. However, currently available cell segmentation and volumetric analysis methods perform poorly on 3D images. Here, we generated ShapeMetrics, a MATLAB-based script that segments cells in 3D and, by performing unbiased clustering using a heatmap, separates the cells into subgroups according to their volumetric and morphological differences. The cells can be accurately segregated according to different biologically meaningful features such as cell ellipticity, longest axis, cell elongation, or the ratio between cell volume and surface area. Our machine learning based script enables dissection of a large amount of novel data from microscope images in addition to the traditional information based on fluorescent biomarkers. Furthermore, the cells in different subgroups can be spatially mapped back to their original locations in the tissue image to help elucidate their roles in their respective morphological contexts. In order to facilitate the transition from bulk analysis to single-cell level accuracy, we emphasize the user-friendliness of our method by providing detailed step-by-step instructions through the pipeline hence aiming to reach users with less experience in computational biology.

Scientific Reports

Kidney mesenchyme (KM) and nephron progenitors (NPs) depend on WNT activity, and their culture in... more Kidney mesenchyme (KM) and nephron progenitors (NPs) depend on WNT activity, and their culture in vitro requires extensive repertoire of recombinant proteins and chemicals. Here we established a robust, simple culture of mouse KM using a combination of 3D Matrigel and growth media supplemented with Fibroblast Growth Factor 2 (FGF2) and Src inhibitor PP2. This allows dissociated KM to spontaneously self-organize into spheres. To reassess the requirement of WNT activity in KM self-organization and NPs maintenance, cells were cultured with short pulse of high-dose GSK3β inhibitor BIO, on a constant low-dose or without BIO. Robust proliferation at 48 hours and differentiation at 1 week were observed in cultures with high BIO pulse. Importantly, dissociated KM cultured without BIO, similarly to that exposed to constant low dose of BIO, maintained NPs up to one week and spontaneously differentiated into nephron tubules at 3 weeks of culture. Our results show that KM is maintained and indu...

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects deriving fr... more Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects deriving from abnormalities in renal differentiation during embryogenesis. CAKUT is the major cause of end-stage renal disease and chronic kidney diseases in children, but its genetic causes remain largely unresolved. Here we discuss advances in the understanding of how MAPK/ERK activity contributes to the regulation of ureteric bud branching morphogenesis, which dictates the final size, shape, and nephron number of the kidney. Recent studies also demonstrate that MAPK/ERK pathway is directly involved in nephrogenesis, regulating both the maintenance and differentiation of the nephrogenic mesenchyme. Interestingly, aberrant MAPK/ERK signaling is linked to many cancers, and recent studies suggest it also plays a role in the most common pediatric renal cancer,…

International Journal of Molecular Sciences

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects derived fro... more Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects derived from abnormalities in renal differentiation during embryogenesis. CAKUT is the major cause of end-stage renal disease and chronic kidney diseases in children, but its genetic causes remain largely unresolved. Here we discuss advances in the understanding of how mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity contributes to the regulation of ureteric bud branching morphogenesis, which dictates the final size, shape, and nephron number of the kidney. Recent studies also demonstrate that the MAPK/ERK pathway is directly involved in nephrogenesis, regulating both the maintenance and differentiation of the nephrogenic mesenchyme. Interestingly, aberrant MAPK/ERK signaling is linked to many cancers, and recent studies suggest it also plays a role in the most common pediatric renal cancer, Wilms’ tumor.

Scientific Reports

Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial ... more Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cellline derived neurotrophic factor (GDNF) induced Ret signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3′ untranslated region (UtR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF's effects on progenitors. Moreover, Gdnf hyper mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3′UtR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUt) and/or congenital infertility cases in humans and pave way to future studies. Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects affecting around 1% of live births, and causing most cases of the chronic kidney disease in children 1. CAKUT covers a wide range of malformations that derive from deficiencies in embryonic kidney and lower urinary tract development, including obstruction of ureteropelvic junction, renal dysplasia, hydro-, ectopic and short ureters. Genetic causes for these malformations remain largely unknown despite the extensive sequencing efforts in gene coding regions 2. The morphogenesis of urogenital system is influenced by a common nominator, the nephric duct (ND), also known as Wolffian duct 3. The ND extends caudally towards the posterior end of the embryo to connect to the cloaca through an endoderm-derived structure called the urogenital sinus. Proper positioning and timing of ND connection to the cloaca are important steps for the later development and function of the kidney and rest of the urogenital system. At least 1-2% of male infertility associates with ND defects 4 and the development of Müllerian ducts, which give rise to oviducts (uterine tubes), the uterus and the upper part of vagina, depends on the normal caudal extension of ND 5. An imperforate hymen is seen in 0.1% of newborn girls 6 and 43% of vaginal and/or hymen agenesis patients co-present malformations in the kidney indicating common etiology between urinary and genital tract development 7. Development of the kidney begins by budding of the ND towards renal mesenchyme to create a ureteric bud (UB), which then undergoes extensive branching morphogenesis to generate the renal collecting duct system 8. According to the "Ureteral Bud Theory" of Mackie and Stephens (1975) and supported by recent transgenic

