FGF8 induces chemokinesis and regulates condensation of mouse nephron progenitor cells.

Kidneys develop via iterative branching of the ureteric epithelial tree and subsequent nephrogenesis at the branch points. Nephrons form in the cap mesenchyme as the metanephric mesenchyme (MM) condenses around the epithelial ureteric buds (UBs). Previous work has demonstrated that FGF8 is important for the survival of nephron progenitor cells (NPCs), and early deletion of Fgf8 leads to the cessation of nephron formation, which results in post-natal lethality. We now reveal a previously unreported function of FGF8. By combining transgenic mouse models, quantitative imaging assays and data-driven computational modelling, we show that FGF8 has a strong chemokinetic effect and that this chemokinetic effect is important for the condensation of NPCs to the UB. The computational model shows that the motility must be lower close to the UB to achieve NPC attachment. We conclude that the FGF8 signalling pathway is crucial for the coordination of NPC condensation at the UB. Chemokinetic effects have also been described for other FGFs and may be generally important for the formation of mesenchymal condensates.

Omics profiling identifies the regulatory functions of the MAPK/ERK pathway in nephron progenitor metabolism.

Nephron endowment is defined by fetal kidney growth and crucially dictates renal health in adults. Defects in the molecular regulation of nephron progenitors contribute to only a fraction of reduced nephron mass cases, suggesting alternative causative mechanisms. The importance of MAPK/ERK activation in nephron progenitor maintenance has been previously demonstrated, and here, we characterized the metabolic consequences of MAPK/ERK deficiency. Liquid chromatography/mass spectrometry-based metabolomics profiling identified 42 reduced metabolites, of which 26 were supported by in vivo transcriptional changes in MAPK/ERK-deficient nephron progenitors. Among these, mitochondria, ribosome and amino acid metabolism, together with diminished pyruvate and proline metabolism, were the most affected pathways. In vitro cultures of mouse kidneys demonstrated a dosage-specific function for pyruvate in controlling the shape of the ureteric bud tip, a regulatory niche for nephron progenitors. In vivo disruption of proline metabolism caused premature nephron progenitor exhaustion through their accelerated differentiation in pyrroline-5-carboxylate reductases 1 (Pycr1) and 2 (Pycr2) double-knockout kidneys. Pycr1/Pycr2-deficient progenitors showed normal cell survival, indicating no changes in cellular stress. Our results suggest that MAPK/ERK-dependent metabolism functionally participates in nephron progenitor maintenance by monitoring pyruvate and proline biogenesis in developing kidneys.

Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF.

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 excess GDNF has a bi-phasic effect on nephron progenitors in mice, which can faithfully respond to GDNF dosage manipulation during the fetal and postnatal period. Our results suggest that sensing the signaling activity level is an important mechanism through which GDNF and other molecules contribute to nephron progenitor lifespan specification.

Hepsin regulates TGFß signaling via fibronectin proteolysis.

Transforming growth factor-beta (TGFß) 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 TGFß activity by acting as a storage compartment of latent-TGFß, but how TGFß 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 TGFß signaling through the release of latent-TGFß from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFß signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGFß1, while overexpression of hepsin in mammary tumors increased TGFß signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGFß and, importantly, that the ability of hepsin to activate TGFß signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFß pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.

Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney.

BACKGROUND: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. RESULTS: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. CONCLUSIONS: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.

ShapeMetrics: A userfriendly pipeline for 3D cell segmentation and spatial tissue analysis.

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.

TT2020 meeting report on the 16th Transgenic Technology Meeting.

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.

Mouse Models of Congenital Kidney Anomalies.

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.

Correction: GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

[This corrects the article DOI: 10.1371/journal.pgen.1005710.].

MAPK/ERK Signaling in Regulation of Renal Differentiation.

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.

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson's disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson's disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3'UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson's disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3'UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3'UTR targeting may constitute a useful tool in analyzing gene function.

Sticklebacks adapted to divergent osmotic environments show differences in plasticity for kidney morphology and candidate gene expression.

