Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney

Signaling by the ureteric bud epithelium is essential for survival, proliferation and differentiation of the metanephric mesenchyme during kidney development. Most studies that have addressed ureteric signaling have focused on the proximal, branching, ureteric epithelium. We demonstrate that sonic hedgehog is expressed in the ureteric epithelium of the distal, non-branching medullary collecting ducts and continues into the epithelium of the ureter -- the urinary outflow tract that connects the kidney with the bladder. Upregulation of patched 1, the sonic hedgehog receptor and a downstream target gene of the signaling pathway in the mesenchyme surrounding the distal collecting ducts and the ureter suggests that sonic hedgehog acts as a paracrine signal. In vivo and in vitro analyses demonstrate that sonic hedgehog promotes mesenchymal cell proliferation, regulates the timing of differentiation of smooth muscle progenitor cells, and sets the pattern of mesenchymal differentiation through its dose-dependent inhibition of smooth muscle formation. In addition, we also show that bone morphogenetic protein 4 is a downstream target gene of sonic hedgehog signaling in kidney stroma and ureteral mesenchyme, but does not mediate the effects of sonic hedgehog in the control of mesenchymal proliferation.     

Follistatin‐like 1 in vertebrate development

Follistatin‐like 1 (Fstl1) is a member of the secreted protein acidic rich in cysteins (SPARC) family and has been implicated in many different signaling pathways, including bone morphogenetic protein (BMP) signaling. In many different developmental processes like, dorso‐ventral axis establishment, skeletal, lung and ureter development, loss of function experiments have unveiled an important role for Fstl1. Fstl1 largely functions through inhibiting interactions with the BMP signaling pathway, although, in various disease models, different signaling pathways, like activation of pAKT, pAMPK, Na/K‐ATPase, or innate immune responses, are linked to Fstl1. How Fstl1 inhibits BMP signaling remains unclear, although it is known that Fstl1 does not function through a scavenging mechanism, like the other known extracellular BMP inhibitors such as noggin. It has been proposed that Fstl1 interferes with BMP receptor complex formation and as such inhibits propagation of the BMP signal into the cell. Future challenges will encompass the identification of the factors that determine the mechanisms that underlie the fact that Fstl1 acts by interfering with BMP signaling during development, but through other signaling pathways during disease. Birth Defects Research (Part C) 99:61–69, 2013. © 2013 Wiley Periodicals, Inc.     

Differential gene expression in the developing mouse ureter

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.     

Tailbud-derived mesenchyme promotes urinary tract segmentation via BMP4 signaling

Urinary tract morphogenesis requires the sub-division of the ureteric bud (UB) into the intra-renal collecting system and ureter, two tissues with unique structural and functional properties. In this report we investigate the cellular and molecular mechanisms that mediate their differentiation. Fate mapping experiments in the developing chick indicate that the UB is surrounded by two distinct mesenchymal populations: nephrogenic mesenchyme derived from the intermediate mesoderm and tailbud-derived mesoderm, which is selectively associated with the domain of the UB that differentiates into the ureter. Functional experiments utilizing murine metanephric kidney explants show that BMP4, a paracrine factor secreted by tailbud-derived mesenchyme, is required for ureter morphogenesis. Conversely, ectopic BMP4 signaling is sufficient to induce ureter morphogenesis in domains of the UB normally fated to differentiate into the intra-renal collecting system. Collectively, these results indicate that the border between the kidney and ureter forms where mesenchymal tissues originating in two different areas of the early embryo meet. These data raise the possibility that the susceptibility of this junction to congenital defects in humans, such as ureteral-pelvic obstructions, may be related to the complex morphogenetic movements that are required to integrate cells from these different lineages into a single functional structure.     

Role of BMP family members during kidney development.