Developmental Cell

Highlights d Fat4 mutant mice have duplex kidney defects due to ectopic bud formation d FAT4 func... more Highlights d Fat4 mutant mice have duplex kidney defects due to ectopic bud formation d FAT4 functions non-autonomously in the mesenchyme to prevent kidney duplication d RET signaling is overactive in Fat4 mutants d FAT4 interacts with RET, perturbs RET-GFRA1-GDNF assembly, and reduces RET signaling

Mechanisms of Development

and oligodendrocyte precursors as adherent cells. Based on high expression level of Pax6, Sox1, S... more and oligodendrocyte precursors as adherent cells. Based on high expression level of Pax6, Sox1, Sox2 and low expression of Pax3 NESCs likely have primitive neural plate identity. Besides spontaneous differentiation in adherent culture, hNESCs are capable of spontaneously forming cerebral organoids or “minibrains” in suspension. These organoids contain functional neurons and glial cells. We also obeserve self-organized structures resembling developing brain cortex with occasional folding. We demonstrate the importance of WNT and FGF2 signaling during organoids expansion phase for the formation of these cortex-like structures. Cultivation of cerebral organoids as floating aggregates on orbital shaker for more than one month leads to downregulation of Sox2 and DCX and to neuronal maturation. After 2 months after aggregation, we were able to detect various types of neurons including GABA, dopaminergic and serotoninergic, as well as astrocytes. Two-photon microscopy revealed an intricate network of MAP2-positive neuronal dendrites. Calcium imaging demonstrated spontaneous calcium waves characteristic of both neuronal and astroglial activity. Regionalization of organoids by FGF8 and SHH-signaling activation lead to significant increase in tyrosine-hydroxylase-positive cell number and to the detection of dopamine in conditioned culture media. Drug testing of marketed drugs demonstrated the adverse effects of one compound on cerebral organoids growth and development. The presented approach allows efficient derivation of hNESC that can differentiate into neuronal cells with high efficiency both in 2D and in 3D culture and can serve as a platform for human brain development studies and for studies of drugs teratogenicity.

Helsinki 2007 3The developmental biology of my life became true in my sons Otto and Oula.

Glial cell line-derived neurotrophic factor signaling through the Ret receptor tyrosine kinase is... more Glial cell line-derived neurotrophic factor signaling through the Ret receptor tyrosine kinase is crucial for ureteric bud branching morphogenesis during kidney development, yet few of the downstream genes are known. Here we show that the ETS transcription factors Etv4 and Etv5 are positively regulated by Ret signaling in the ureteric bud tips. Mice lacking both Etv4 alleles and one Etv5 allele show either renal agenesis or severe hypodysplasia, whereas kidney development fails completely in double homozygotes. We identified several genes whose expression in the ureteric bud depends on Etv4 and Etv5, including Cxcr4, Myb, Met and Mmp14. Thus, Etv4 and Etv5 are key components of a gene network downstream of Ret that promotes and controls renal branching morphogenesis.

Transgenic Research

The 16th transgenic technology (TT) meeting of the International Society of Transgenic technology... more The 16th transgenic technology (TT) meeting of the International Society of Transgenic technology (ISTT) took place on October 26–29th 2020 and was quite unique as it was the first-ever virtual meeting in the history of ISTT events. Dr. Rebecca Haffner-Krausz at Weizmann Institute of Science, Israel, was the local organizer of the meeting, which attracted 756 registered participants from 32 different countries.

The 16th transgenic technology (TT) meeting of the International Society of Transgenic technology... more The 16th transgenic technology (TT) meeting of the International Society of Transgenic technology (ISTT) took place on October 26–29th 2020 and was quite unique as it was the first-ever virtual meeting in the history of ISTT events. Dr. Rebecca Haffner-Krausz at Weizmann Institute of Science, Israel, was the local organizer of the meeting, which attracted 756 registered participants from 32 different countries.