Novel physiological challenges in different environments can promote the evolution of divergent phenotypes, either through plastic or genetic changes. Environmental salinity serves as a key barrier to the distribution of nearly all aquatic organisms, and species diversification is likely to be enabled by adaptation to alternative osmotic environments. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations found both in marine and freshwater environments. It has evolved both highly plastic and locally adapted phenotypes due to salinity-derived selection, but the physiological and genetic basis of adaptation to salinity is not fully understood. We integrated comparative cellular morphology of the kidney, a key organ for osmoregulation, and candidate gene expression to explore the underpinnings of evolved variation in osmotic plasticity within two populations of sticklebacks from distinct salinity zones in the Baltic Sea: the high salinity Kattegat, representative of the ancestral marine habitat; and the low salinity Bay of Bothnia. A common-garden experiment revealed that kidney morphology in the ancestral high-salinity population had a highly plastic response to salinity conditions whereas this plastic response was reduced in the low-salinity population. Candidate gene expression in kidney tissue revealed a similar pattern of population-specific differences, with a higher degree of plasticity in the native high-salinity population. Together these results suggest that renal cellular morphology has become canalized to low salinity, and that these structural differences may have functional implications for osmoregulation.

Mitogen-activated protein kinase (MAPK) pathway regulates branching by remodeling epithelial cell adhesion.

Although the growth factor (GF) signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK) pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA) protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions.

ETS-related transcription factors ETV4 and ETV5 are involved in proliferation and induction of differentiation-associated genes in embryonic stem (ES) cells.

The pluripotency and self-renewal capacity of embryonic stem (ES) cells is regulated by several transcription factors. Here, we show that the ETS-related transcription factors Etv4 and Etv5 (Etv4/5) are specifically expressed in undifferentiated ES cells, and suppression of Oct3/4 results in down-regulation of Etv4/5. Simultaneous deletion of Etv4 and Etv5 (Etv4/5 double knock-out (dKO)) in ES cells resulted in a flat, epithelial cell-like appearance, whereas the morphology changed into compact colonies in a 2i medium (containing two inhibitors for GSK3 and MEK/ERK). Expression levels of self-renewal marker genes, including Oct3/4 and Nanog, were similar between wild-type and dKO ES cells, whereas proliferation of Etv4/5 dKO ES cells was decreased with overexpression of cyclin-dependent kinase inhibitors (p16/p19, p15, and p57). A differentiation assay revealed that the embryoid bodies derived from Etv4/5 dKO ES cells were smaller than the control, and expression of ectoderm marker genes, including Fgf5, Sox1, and Pax3, was not induced in dKO-derived embryoid bodies. Microarray analysis demonstrated that stem cell-related genes, including Tcf15, Gbx2, Lrh1, Zic3, and Baf60c, were significantly repressed in Etv4/5 dKO ES cells. The artificial expression of Etv4 and/or Etv5 in Etv4/5 dKO ES cells induced re-expression of Tcf15 and Gbx2. These results indicate that Etv4 and Etv5, potentially through regulation of Gbx2 and Tcf15, are involved in the ES cell proliferation and induction of differentiation-associated genes in ES cells.

Developing therapeutically more efficient Neurturin variants for treatment of Parkinson's disease.

In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue.

The transcription factors Etv4 and Etv5 mediate formation of the ureteric bud tip domain during kidney development.

Signaling by the Ret receptor tyrosine kinase promotes cell movements in the Wolffian duct that give rise to the first ureteric bud tip, initiating kidney development. Although the ETS transcription factors Etv4 and Etv5 are known to be required for mouse kidney development and to act downstream of Ret, their specific functions are unclear. Here, we examine their role by analyzing the ability of Etv4 Etv5 compound mutant cells to contribute to chimeric kidneys. Etv4(-/-);Etv5(+/-) cells show a limited distribution in the caudal Wolffian duct and ureteric bud, similar to Ret(-/-) cells, revealing a cell-autonomous role for Etv4 and Etv5 in the cell rearrangements promoted by Ret. By contrast, Etv4(-/-);Etv5(-/-) cells display more severe developmental limitations, suggesting a broad role for Etv4 and Etv5 downstream of multiple signals, which are together important for Wolffian duct and ureteric bud morphogenesis.