Members of the Bone morphogenetic protein (BMP) family have been shown to be important signaling molecules throughout mouse development. Accordingly, many BMPs are also expressed during organogenesis of the metanephric kidney. However, only BMP7 has been shown to be absolutely required for proper formation of the kidney, thus the majority of information known involves this family member. BMP7 is expressed in both the ureteric epithelium and the mesenchyme throughout embryonic development and has been shown to function as a survival factor for the nephrogenic mesenchyme. However, there has been some controversy over the role of BMP7 as an inducing molecule for the metanephric mesenchyme. Recent studies have shown that BMP7 functions as an anti-differentiation factor for this mesenchyme cell population. The function of BMPs in the ureter and in the more differentiated epithelial structures of the nephron is less well understood.     

Epithelial BMP signaling is required for proper specification of epithelial cell lineages and gastric endocrine cells

Bone morphogenetic protein (BMP) signaling within the gastrointestinal tract is complex. BMP ligands and their receptors are expressed in both epithelial and mesenchymal compartments, suggesting bidirectional signaling between these two entities. Despite an increasing interest in BMP signaling in gut physiology and pathologies, the distinct contribution of BMP signaling in the epithelium vs. the mesenchyme in gastrointestinal homeostasis remains to be established. We aimed to investigate the role of epithelial BMP signaling in gastric organogenesis, gland morphogenesis, and maintenance of epithelial cell functions. Using the Cre/loxP system, we generated a mouse model with an early deletion during development of BMP receptor 1A (Bmpr1a) exclusively in the foregut endoderm. Bmpr1a(ΔGEC) mice showed no severe abnormalities in gastric organogenesis, gland epithelial proliferation, or morphogenesis, suggesting only a minor role for epithelial BMP signaling in these processes. However, early loss of BMP signaling in foregut endoderm did impact on gastric patterning, leading to an anteriorization of the stomach. In addition, numbers of parietal cells were reduced in Bmpr1a(ΔGEC) mice. Epithelial BMP deletion significantly increased the numbers of chromogranin A-, ghrelin-, somatostatin-, gastrin-, and serotonin-expressing gastric endocrine cells. Cancer never developed in young adult (<100 days) Bmpr1a-inactivated mice although a marker of spasmolytic polypeptide-expressing metaplasia was upregulated. Using this model, we have uncovered that BMP signaling negatively regulates the proliferation and commitment of endocrine precursor cells. Our data also indicate that loss of BMP signaling in epithelial gastric cells alone is not sufficient to induce gastric neoplasia.     

Impaired Elastin Deposition in Fstl1?/? Lung Allograft under the Renal Capsule

Lung alveolar development in late gestation is a process important to postnatal survival. Follistatin-like 1 (Fstl1) is a matricellular protein of the Bmp antagonist class, which is involved in the differentiation/maturation of alveolar epithelial cells during saccular stage of lung development. This study investigates the role of Fstl1 on elastin deposition in mesenchyme and subsequent secondary septation in the late gestation stage of terminal saccular formation. To this aim, we modified the renal capsule allograft model for lung organ culture by grafting diced E15.5 distal lung underneath the renal capsule of syngeneic host and cultured up to 7 days. The saccular development of the diced lung allografts, as indicated by the morphology, epithelial and vascular developments, occurred in a manner similar to that in utero. Fstl1 deficiency caused atelectatic phenotype companied by impaired epithelial differentiation in D3 Fstl1^−/− lung allografts, which is similar to that of E18.5 Fstl1^−/− lungs, supporting the role of Fstl1 during saccular stage. Inhibition of Bmp signaling by intraperitoneal injection of dorsomorphin in the host mice rescued the pulmonary atelectasis of D3 Fstl1^−/− allografts. Furthermore, a marked reduction in elastin expression and deposition was observed in walls of air sacs of E18.5 Fstl1^−/− lungs and at the tips of the developing alveolar septae of D7 Fstl1^−/− allografts. Thus, in addition to its role on alveolar epithelium, Fstl1 is crucial for elastin expression and deposition in mesenchyme during lung alveologenesis. Our data demonstrates that the modified renal capsule allograft model for lung organ culture is a robust and efficient technique to increase our understanding of saccular stage of lung development.     