GSK3 inactivation and stabilisation of-catenin induce nephron differentiation in isolated mouse a... more GSK3 inactivation and stabilisation of-catenin induce nephron differentiation in isolated mouse and rat kidney mesenchymes.

Cells

The modification of genes in animal models has evidently and comprehensively improved our knowled... more The modification of genes in animal models has evidently and comprehensively improved our knowledge on proteins and signaling pathways in human physiology and pathology. In this review, we discuss almost 40 monogenic rare diseases that are enriched in the Finnish population and defined as the Finnish disease heritage (FDH). We will highlight how gene-modified mouse models have greatly facilitated the understanding of the pathological manifestations of these diseases and how some of the diseases still lack proper models. We urge the establishment of subsequent international consortiums to cooperatively plan and carry out future human disease modeling strategies. Detailed information on disease mechanisms brings along broader understanding of the molecular pathways they act along both parallel and transverse to the proteins affected in rare diseases, therefore also aiding understanding of common disease pathologies.

Advances in Experimental Medicine and Biology

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects, which caus... more Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects, which cause the majority of chronic kidney diseases in children. CAKUT covers a wide range of malformations that derive from deficiencies in embryonic kidney and lower urinary tract development, including renal aplasia, hypodysplasia, hypoplasia, ectopia, and different forms of ureter abnormalities. The majority of the genetic causes of CAKUT remain unknown. Research on mutant mice has identified multiple genes that critically regulate renal differentiation. The data generated from this research have served as an excellent resource to identify the genetic bases of human kidney defects and have led to significantly improved diagnostics. Furthermore, genetic data from human CAKUT studies have also revealed novel genes regulating kidney differentiation.

Development

Nephron endowment, defined during the fetal period, dictates renal and related cardiovascular hea... more Nephron endowment, defined during the fetal period, dictates renal and related cardiovascular health throughout life. We show here that, despite its negative effects on kidney growth, genetic increase of GDNF prolongs the nephrogenic program beyond its normal cessation. Multi-stage mechanistic analysis revealed that excess GDNF maintains nephron progenitors and nephrogenesis through increased expression of its secreted targets and augmented WNT signaling, leading to a two-part effect on nephron progenitor maintenance. Abnormally high GDNF in embryonic kidneys upregulates its known targets but also Wnt9b and Axin2, with concomitant deceleration of nephron progenitor proliferation. Decline of GDNF levels in postnatal kidneys normalizes the ureteric bud and creates a permissive environment for continuation of the nephrogenic program, as demonstrated by morphologically and molecularly normal postnatal nephron progenitor self-renewal and differentiation. These results establish that exce...

Methods in Molecular Biology

Kidney organogenesis has been a widely used classical model system to study inductive tissue inte... more Kidney organogenesis has been a widely used classical model system to study inductive tissue interactions that guide differentiation of many organs. The basis for this is in the pioneering work done during the early 1950s when the conditions of how to support ex vivo growth and differentiation of developing kidneys were revealed. Importantly, culturing developing kidneys remains as an essential instrument to advance our understanding of molecular and cellular regulation of morphogenesis even today. Despite the fact that embryonic kidneys have been cultured for decades, it is not a trivial method and requires specific anatomical and developmental biology knowledge. This chapter outlines the general steps in organ culture and details the requirements for successful kidney explant differentiation.

Genes

The adult mammalian kidney is a poorly regenerating organ that lacks the stem cells that could re... more The adult mammalian kidney is a poorly regenerating organ that lacks the stem cells that could replenish functional homeostasis similarly to, e.g., skin or the hematopoietic system. Unlike a mature kidney, the embryonic kidney hosts at least three types of lineage-specific stem cells that give rise to (a) a ureter and collecting duct system, (b) nephrons, and (c) mesangial cells together with connective tissue of the stroma. Extensive interest has been raised towards these embryonic progenitor cells, which are normally lost before birth in humans but remain part of the undifferentiated nephrogenic rests in the pediatric renal cancer Wilms tumor. Here, we discuss the current understanding of kidney-specific embryonic progenitor regulation in the innate environment of the developing kidney and the types of disruptions in their balanced regulation that lead to the formation of Wilms tumor.