Actin depolymerizing factors cofilin1 and destrin are required for ureteric bud branching morphogenesis.

The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.

Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.

The immunoglobulin (Ig) superfamily members are involved in cell adhesion and migration, complex multistep processes that play critical roles in embryogenesis, wound healing, tissue formation, and many other processes, but their specific functions during embryonic development remain unclear. Here, we have studied the function of the immunoglobulin superfamily member 3 (IGSF3) by generating an Igsf3 knockout (KO) mouse model with CRISPR/Cas9-mediated genome engineering. By combining RNA and protein detection methodology, we show that during development, IGSF3 localizes to the neural crest and a subset of its derivatives, suggesting a role in normal embryonic and early postnatal development. Indeed, inactivation of Igsf3 impairs the ability of the vagal neural crest cells to migrate and normally innervate the intestine. The small intestine of Igsf3 KO mice shows reduced thickness of the muscularis externa and diminished number of enteric neurons. Also, misalignment of neurons and smooth muscle cells in the developing intestinal villi is detected. Taken together, our results suggest that IGSF3 functions contribute to the formation of the enteric nervous system. Given the essential role of the enteric nervous system in maintaining normal gastrointestinal function, our study adds to the pool of information required for further understanding the mechanisms of gut innervation and etiology behind bowel motility disorders.

The GDNF target Vsnl1 marks the ureteric tip.

Glial cell line-derived neurotrophic factor (GDNF) is indispensable for ureteric budding and branching. If applied exogenously, GDNF promotes ectopic ureteric buds from the Wolffian duct. Although several downstream effectors of GDNF are known, the identification of early response genes is incomplete. Here, microarray screening detected several GDNF-regulated genes in the Wolffian duct, including Visinin like 1 (Vsnl1), which encodes a neuronal calcium-sensor protein. We observed renal Vsnl1 expression exclusively in the ureteric epithelium, but not in Gdnf-null kidneys. In the tissue culture of Gdnf-deficient kidney primordium, exogenous GDNF and alternative bud inducers (FGF7 and follistatin) restored Vsnl1 expression. Hence, Vsnl1 characterizes the tip of the ureteric bud epithelium regardless of the inducer. In the tips, Vsnl1 showed a mosaic expression pattern that was mutually exclusive with ß-catenin transcriptional activation. Vsnl1 was downregulated in both ß-catenin-stabilized and ß-catenin-deficient kidneys. Moreover, in a mouse collecting duct cell line, Vsnl1 compromised ß-catenin stability, suggesting a counteracting relationship between Vsnl1 and ß-catenin. In summary, Vsnl1 marks ureteric bud tips in embryonic kidneys, and its mosaic pattern demonstrates a heterogeneity of cell types that may be critical for normal ureteric branching.

The Finnish genetic heritage in 2022 - from diagnosis to translational research.

Isolated populations have been valuable for the discovery of rare monogenic diseases and their causative genetic variants. Finnish disease heritage (FDH) is an example of a group of hereditary monogenic disorders caused by single major, usually autosomal-recessive, variants enriched in the population due to several past genetic drift events. Interestingly, distinct subpopulations have remained in Finland and have maintained their unique genetic repertoire. Thus, FDH diseases have persisted, facilitating vigorous research on the underlying molecular mechanisms and development of treatment options. This Review summarizes the current status of FDH, including the most recently discovered FDH disorders, and introduces a set of other recently identified diseases that share common features with the traditional FDH diseases. The Review also discusses a new era for population-based studies, which combine various forms of big data to identify novel genotype-phenotype associations behind more complex conditions, as exemplified here by the FinnGen project. In addition to the pathogenic variants with an unequivocal causative role in the disease phenotype, several risk alleles that correlate with certain phenotypic features have been identified among the Finns, further emphasizing the broad value of studying genetically isolated populations.