Effects of TCDD on embryonic ureteric epithelial EGF receptor expression and cell proliferation

The potent toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing hydronephrosis and cleft palate. Because of the long half-life of TCDD, the urinary tract is exposed throughout development after a single dose on gestation day (GD) 10 or earlier. TCDD-induced hydronephrosis is a consequence of occlusion of the ureter by epithelial cells. Since embryonic growth factors and the epidermal growth factor (EGF) receptor are probably involved in regulation of embryonic cell proliferation, this study examines the effects of TCDD on expression of EGF receptors and proliferation of ureteric epithelial cells in vivo and in culture. After exposure to TCDD by gavage (12, 24, or 30 micrograms/kg on GD 10; 6 or 24 micrograms/kg on GD 12) the mean cell depth of the ureteric and bladder epithelia was increased. EGF receptors were detected immunohistochemically in sectioned urinary tracts. The expression of receptors decreased with advancing development in control ureteric epithelia. However, after TCDD exposure the level of EGF receptors failed to decline. The incorporation of 3H-TdR was observed in sections by autoradiography, and after exposure to TCDD more epithelial cells showed incorporation than was apparent in controls. Transmission electron microscopy (TEM) of embryonic ureters from fetuses exposed to TCDD in vivo showed no cytotoxicity in basal cells and the cells remained undifferentiated, as in controls. Ureters taken from GD 12 embryos and cultured with 1 x 10(-10)M TCDD showed ureteric epithelial hyperplasia without cytotoxicity, but at 1 x 10(-8)M TCDD evidence of cytotoxicity was observed by TEM. The levels of TCDD found in fetuses after in vivo exposure (204-307 pg/fetus, with 1-2 pg in the urinary tract) compare well with the in vitro level (32 pg/ml), which was most effective in producing hyperplasia of the epithelial cells. The present study correlates a TCDD-induced increase in cell depth with altered regulation of EGF receptors and excessive proliferation, both in vivo and in cultured embryonic ureters.     

Cell type specific expression of Follistatin-like 1 (Fstl1) in mouse embryonic lung development

Follistatin like-1 (Fstl1) is a secreted glycoprotein and can be up-regulated by TGF-β1. To better study the function of Fstl1 in lung development, we examined Fstl1 expression in the developing lung, in a cell type specific manner, using a tamoxifen inducible Fstl1-reporter mouse strain. Our results show that Fstl1 is ubiquitously expressed at saccular stage in the developing lung. At E18.5, Fstl1 expression is robust in most type of mesenchymal cells, including airway smooth muscle cells surrounding airways, vascular smooth muscle cells, endothelial cells, and vascular pericytes from blood vessel, but not PDGFRα⁺ fibroblasts in the distal alveolar sacs. Meanwhile, relative weak and sporadic signals of Fstl1 expression are observed in epithelium, including a subgroup of club cells in proximal airways and a few type II alveolar epithelial cells in distal airways. Our data help to understand the critical role of Fstl1 in lung development and lung disease pathogenesis.     

Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis

Antagonists act to restrict and negatively modulate the activity of secreted signals during progression of embryogenesis. In mouse embryos lacking the extra-cellular BMP antagonist gremlin 1 (Grem1), metanephric development is disrupted at the stage of initiating ureteric bud outgrowth. Treatment of mutant kidney rudiments in culture with recombinant gremlin 1 protein induces additional epithelial buds and restores outgrowth and branching. All epithelial buds express Wnt11, and Gdnf is significantly upregulated in the surrounding mesenchyme, indicating that epithelial-mesenchymal (e-m) feedback signalling is restored. In the wild type, Bmp4 is expressed by the mesenchyme enveloping the Wolffian duct and ureteric bud and Grem1 is upregulated in the mesenchyme around the nascent ureteric bud prior to initiation of its outgrowth. In agreement, BMP activity is reduced locally as revealed by lower levels of nuclear pSMAD protein in the mesenchyme. By contrast, in Grem1-deficient kidney rudiments, pSMAD proteins are detected in many cell nuclei in the metanephric mesenchyme, indicative of excessive BMP signal transduction. Indeed, genetic lowering of BMP4 levels in Grem1-deficient mouse embryos completely restores ureteric bud outgrowth and branching morphogenesis. The reduction of BMP4 levels in Grem1 mutant embryos enables normal progression of renal development and restores adult kidney morphology and functions. This study establishes that initiation of metanephric kidney development requires the reduction of BMP4 activity by the antagonist gremlin 1 in the mesenchyme, which in turn enables ureteric bud outgrowth and establishment of autoregulatory GDNF/WNT11 feedback signalling.     