ABSTRACTNephron endowment is defined by fetal kidney growth and critically dictates renal health ... more ABSTRACTNephron endowment is defined by fetal kidney growth and critically dictates renal health in adults. Despite the advances in understanding the molecular regulation of nephron progenitors, the causes for low congenital nephron count and contribution of basic metabolism to nephron progenitor biology remain poorly understood. Here we characterized the metabolic consequences of MAPK/ERK-deficiency in nephron progenitors, whose maintenance and propagation in developing kidney critically depends on ERK activation. Our LC/MS-based metabolomics profiling identified 42 reduced metabolites, of which 26 were further supported by in vivo transcriptional characterization of MAPK/ERK-deficient nephron progenitors. This revealed a severe shortage of energy and nucleotide biosynthesis precursors, blockage in glycolysis and diminished pyruvate and proline metabolism. Utilization of in vitro kidney cultures demonstrated a dosage-specific function for glycolytic pyruvate as an energy source tha...

EMBO reports

Transforming growth factor-beta (TGFβ) is a multifunctional cytokine with a well-established role... more Transforming growth factor-beta (TGFβ) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent-TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent-TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.

ABSTRACTDue to poor regenerative capacity of adult kidneys, nephron endowment defined by the neph... more ABSTRACTDue to poor regenerative capacity of adult kidneys, nephron endowment defined by the nephrogenic program during the fetal period dictates renal and related cardiovascular health throughout life. We show that the neurotropic factor GDNF, which is in clinical trials for Parkinson’s disease, is capable of prolonging the nephrogenic program beyond its normal cessation without increasing the risk of kidney tumors. Our data demonstrates that excess GDNF expands the nephrogenic program by maintaining nephron progenitors and nephrogenesis in postnatal mouse kidneys. GDNF, through its transcriptional targets excreted from the adjacent epithelium, has a major effect on nephron progenitor self-renewal and maintenance; abnormally high GDNF inhibits nephron progenitor proliferation, but lowering its level normalizes the nephrogenic program to that permissive for nephron progenitor self-renewal and differentiation. Based on our results, we propose that the lifespan of nephron progenitors ...

Developmental Biology

The demand for single-cell level data is constantly increasing within life sciences. In order to ... more The demand for single-cell level data is constantly increasing within life sciences. In order to meet this demand, robust cell segmentation methods that can tackle challenging in vivo tissues with complex morphology are required. However, currently available cell segmentation and volumetric analysis methods perform poorly on 3D images. Here, we generated ShapeMetrics, a MATLAB-based script that segments cells in 3D and, by performing unbiased clustering using a heatmap, separates the cells into subgroups according to their volumetric and morphological differences. The cells can be accurately segregated according to different biologically meaningful features such as cell ellipticity, longest axis, cell elongation, or the ratio between cell volume and surface area. Our machine learning based script enables dissection of a large amount of novel data from microscope images in addition to the traditional information based on fluorescent biomarkers. Furthermore, the cells in different subgroups can be spatially mapped back to their original locations in the tissue image to help elucidate their roles in their respective morphological contexts. In order to facilitate the transition from bulk analysis to single-cell level accuracy, we emphasize the user-friendliness of our method by providing detailed step-by-step instructions through the pipeline hence aiming to reach users with less experience in computational biology.

Scientific Reports

Kidney mesenchyme (KM) and nephron progenitors (NPs) depend on WNT activity, and their culture in... more Kidney mesenchyme (KM) and nephron progenitors (NPs) depend on WNT activity, and their culture in vitro requires extensive repertoire of recombinant proteins and chemicals. Here we established a robust, simple culture of mouse KM using a combination of 3D Matrigel and growth media supplemented with Fibroblast Growth Factor 2 (FGF2) and Src inhibitor PP2. This allows dissociated KM to spontaneously self-organize into spheres. To reassess the requirement of WNT activity in KM self-organization and NPs maintenance, cells were cultured with short pulse of high-dose GSK3β inhibitor BIO, on a constant low-dose or without BIO. Robust proliferation at 48 hours and differentiation at 1 week were observed in cultures with high BIO pulse. Importantly, dissociated KM cultured without BIO, similarly to that exposed to constant low dose of BIO, maintained NPs up to one week and spontaneously differentiated into nephron tubules at 3 weeks of culture. Our results show that KM is maintained and indu...

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects deriving fr... more Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects deriving from abnormalities in renal differentiation during embryogenesis. CAKUT is the major cause of end-stage renal disease and chronic kidney diseases in children, but its genetic causes remain largely unresolved. Here we discuss advances in the understanding of how MAPK/ERK activity contributes to the regulation of ureteric bud branching morphogenesis, which dictates the final size, shape, and nephron number of the kidney. Recent studies also demonstrate that MAPK/ERK pathway is directly involved in nephrogenesis, regulating both the maintenance and differentiation of the nephrogenic mesenchyme. Interestingly, aberrant MAPK/ERK signaling is linked to many cancers, and recent studies suggest it also plays a role in the most common pediatric renal cancer,…

