c-kit delineates a distinct domain of progenitors in the developing kidney

Early inductive events in mammalian nephrogenesis depend on an interaction between the ureteric bud and the metanephric mesenchyme. However, mounting evidence points towards an involvement of additional cell types--such as stromal cells and angioblasts--in growth and patterning of the nephron. In this study, through analysis of the stem cell factor (SCF)/c-kit ligand receptor pair, we describe an additional distinct cell population in the early developing kidney. While SCF is restricted to the ureteric bud, c-kit-positive cells are located within the renal interstitium, but are negative for Foxd1, an established marker of stromal cells. In fact, the c-kit-positive domain is continuous with a central mesodermal cell mass ventral and lateral to the dorsal aorta, while Foxd1-expressing stromal cells are continuous with a dorsal perisomitic cell population suggesting distinct intraembryonic origins for these cell types. A subset of c-kit-positive cells expresses Flk-1 and podocalyxin, suggesting that this cell population includes angioblasts and their progenitors. c-kit activation is not required for the survival of these cells in vivo, because white spotting (c-kit(W/W)) mice, carrying a natural inactivating mutation of c-kit, display normal intrarenal distribution of the c-kit-positive cells at E13.5. In addition, early kidney development in these mutants is preserved up to the stage when anemia compromises global embryonic development. In contrast, under defined conditions in organ cultures of metanephric kidneys, c-kit-positive cells, including the Flk-1-positive subset, undergo apoptosis after treatment with STI-571, an inhibitor of c-kit tyrosine phosphorylation. This is associated with reductions in ureteric bud branching and nephron number. Conversely, exogenous SCF expands the c-kit-positive population, including Flk-1-positive angioblasts, and accelerates kidney development in vitro. These data suggest that ureteric bud-derived SCF elicits growth-promoting effects in the metanephric kidney by expanding one or more components of the interstitial c-kit-positive progenitor pool.     

Frizzled7 as an emerging target for cancer therapy

Wnt proteins are secreted glycoproteins that bind to the N-terminal extra-cellular cysteine-rich domain of the Frizzled (Fzd) receptor family. The Fzd receptors can respond to Wnt proteins in the presence of Wnt co-receptors to activate the canonical and non-canonical Wnt pathways. Recent studies indicated that, among the Fzd family, Fzd7 is the Wnt receptor most commonly upregulated in a variety of cancers including colorectal cancer, hepatocellular carcinoma and triple negative breast cancer. Fzd7 plays an important role in stem cell biology and cancer development and progression. In addition, it has been demonstrated that siRNA knockdown of Fzd7, the anti-Fzd7 antibody or the extracellular peptide of Fzd7 (soluble Fzd7 peptide) displayed anti-cancer activity in vitro and in vivo mainly due to the inhibition of the canonical Wnt signaling pathway. Furthermore, pharmacological inhibition of Fzd7 by small interfering peptides or a small molecule inhibitor suppressed β-catenin-dependent tumor cell growth. Therefore, targeted inhibition of Fzd7 represents a rational and promising new approach for cancer therapy.     

Inhibition of canonical Wnt signaling promotes gliogenesis in P0-NSCs

Wnt signaling plays an essential role in the development of mammalian central nervous system. We investigated the impact of activation/inhibition of the Wnt signaling pathway on neuronal/glial differentiation in neurospheres derived from neonatal mouse forebrains. For short term alterations, neurospheres were stimulated with recombinant Wnt-3a, Wnt-5a and the Wnt inhibitor Dickkopf-1 (Dkk1). Furthermore, neurospheres were transduced with retroviral vectors encoding Wnt-3a, Wnt-7a and their inhibitors Dkk1 and soluble Frizzled related protein-5 (sFRP5). Long-term activation of Wnt pathway by Wnt-7a or by treatment with GSK3 inhibitors promoted a moderate increase of the neuronal differentiation and blocked gliogenesis. In contrast, Wnt pathway inhibition in neurospheres, induced by retroviral overexpression of either Dkk1 or sFRP5, robustly increased the gliogenesis at the expense of neurogenesis. In summary, our data demonstrate that activation or inhibition of Wnt/β-catenin signaling in neurospheres regulates neuronal and glial differentiation, respectively. Thus, our results suggest that Wnt signaling may also contribute to regulate these processes in the neonatal brain.     