The BMP antagonist follistatin-like 1 is required for skeletal and lung organogenesis

Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and zebrafish demonstrated a role as a BMP antagonist in early development. To investigate the role of Fstl1 during mouse development, a conditional Fstl1 KO allele as well as a Fstl1-GFP reporter mouse were created. KO mice die at birth from respiratory distress and show multiple defects in lung development. Also, skeletal development is affected. Endochondral bone development, limb patterning as well as patterning of the axial skeleton are perturbed in the absence of Fstl1. Taken together, these observations show that Fstl1 is a crucial regulator in BMP signalling during mouse development.     

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system

Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.     

BMP signaling modulates hedgehog-induced secondary heart field proliferation

Sonic hedgehog signaling in the secondary heart field has a clear role in cardiac arterial pole development. In the absence of hedgehog signaling, proliferation is reduced in secondary heart field progenitors, and embryos predominantly develop pulmonary atresia. While it is expected that proliferation in the secondary heart field would be increased with elevated hedgehog signaling, this idea has never been tested. We hypothesized that up-regulating hedgehog signaling would increase secondary heart field proliferation, which would lead to arterial pole defects. In culture, secondary heart field explants proliferated up to 6-fold more in response to the hedgehog signaling agonist SAG, while myocardial differentiation and migration were unaffected. Treatment of chick embryos with SAG at HH14, just before the peak in secondary heart field proliferation, resulted unexpectedly in stenosis of both the aortic and pulmonary outlets. We examined proliferation in the secondary heart field and found that SAG-treated embryos exhibited a much milder increase in proliferation than was indicated by the in vitro experiments. To determine the source of other signaling factors that could modulate increased hedgehog signaling, we co-cultured secondary heart field explants with isolated pharyngeal endoderm or outflow tract and found that outflow tract co-cultures prevented SAG-induced proliferation. BMP2 is made and secreted by the outflow tract myocardium. To determine whether BMP signaling could prevent SAG-induced proliferation, we treated explants with SAG and BMP2 and found that BMP2 inhibited SAG-induced proliferation. In vivo, SAG-treated embryos showed up-regulated BMP2 expression and signaling. Together, these results indicate that BMP signaling from the outflow tract modulates hedgehog-induced proliferation in the secondary heart field.     

BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions

Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER^-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER^-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER^-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.     

Genetics of Vesicoureteral Reflux

Vesicoureteral reflux (VUR) is the retrograde passage of urine from the bladder to the upper urinary tract. It is the most common congenital urological anomaly affecting 1-2% of children and 30-40% of patients with urinary tract infections. VUR is a major risk factor for pyelonephritic scarring and chronic renal failure in children. It is the result of a shortened intravesical ureter with an enlarged or malpositioned ureteric orifice. An ectopic embryonal ureteric budding development is implicated in the pathogenesis of VUR, which is a complex genetic developmental disorder. Many genes are involved in the ureteric budding formation and subsequently in the urinary tract and kidney development. Previous studies demonstrate an heterogeneous genetic pattern of VUR. In fact no single major locus or gene for primary VUR has been identified. It is likely that different forms of VUR with different genetic determinantes are present. Moreover genetic studies of syndromes with associated VUR have revealed several possible candidate genes involved in the pathogenesis of VUR and related urinary tract malformations. Mutations in genes essential for urinary tract morphogenesis are linked to numerous congenital syndromes, and in most of those VUR is a feature. The Authors provide an overview of the developmental processes leading to the VUR. The different genes and signaling pathways controlling the embryonal urinary tract development are analyzed. A better understanding of VUR genetic bases could improve the management of this condition in children.