International Journal of Molecular Sciences

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects derived fro... more Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects derived from abnormalities in renal differentiation during embryogenesis. CAKUT is the major cause of end-stage renal disease and chronic kidney diseases in children, but its genetic causes remain largely unresolved. Here we discuss advances in the understanding of how mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity contributes to the regulation of ureteric bud branching morphogenesis, which dictates the final size, shape, and nephron number of the kidney. Recent studies also demonstrate that the MAPK/ERK pathway is directly involved in nephrogenesis, regulating both the maintenance and differentiation of the nephrogenic mesenchyme. Interestingly, aberrant MAPK/ERK signaling is linked to many cancers, and recent studies suggest it also plays a role in the most common pediatric renal cancer, Wilms’ tumor.

Scientific Reports

Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial ... more Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cellline derived neurotrophic factor (GDNF) induced Ret signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3′ untranslated region (UtR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF's effects on progenitors. Moreover, Gdnf hyper mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3′UtR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUt) and/or congenital infertility cases in humans and pave way to future studies. Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects affecting around 1% of live births, and causing most cases of the chronic kidney disease in children 1. CAKUT covers a wide range of malformations that derive from deficiencies in embryonic kidney and lower urinary tract development, including obstruction of ureteropelvic junction, renal dysplasia, hydro-, ectopic and short ureters. Genetic causes for these malformations remain largely unknown despite the extensive sequencing efforts in gene coding regions 2. The morphogenesis of urogenital system is influenced by a common nominator, the nephric duct (ND), also known as Wolffian duct 3. The ND extends caudally towards the posterior end of the embryo to connect to the cloaca through an endoderm-derived structure called the urogenital sinus. Proper positioning and timing of ND connection to the cloaca are important steps for the later development and function of the kidney and rest of the urogenital system. At least 1-2% of male infertility associates with ND defects 4 and the development of Müllerian ducts, which give rise to oviducts (uterine tubes), the uterus and the upper part of vagina, depends on the normal caudal extension of ND 5. An imperforate hymen is seen in 0.1% of newborn girls 6 and 43% of vaginal and/or hymen agenesis patients co-present malformations in the kidney indicating common etiology between urinary and genital tract development 7. Development of the kidney begins by budding of the ND towards renal mesenchyme to create a ureteric bud (UB), which then undergoes extensive branching morphogenesis to generate the renal collecting duct system 8. According to the "Ureteral Bud Theory" of Mackie and Stephens (1975) and supported by recent transgenic

Developmental Cell

Highlights d Fat4 mutant mice have duplex kidney defects due to ectopic bud formation d FAT4 func... more Highlights d Fat4 mutant mice have duplex kidney defects due to ectopic bud formation d FAT4 functions non-autonomously in the mesenchyme to prevent kidney duplication d RET signaling is overactive in Fat4 mutants d FAT4 interacts with RET, perturbs RET-GFRA1-GDNF assembly, and reduces RET signaling

Mechanisms of Development

and oligodendrocyte precursors as adherent cells. Based on high expression level of Pax6, Sox1, S... more and oligodendrocyte precursors as adherent cells. Based on high expression level of Pax6, Sox1, Sox2 and low expression of Pax3 NESCs likely have primitive neural plate identity. Besides spontaneous differentiation in adherent culture, hNESCs are capable of spontaneously forming cerebral organoids or “minibrains” in suspension. These organoids contain functional neurons and glial cells. We also obeserve self-organized structures resembling developing brain cortex with occasional folding. We demonstrate the importance of WNT and FGF2 signaling during organoids expansion phase for the formation of these cortex-like structures. Cultivation of cerebral organoids as floating aggregates on orbital shaker for more than one month leads to downregulation of Sox2 and DCX and to neuronal maturation. After 2 months after aggregation, we were able to detect various types of neurons including GABA, dopaminergic and serotoninergic, as well as astrocytes. Two-photon microscopy revealed an intricate network of MAP2-positive neuronal dendrites. Calcium imaging demonstrated spontaneous calcium waves characteristic of both neuronal and astroglial activity. Regionalization of organoids by FGF8 and SHH-signaling activation lead to significant increase in tyrosine-hydroxylase-positive cell number and to the detection of dopamine in conditioned culture media. Drug testing of marketed drugs demonstrated the adverse effects of one compound on cerebral organoids growth and development. The presented approach allows efficient derivation of hNESC that can differentiate into neuronal cells with high efficiency both in 2D and in 3D culture and can serve as a platform for human brain development studies and for studies of drugs teratogenicity.