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Duodenogastric reflux and pyloric sphincter abnormalities in Six2-cre tg/+ , Wnt9b tg/+ double heterozygotes. (A, B) Yellow amniotic fluid refluxed into the stomach of E18.5 double heterozygotes, but was properly retained in the intestine in control animals. A lack of constriction (arrow) and an increased diameter (inset) suggests that the reflux is due to a malformed pyloric sphincter. (C, D) Whole mount staining for Nephrocan mRNA delineates a thin band demarking the sphincter at E16.5 that is expanded in the double heterozygotes. (E, F) Smooth muscle actin staining in sections from control adult stomach demonstrates a thick muscular layer that leads to the sphincter. Double heterozygotes exhibit a thickened muscular layer; however it does not coalesce at the point of highest constriction. Vertical lines mark the transition from antral stomach (left side) to duodenum (right side) in E-H. (G, H) Alcian blue stains mucosal cells in the antral stomach in control sections and these cells are absent in the double heterozygotes. Mucosal cells in the intestine are stained normally. (I, J) Staining for the H + /K + ATPase detects parietal cells in the forestomach that are excluded from the distal stomach. These cells are found throughout the distal stomach and pylorus in double heterozygotes. Sections depicted in panels I and J highlight the transition between forestomach and distal stomach (diagonal line) and do not include the duodenum. doi:10.1371/journal.pone.0043098.g006
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During kidney development, canonical Wnt signaling activates differentiation, while the transcription factor Six2 maintains the progenitor pool. These opposing signals help to regulate nephron formation and ensure the full complement of nephrons are formed. Since these two factors control differing fates in kidney mesenchyme, we hypothesized that o...
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Context 1
... second phenotype contributing to reduced body weight in Six2-cre tg/+ , Wnt9b tg/+ double heterozygotes was gastric distress. Yellow amniotic fluid was observed in the stomach of double transgenics and not in single transgenic littermates (n = 6, Fig. ...
Context 2
... indicated that duodenogastric reflux was occurring and could be due to abnormal pyloric sphincter formation. The sphincter between the stomach and the duodenum was elongated and widened in the double transgenic (Fig. 6, inset). The TGFb antagonist, Nephrocan, which is normally expressed in a very restricted pattern at E16.5, was expanded in double transgenics, suggesting that the sphincter region was not properly constrained. This elongated morphology was supported by immunohistochem- ical staining of the smooth muscle layer that forms the dense muscular ...
Context 3
... Nephrocan, which is normally expressed in a very restricted pattern at E16.5, was expanded in double transgenics, suggesting that the sphincter region was not properly constrained. This elongated morphology was supported by immunohistochem- ical staining of the smooth muscle layer that forms the dense muscular wall surrounding the pylorus (Fig. 6 E, F). In single transgenic controls, the thickest musculature was found where the tissue exhibits the most constriction at the sphincter (line). In the double transgenics, thick muscularity was observed, but this region was offset from the area that was the most ...
Citations
... Six2 is located on mouse chromosome 17, encoding the protein sine oculis-related homeobox 2. It plays an important role in stomach, kidney and craniofacial development (Li et al., 2020, Kiefer et al., 2012. In the kidney is it know to maintain mesenchyme multipotent nephron progenitor cells in an undifferentiated state, and renew their proliferation through Wnt signalling (Li et al., 2016b). ...
The cause of duodenal atresia (DA) is not known. Tandler’s “solid cord” hypothesis conflicts
with current biological evidence. In humans, a genetic aetiology is supported by the
association with Trisomy 21. Interruption of Fgf10 is the strongest genetic link to DA in mice,
but the lethality of associated defects means it is an unlikely cause of DA in humans. It is
hypothesised that DA is caused by a genetic change downstream of the
FGF10/FGFR2b signalling pathway. This thesis aims to identify gene signalling changes
that may cause DA.
Aim 1 was to validate the spectrum of DA present in CRISPR-derived Fgf10-/- embryos.
These embryos demonstrated a higher penetrance of DA than previously reported, with
morphology distribution closely reiterating that observed in humans. DA penetrance and
phenotype severity increased with gestational age, suggesting an evolutionary nature.
