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We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor, cyclopamine, and the activators, Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes.

Down syndrome (DS) is among the most frequent genetic causes of intellectual disability, and ameliorating this deficit is a major goal in support of people with trisomy 21. The Ts65Dn mouse recapitulates some major brain structural and behavioral phenotypes of DS, including reduced size and cellularity of the cerebellum and learning deficits associated with the hippocampus. We show that a single treatment of newborn mice with the Sonic hedgehog pathway agonist SAG 1.1 (SAG) results in normal cerebellar morphology in adults. Further, SAG treatment at birth rescued phenotypes associated with hippocampal deficits that occur in untreated adult Ts65Dn mice. This treatment resulted in behavioral improvements and normalized performance in the Morris water maze task for learning and memory. SAG treatment also produced physiological effects and partially rescued both N-methyl-d-aspartate (NMDA) receptor-dependent synaptic plasticity and NMDA/AMPA receptor ratio, physiological measures associated with memory. These outcomes confirm an important role for the hedgehog pathway in cerebellar development and raise the possibility for its direct influence in hippocampal function. The positive results from this approach suggest a possible direction for therapeutic intervention to improve cognitive function for this population.

Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.