Single nucleotide polymorphism scanning and expression of the FRZB gene in pig populations

Secreted frizzled-related protein 3 (sFRP3), encoded by the gene FRZB, is a member of the sFRP family with important roles in inhibition of the Wnt signalling pathway through competitive binding of the Wnt receptor. Here, we investigated pig FRZB as a candidate gene for growth traits and identified three polymorphic sites, an insertion (A-532B) and two SNPs (G636A and C650T) in its 5′-UTR. The genotype distributions of G636A and C650T were significantly different among mini-type indigenous (Diannan Small-ear and Tibetan), normal indigenous (Laiwu and Huai), and introduced (Large Yorkshire and Landrace) breeds. In semi-quantitative PCR expression analysis, expression of FRZB mRNA was abundant in tissues of hypophysis, longissimus dorsi muscle, and adipose tissues, and low in the heart, hypothalamus, and brain. Quantitative determination of mRNA level and protein expression analysis were corresponding. The results demonstrated that FRZB gene expression in longissimus dorsi muscle and liver tissue was significantly higher in Diannan Small-ear and Tibetan pigs than in the Large Yorkshire breed (P < 0.05); however, in back fat tissue, the expression was significantly higher in Diannan Small-ear pig than in Tibetan or Large Yorkshire breeds (P < 0.05). Given the known growth and fat characteristics of the breeds, these results indicate that FRZB expression has a negative association with muscle growth and a positive association with fat deposition. In conclusion, FRZB may be a major candidate gene for growth traits in pigs.     

Structure-function analysis of secreted frizzled-related protein-1 for its Wnt antagonist function

Secreted frizzled-related proteins (sFRPs) are glycoproteins that are recognized as Wnt antagonists. To identify the functional domains that are involved in Wnt antagonist function, several sFRP-1 mutants and sFRP-1/sFRP-2 chimeras were generated. These mutants were characterized in an optimized T-cell factor (TCF)-luciferase based assay in U2OS human osteosarcoma cells. Deletions of the sFRP-1 cysteine rich domain (CRD) lead to the complete loss of Wnt antagonist function. A region between amino acids 73-86 within the second loop of the CRD of sFRP-1 was necessary for the optimal Wnt inhibitory function. Within this region, a conserved tyrosine residue played a critical role, and its change to neutral or polar amino acids lead to decreased Wnt inhibitory activity. The sFRP-1/sFRP-2 chimeras with the netrin domain of sFRP-1 replaced by corresponding sFRP-2 sequences showed 40-70% loss of Wnt antagonist function. The sFRP-1/sFRP-2 chimera with the replacement of C-terminal 19 amino acids of sFRP-1 with 11 amino acids of sFRP-2 resulted in 70% loss of activity indicating that carboxyl-terminal region of sFRP-1 is important for its Wnt inhibitory activity. The structure-function analysis studies of sFRP-1 clearly demonstrate the interaction of several functional domains for its optimal Wnt antagonist function.     

Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 Å resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.     

A critical role for sFRP proteins in maintaining caudal neural tube closure in mice via inhibition of BMP signaling

Both the BMP and Wnt pathways have been implicated in directing aspects of dorsal neural tube closure and cell fate specification. However, the mechanisms that control the diverse responses to these signals are poorly understood. In this study, we provide genetic and functional evidence that the secreted sFRP1 and sFRP2 proteins, which have been primarily implicated as negative regulators of Wnt signaling, can also antagonize BMP signaling in the caudal neural tube and that this function is critical to maintain proper neural tube closure and dorsal cell fate segregation. Our studies thus reveal a novel role for specific sFRP proteins in balancing the response of cells to two critical extracellular signaling pathways.     

Coordination of epithelial branching and salivary gland lumen formation by Wnt and FGF signals

Branching morphogenesis is a molecularly conserved mechanism that is adopted by several organs, such as the lung, kidney, mammary gland and salivary gland, to maximize the surface area of a tissue within a small volume. Branching occurs through repetitive clefting and elongation of spherical epithelial structures, called endbuds, which invade the surrounding mesenchyme. In the salivary gland, lumen formation takes place alongside branching morphogenesis, but in a controlled manner, so that branching is active at the distal ends of epithelial branches while lumen formation initiates at the proximal ends, and spreads distally. We present here data showing that interaction between FGF signaling and the canonical (β-catenin dependent) and non-canonical branches of Wnt signaling coordinates these two processes. Using the Axin2^lacZ reporter mice, we find Wnt/β-catenin signaling activity first in the mesenchyme and later, at the time of lumen formation, in the ductal epithelium. Gain and loss of function experiments reveal that this pathway exerts an inhibitory effect on salivary gland branching morphogenesis. We have found that endbuds remain devoid of Wnt/β-catenin signaling activity, a hallmark of ductal structures, through FGF-mediated inhibition of this pathway. Our data also show that FGF signaling has a major role in the control of lumen formation by preventing premature hollowing of epithelial endbuds and slowing down the canalization of presumptive ducts. Concomitantly, FGF signaling strongly represses the ductal marker Cp2l1, most likely via repression of Wnt5b and non-canonical Wnt signaling. Inhibition of canonical and non-canonical Wnt signaling in endbuds by FGF signaling occurs at least in part through sFRP1, a secreted inhibitor of Wnt signaling and downstream target of FGF signaling. Altogether, these findings point to a key function of FGF signaling in the maintenance of an undifferentiated state in endbud cells by inhibition of a ductal fate.