Cyclopamine

The study of the tetrapod limb has contributed greatly to our understanding of developmental pathways and how changes to these pathways affect the evolution of morphology. Most of our understanding of tetrapod limb development comes from research on amniotes, with far less known about mechanisms of limb development in amphibians. To better understand the mechanisms of limb development in anuran amphibians, we used cyclopamine to inhibit Hedgehog signaling at various stages of development in the western clawed frog, Xenopus tropicalis, and observed resulting morphologies. We also analyzed gene expression changes resulting from similar experiments in Xenopus laevis. Inhibition of Hedgehog signaling in X. tropicalis results in limb abnormalities including reduced digit number, missing skeletal elements, and complete absence of limbs. In addition, posterior digits assume an anterior identity by developing claws that are usually only found on anterior digits, confirming Sonic hedgehog's role in digit identity determination.

The present study aimed to investigate the effect of palmatine (Pal) in a rabbit osteoarthritis (OA) model in vivo and rabbit interleukin-1β (IL-1β)-stimulated chondrocytes in vitro. Appropriate concentrations of Pal were identified by the MTT assay and used to preincubate IL-1β-induced chondrocytes, as well as an activator or inhibitor of Wnt and Hedgehog signaling pathways. Matrix metalloproteinase (MMP)-1, 3, and 13; tissue inhibitor of metalloproteinase (TIMP)-1; collagenase II; aggrecan; and the related molecules of the Wnt/β-catenin and Hedgehog signaling pathways were investigated. Protein expression was detected by Western blot analysis and messenger RNA (mRNA) expression was examined by PCR analysis. Pal (0.3mL, 100mg/L) was injected into rabbit knee joints and histological examination, immunohistochemistry, and Mankin scoring of the articular cartilage were performed. Pal (10-100mg/L) had no effect on chondrocyte viability, decreased the expression of the MMPs, and increased the synthesis of TIMP-1whereas collagenase II and aggrecan were inhibited by IL-1β.

Although recovery following a stroke is limited, undamaged neurons under the right conditions can establish new connections and take on-board lost functions. Sonic hedgehog (Shh) signaling is integral for developmental axon growth, but its role after injury has not been fully examined. To investigate the effects of Shh on neuronal sprouting after injury, we used an in vitro model of glial scar, whereby cortical astrocytes were mechanically traumatized to mimic reactive astrogliosis observed after stroke. This mechanical trauma impaired neurite outgrowth from post-natal cortical neurons plated on top of reactive astrocytes. Addition of Shh to the media, however, resulted in a concentration-dependent increase in neurite outgrowth. This response was inhibited by cyclopamine and activated by oxysterol 20(S)-hydroxycholesterol, both of which modulate the activity of the Shh co-receptor Smoothened (Smo), demonstrating that Shh-mediated neurite outgrowth is Smo-dependent.

Ischemic stroke is often associated with loss of cortical neurons leading to various neurological deficits. A cell replacement based on stem cell transplantation to repair the damaged brain requires the generation of specific neuronal subtypes. Recently, induced pluripotent stem cells have been used to generate various subtypes of neurons in vitro for transplantation in stroke-damaged brains. However, whether these cells can be primed as neuronal precursors to become cortical projection neurons by means of biomaterials releasing differentiation factors is not known. Here, we report that microspheres of biodegradable poly(ester-amide) composed of adipic acid, L-phenyl-alanine and 1,4-butanediol, loaded with differentiation factors, can be used to fate human induced pluripotent stem cell-derived long-term expandable neuroepithelial-like stem cells to cortical projection neurons. The three factors, Wnt3A, BMP4 and cyclopamine, were released from loaded microspheres over at least one month following biphasic dynamic time course, promoting cortical differentiation of the cells in vitro.