Hedgehog signaling regulates the amount of hypaxial muscle development during Xenopus myogenesis

Hedgehog (Hh) signaling is proposed to have different roles on differentiation of hypaxial myoblasts of amniotes. Within the somitic environment, Hh signals restrict hypaxial development and promote epaxial muscle formation. On the other hand, in the limb bud, Hh signaling represses hypaxial myoblast differentiation. This poses the question of whether differences in response to Hh signaling are due to variations in local environment or are intrinsic differences between pre- and post-migratory hypaxial myoblasts. We have approached this question by examining the role of Hh signaling on myoblast development in Xenopus laevis, which, due to its unique mode of hypaxial muscle development, allows us to examine myoblast development in vivo in the absence of the limb environment. Cyclopamine and sonic hedgehog (shh) mRNA overexpression were used to inhibit or activate the Hh pathway, respectively. We find that hypaxial myoblasts respond similarly to Hh manipulations regardless of their location, and that this response is the same for epaxial myoblasts. Overexpression of shh mRNA causes a premature differentiation of the dermomyotome, subsequently inhibiting all further growth of the epaxial and hypaxial myotome. Cyclopamine treatment has the opposite effect, causing an increase in dermomyotome and a shift in myoblast fate from epaxial to hypaxial, eventually leading to an excess of hypaxial body wall muscle. Cyclopamine treatment before stage 20 can rescue the effects of shh overexpression, indicating that early Hh signaling plays an essential role in maintaining the balance between epaxial and hypaxial muscle mass. After stage 20, the premature differentiation of the dermomyotome caused by shh overexpression cannot be rescued by cyclopamine, and no further embryonic muscle growth occurs.     

Inhibition of the anti-adipogenic Hedgehog signaling pathway by cyclopamine does not trigger adipocyte differentiation

Dysregulation of Hedgehog signaling can lead to several pathologies such as congenital defects and cancer. Here, we show that Hedgehog signaling is active in undifferentiated 3T3-L1 cells and decreases during adipocyte differentiation. Interestingly, this is paralleled by a decrease in Indian Hedgehog expression. We then tested if this down-regulation was sufficient to induce adipocyte differentiation. To this end, we demonstrate that the well-characterized Hedgehog inhibitor cyclopamine induced a decrease in Hedgehog signaling, similar to the one observed during adipocyte differentiation. However, cyclopamine did not induce nor potentiate adipocyte differentiation, as monitored by triglyceride staining and by the expression of several adipocyte markers: aP2, adipsin, C/EBPalpha, and Pref-1. Moreover, cyclopamine cannot substitute for other components of the differentiation medium: insulin, dexamethasone or IBMX. These results indicate that although Hedgehog signaling decreases during adipocyte differentiation, this down-regulation is not sufficient to trigger adipocyte differentiation. This suggests that Hedgehog signaling is an inadequate pharmacological target for patient suffering from syndromes associated with a decrease in fat mass, such as the ones observed in lipodystrophies.     

Isolation,identification, and bioactivity of microbial metabolites of cyclopamine and its congeners

The biotransformation of cyclopamine (1) and its congeners (2, 3 and 4) by Cunninghamella echinulata (ACCC 30369) was investigated. The chemical structures of two new congeners (2 and 4) and nine new metabolites were elucidated by 1D NMR (¹H, ¹³C and DEPT), 2D NMR (COSY, HMBC, HSQC and NOESY) and HRESIMS analyses and further confirmed by X-ray diffraction studies. Among these compounds, 2 and 4 showed moderate cytotoxicity in HepG2 cells (2, IC₅₀ = 8.03 ± 1.92 μM) and A549 cells (4, IC₅₀ = 10.19 ± 2.18 μM). Conversely, the cytotoxicity of the nine metabolites was sharply reduced. Similar to 1, compound 4 induced a cyclopia phenotype in zebrafish embryos at 20 μM. Moreover, compound 4 was more stable than 1 in an acidic environment.     

Dhh signaling pathway regulates reconstruction of seminiferous tubule-like structure

The reconstruction of a tubule-like structure in vitro has provided a promising system to analyze factors that drive morphogenesis and the underlying mechanisms. In this study, we took advantage of the inhibitor cyclopamine and a smoothened agonist to detect the role of the Dhh signaling pathway in the reconstructed tubule-like structure. Sertoli cells did not show polarity, rounded peritubular myoid cells and scattered Leydig cells were observed, combined with less laminin and lower proliferation of Leydig, peritubular myoid, germ, and Sertoli cells. However, in the presence of SAG, elongated peritubular myoid cells gathered at the bottom of polarized Sertoli cells, and most of the Leydig cells gathered at the outer part of the elongated peritubular myoid cells. Moreover, SAG promoted the secretion of laminin, assisting in the formation of the basal membrane and promoting the proliferation of Leydig, peritubular myoid, and germ cells. The level of Gli1 was significantly downregulated when treated with cyclopamine, whereas it was significantly upregulated when treated with SAG. These results indicate that the Dhh signaling pathway regulates the reconstruction of tubule-like structures by regulating the expression of Gli1.     

Cyclopamine bioactivity by extraction method from Veratrum californicum

Veratrum californicum, commonly referred to as corn lily or Californian false hellebore, grows in high mountain meadows and produces the steroidal alkaloid cyclopamine, a potent inhibitor of the Hedgehog (Hh) signaling pathway. The Hh pathway is a crucial regulator of many fundamental processes during vertebrate embryonic development. However, constitutive activation of the Hh pathway contributes to the progression of various cancers. In the present study, a direct correlation was made between the extraction efficiency for cyclopamine from root and rhizome by eight methods, and the associated biological activity in Shh-Light II cells using the Dual-Glo® Luciferase Assay System. Alkaloid recovery ranged from 0.39 to 8.03 mg/g, with ethanol soak being determined to be the superior method for obtaining biologically active cyclopamine. Acidic ethanol and supercritical extractions yielded degraded or contaminated cyclopamine with lower antagonistic activity towards Hh signaling.