Aim 2 was to evaluate gene expression associated with DA, through the use of RNA-seq
and RT-qPCR. Whilst multiple genes were up- and downregulated in the absence of Fgf10,
a cluster, otherwise associated with pyloric muscle specification, was of particular interest.
This possible interplay between pyloric development and DA unveils a new hypothesis, that:
in the duodenum of Fgf10-/- mice, anterior-posterior mis-patterning with ectopic
pyloric specification may be the basis of DA formation, and loss luminal continuity.
Aim 3 was to develop an organoid model to facilitate future ex vivo DA research. Normal
organoids were shown to grow from wild-type duodenum and growth patterns differed
between Fgf10 genotypes.
This thesis forms a foundation for future research into DA aetiology. Further animal research
must combine gene analysis of human patients born with DA, to identify putative causative
genes/pathways. Determining the cause of DA in humans is a clinical and scientific
imperative, with immediate therapeutic relevance
... In the absence of Six2, ectopic renal vesicles form on the dorsal (cortical) side of the ureteric bud (UB), and the NPC pool depletes rapidly, terminating nephrogenesis after induction of only a few nephrons [32]. Likewise, uniform activation of WNT signalling in the cap mesenchyme (CM) by Six2-Credriven expression of stabilised β-catenin or by co-culture with glycogen synthase kinase (GSK) inhibitors results in the uniform emergence of ectopic PTA [16,18,28]. WNT9b induces nephrogenesis also in isolated metanephric mesenchyme [3], but Six2-Cre-driven expression of Wnt9b does not result in ectopic RV [16]. ...
... Likewise, uniform activation of WNT signalling in the cap mesenchyme (CM) by Six2-Credriven expression of stabilised β-catenin or by co-culture with glycogen synthase kinase (GSK) inhibitors results in the uniform emergence of ectopic PTA [16,18,28]. WNT9b induces nephrogenesis also in isolated metanephric mesenchyme [3], but Six2-Cre-driven expression of Wnt9b does not result in ectopic RV [16]. ...
... According to our model, the increased WNT9b concentrations in the branch corners induce PTA/RVs. However, previous studies reported that Six2-Cre-driven expression of Wnt9b does not lead to ectopic RV induction, while Six2-Cre-driven expression of stabilised β-catenin (Ctnnb1 ) or addition of GSK inhibitors both do [16,18,28]. To analyse the impact of a local increase in the WNT9b concentration, we compared cultures in which 1) the WNT9b concentration was increased locally via WNT9b-soaked beads, 2) the WNT9b concentration was increased globally by adding recombinant WNT9b to the explant cultures, and 3) ...
During kidney development, reciprocal signalling between the epithelium and the mesenchyme coordinates nephrogenesis with branching morphogenesis of the collecting ducts. The mechanism that positions the renal vesicles, and thus the nephrons, relative to the branching ureteric buds has remained elusive. By combining computational modelling and experiments, we show that geometric effects concentrate the key regulator, WNT9b, at the junctions between parent and daughter branches where renal vesicles emerge, despite its uniform expression in the ureteric epithelium. This curvature effect might be a general paradigm to create non-uniform signalling in development.
... 24,30 It is known that Six2-Cre induces recombination in the kidney and stomach; however, in the stomach, Six2-Cre activity has not been fully characterized. 31 To analyze spatiotemporal activity of Six2-Cre in the stomach, Six2-Cre þ/-R-tdTomato fl/wt mice were generated in which Six2 þ cells and their derivatives were labeled permanently with tdTomato ( Figure 2, A-B). Stomach sections from newborn Six2-Cre þ/-R-tdTomato fl/wt mice were co-stained with red fluorescent protein (RFP) antibody (that detects tdTomato) and either E-cadherin, an epithelial marker, or ACTA2, a mesenchymal marker ( Figure 3, A-L). ...
... 24,29,79 Six2-Cre activity also was reported previously in the stomach, but, not fully characterized. 31,80 In addition to kidney and stomach, Six2-cre activity was also reported in the developing head, ear, limb, and osteoprogenitor cells. 24,81 Six2-Cre-mediated recombination has not been observed in the thymus, brain, lung, intestine, liver, spleen, and myeloid cells. ...
Background and aims:
Mesenchymal-epithelial crosstalk (MEC) in the stomach is executed by pathways such as bone morphogenetic protein (BMP) and extracellular signal-regulated kinase (ERK). Mis-regulation of MEC disrupts gastric homeostasis and causes tumorigenesis. Protein Kinase A (PKA) crosstalks with BMP and ERK signaling; however, PKA function(s) in stomach development and homeostasis remains undefined.
Methods:
We generated a novel Six2-Cre+/-PKAcαRfl/wt (CA-PKA) mouse in which expression of constitutive-active PKAcαR was induced in gastric mesenchyme progenitors. Lineage tracing determined spatiotemporal activity of Six2-Cre in the stomach. For phenotyping CA-PKA mice histological, co-immunofluorescence (Co-IF), immunoblotting, mRNA sequencing and bioinformatics analyses were performed.
Results:
Lineage tracing showed that Six2-Cre activity in the stomach is restricted to the mesenchymal compartment. CA-PKA mice showed disruption of gastric homeostasis characterized by aberrant mucosal development and epithelial hyperproliferation; ultimately developing multiple features of gastric corpus preneoplasia including decreased parietal cells, mucous cell hyperplasia, spasmolytic peptide expressing metaplasia (SPEM) with intestinal characteristics and dysplastic and invasive cystic glands. Furthermore, mutant corpus showed marked chronic inflammation characterized by infiltration of lymphocytes and myeloid-derived suppressor cells along with the upregulation of innate and adaptive immune system components. Striking upregulation of inflammatory mediators and STAT3 activation was observed. Mechanistically, we determined there is an activation of ERK1/2 and downregulation of BMP/SMAD signaling characterized by marked upregulation of BMP inhibitor gremlin 1.
Conclusions:
We report a novel role of PKA signaling in gastric MEC execution and show that PKA activation in the gastric mesenchyme drives preneoplasia by creating a proinflammatory and proproliferative microenvironment associated with the downregulation of BMP/SMAD signaling and activation of ERK1/2.
... GDNF-regulated transcripts also comprise secreted proteins, such as WNT11 and CRLF1, with demonstrated potential to regulate the nephrogenic program Schmidt-Ott et al., 2005). Other UB-derived nephrogenesis regulators are WNT9b and FGF-ligands influencing NP maintenance and differentiation (Barak et al., 2012;Carroll et al., 2005;Kiefer et al., 2012). ...
... whereas NP-expressed WNT targets (Karner et al., 2011) showed a trend of downregulation, likely reflecting the reduced overall number of NPCs (Fig. S7A). Together, these indicate that not only augmented GDNF-induced signaling but also increased Wnt9-and Wnt11-induced signaling with known functions in NPC regulation participate in mediating reciprocal communication from mutant UB to the adjacent NP pool at E14.5 (Karner et al., 2011;Kiefer et al., 2012;Nagy et al., 2016;O'Brien et al., 2018). Gdnf hyper/hyper kidney shows very few LEF1-positive differentiating nephron precursors and typical abundance of ureteric bud (UB) epithelium as detected by calbindin staining (green). ...
... Our data demonstrate that the early decline in embryonic NPs of Gdnf hyper/hyper kidneys is due to decreased cell proliferation rather than increased premature differentiation. This cellular mechanism is supported by molecular changes detected in known GDNF targets (Crlf1, Wnt11 and Srgn) Ola et al., 2011) and in important regulators of NP cell maintenance signaling (Wnt9b, Wnt11 and their transcriptional targets) (Karner et al., 2011;Kiefer et al., 2012;O'Brien et al., 2018). Previous examination of longevity and resilience of NP cells by ablating the NP population with diphtheria toxin A, or by disturbing MAPK/ERK activation, which both resulted in rapid decline in NP cells without a positive effect on their lifespan (Cebrián et al., 2014;Ihermann-Hella et al., 2018), together with our results suggest that NP cell proliferation inherently plays a major role in defining their total lifetime. ...
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.
... Accordingly, delicate changes in signaling activity levels appear to critically dictate the fate decision in the nephron progenitor pool, as Wnt9b has also been shown to support progenitor maintenance by positively regulating proliferation [154,155]. Another UB-derived WNT ligand, Wnt11, is essential for the normal maintenance of nephron progenitors through its function in mediating the interaction between progenitors and UB tip cells [128]. The modulation of WNT signaling activity through R-spondins 1 and 3 is likely involved in signaling-level regulation, but the exact mechanisms remain to be studied as inactivation of R-Spondins only has a mild effect on progenitor propagation [156]. ...
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.
... cDNA was prepared using the High Capacity RNA-to-cDNA kit (Life Technologies). Primer sequences are in Table S2 qRT-PCR was performed using a Quant Studio 3 (Applied Biosystems) Thermocycler and SYBR Green PCR Master Mix (Life Technologies) as described previously (Kiefer et al., 2012). Real-time reactions were performed in triplicate and relative expression was calculated using the delta CT method and normalized to Gapdh or Hprt1 control transcripts (Kiefer et al., 2012). ...
... Primer sequences are in Table S2 qRT-PCR was performed using a Quant Studio 3 (Applied Biosystems) Thermocycler and SYBR Green PCR Master Mix (Life Technologies) as described previously (Kiefer et al., 2012). Real-time reactions were performed in triplicate and relative expression was calculated using the delta CT method and normalized to Gapdh or Hprt1 control transcripts (Kiefer et al., 2012). ...
Chromatin-remodeling complexes play critical roles in establishing gene expression patterns in response to developmental signals. How these epigenetic regulators determine the fate of progenitor cells during development of specific organs is not well understood. We found that genetic deletion of Brg1 (Smarca4), the core enzymatic protein in SWI/SNF, in nephron progenitor cells leads to severe renal hypoplasia. Nephron progenitor cells were depleted in Six2-Cre, Brg1flx/flx mice due to reduced cell proliferation. This defect in self-renewal, together with impaired differentiation resulted in a profound nephron deficit in Brg1 mutant kidneys. Sall1, a transcription factor that is required for expansion and maintenance of nephron progenitors, associates with SWI/SNF. Brg1 and Sall1 bind promoters of many progenitor cell genes and regulate expression of key targets that promote their proliferation.
... GDNF-regulated transcripts also comprise secreted proteins, such as WNT11 and CRLF1, with demonstrated potential to regulate the nephrogenic program Schmidt-Ott et al., 2005). Other UB-derived nephrogenesis regulators are WNT9b and FGF-ligands influencing NP maintenance and differentiation (Barak et al., 2012;Carroll et al., 2005;Kiefer et al., 2012). ...
... whereas NP-expressed WNT targets (Karner et al., 2011) showed a trend of downregulation, likely reflecting the reduced overall number of NPCs (Fig. S7A). Together, these indicate that not only augmented GDNF-induced signaling but also increased Wnt9-and Wnt11-induced signaling with known functions in NPC regulation participate in mediating reciprocal communication from mutant UB to the adjacent NP pool at E14.5 (Karner et al., 2011;Kiefer et al., 2012;Nagy et al., 2016;O'Brien et al., 2018). Gdnf hyper/hyper kidney shows very few LEF1-positive differentiating nephron precursors and typical abundance of ureteric bud (UB) epithelium as detected by calbindin staining (green). ...
... Our data demonstrate that the early decline in embryonic NPs of Gdnf hyper/hyper kidneys is due to decreased cell proliferation rather than increased premature differentiation. This cellular mechanism is supported by molecular changes detected in known GDNF targets (Crlf1, Wnt11 and Srgn) Ola et al., 2011) and in important regulators of NP cell maintenance signaling (Wnt9b, Wnt11 and their transcriptional targets) (Karner et al., 2011;Kiefer et al., 2012;O'Brien et al., 2018). Previous examination of longevity and resilience of NP cells by ablating the NP population with diphtheria toxin A, or by disturbing MAPK/ERK activation, which both resulted in rapid decline in NP cells without a positive effect on their lifespan (Cebrián et al., 2014;Ihermann-Hella et al., 2018), together with our results suggest that NP cell proliferation inherently plays a major role in defining their total lifetime. ...
Due 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 is determined by mechanisms related to perception of GDNF and other signaling levels.
... Wnt9b is a crucial Wnt ligand that plays a role in both canonical Wnt signalling and noncanonical Wnt/PCP signalling. This comes from evidence that displays that mis-expression of Wnt9b in Six2-positive cells disrupts kidney function by activating canonical Wnt signalling in Wnt9b transgenic mice [48]. As well as this, researchers highlighted the upregulation of canonical Wnt signalling, together with the presence of renal cysts throughout the nephron, further supporting the idea that disruption of canonical Wnt signalling can result in cystic disease [48]. ...
... This comes from evidence that displays that mis-expression of Wnt9b in Six2-positive cells disrupts kidney function by activating canonical Wnt signalling in Wnt9b transgenic mice [48]. As well as this, researchers highlighted the upregulation of canonical Wnt signalling, together with the presence of renal cysts throughout the nephron, further supporting the idea that disruption of canonical Wnt signalling can result in cystic disease [48]. Despite this, other evidence has also highlighted that regulation of Wnt9b is crucial for PCP in the epithelium of the kidney and that depleted Wnt9b resulted in an increased diameter in tubules [49]. ...
Chronic kidney disease (CKD) encompasses a group of diverse diseases that are associated with accumulating kidney damage and a decline in glomerular filtration rate (GFR). These conditions can be of an acquired or genetic nature and, in many cases, interactions between genetics and the environment also play a role in disease manifestation and severity. In this review, we focus on genetically inherited chronic kidney diseases and dissect the links between canonical and non-canonical Wnt signalling, and this umbrella of conditions that result in kidney damage. Most of the current evidence on the role of Wnt signalling in CKD is gathered from studies in polycystic kidney disease (PKD) and nephronophthisis (NPHP) and reveals the involvement of beta-catenin. Nevertheless, recent findings have also linked planar cell polarity (PCP) signalling to CKD, with further studies being required to fully understand the links and molecular mechanisms.
... Inactivation of wingless-type MMTV integration site family (WNT) signaling by deleting Wnt9b results in renal aplasia in mouse [80]. Unlike many other aplasia models with early UB arrest, the loss of Wnt9b primarily affects the MM, which fails to maintain nephron progenitors and induce them for differentiation [81,82]. Canonical WNT signaling is mediated by inactivation of glycogen synthase 3 (GSK3) and causes stabilization of cytoplasmic β-catenin, which leads to activation of TCF/LEF1 transcription factors. ...
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.
... 5′-GTTTAGCTCAGAGGGTCCATCTAT-3′ and 5′-AGTGC CAAGACAGAGCGACT-3′, Pdgrfb 5′-AACTGTCACCC ACACCCTTG-3′ and 5′-ACCACCACTTTGAAGGGCAA-3′, Mmp2 5′-GATAACCTGGATGCCGTCGT-3′ and 5′-TGGTGTGCAGCGATGAAGAT-3′, Ctgf 5′-AGAGTGGAG CGCCTGTTCTA-3′ and 5′-GGCTTGGCGATTTTAGGTG TC-3′, Shroom3 5′-AATTTGGGGAGACACAGCCT-3′ and 5′-GCTCCGCCTCAGATAAGCAT-3′, Itga5 5′-ATCCAGT GCACCACCATTCA-3′ and 5′-TCCGAACCACTGCAAGG AC-3′, and Itgb5 5′-CACCCAAAATGTGCCTGGTG-3′ and 5′-AGAGGTAGGTTCCGGAGGAC-3′. Real-time reactions were performed in triplicate, and relative expression was calculated using the delta CT method and normalized to Gapdh 5′-AGGTCGGTGTGAACGGATTTG-3′ and 5′-TGTAGACCATGTAGTTGAGGTCA-3′ or Hprt1 5′-TCA GTCAACGGGGGACATAAA-3′ and 5′-GGGGCTGTACTG CTTAACCAG-3′ control transcripts (Kiefer, Robbins, & Rauchman, 2012). ...
Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-β at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease.
