mTORC1-Mediated Inhibition of 4EBP1 Is Essential for Hedgehog Signaling-Driven Translation and Medulloblastoma.

Mechanistic target of rapamycin (MTOR) cooperates with Hedgehog (HH) signaling, but the underlying mechanisms are incompletely understood. Here we provide genetic, biochemical, and pharmacologic evidence that MTOR complex 1 (mTORC1)-dependent translation is a prerequisite for HH signaling. The genetic loss of mTORC1 function inhibited HH signaling-driven growth of the cerebellum and medulloblastoma. Inhibiting translation or mTORC1 blocked HH signaling. Depleting 4EBP1, an mTORC1 target that inhibits translation, alleviated the dependence of HH signaling on mTORC1. Consistent with this, phosphorylated 4EBP1 levels were elevated in HH signaling-driven medulloblastomas in mice and humans. In mice, an mTORC1 inhibitor suppressed medulloblastoma driven by a mutant SMO that is inherently resistant to existing SMO inhibitors, prolonging the survival of the mice. Our study reveals that mTORC1-mediated translation is a key component of HH signaling and an important target for treating medulloblastoma and other cancers driven by HH signaling.

In Vivo Cochlear Hair Cell Generation and Survival by Coactivation of ß-Catenin and Atoh1.

The mammalian cochlea exhibit minimal spontaneous regeneration, and loss of sensory hair cells (HCs) results in permanent hearing loss. In nonmammalian vertebrates, spontaneous HC regeneration occurs through both proliferation and differentiation of surrounding supporting cells (SCs). HC regeneration in postnatal mammalian cochleae in vivo remains limited by the small HC number and subsequent death of regenerated HCs. Here, we describe in vivo generation of 10-fold more new HCs in the mouse cochlea than previously reported, most of which survive to adulthood. We achieved this by combining the expression of a constitutively active form of ß-catenin (a canonical Wnt activator) with ectopic expression of Atoh1 (a HC fate determination factor) in neonatal Lgr5+ cells (the presumed SC and HC progenitors of the postnatal mouse cochlea), and discovered synergistic increases in proliferation and differentiation. The new HCs were predominantly located near the endogenous inner HCs, expressed early HC differentiation markers, and were innervated despite incomplete alignment of presynaptic and postsynaptic markers. Surprisingly, genetic tracing revealed that only a subset of Lgr5+ cells that lie medial to the inner HCs respond to this combination, highlighting a previously unknown heterogeneity that exists among Lgr5+ cells. Together, our data indicate that ß-catenin and Atoh1 mediate synergistic effects on both proliferation and differentiation of a subset of neonatal cochlear Lgr5+ cells, thus overcoming major limitations of HC regeneration in postnatal mouse cochleae in vivo. These results provide a basis for combinatorial therapeutics for hearing restoration. SIGNIFICANCE STATEMENT: Hearing loss in humans from aging, noise exposure, or ototoxic drugs (i.e., cisplatin or some antibiotics) is permanent and affects every segments of the population, worldwide. However, birds, frog, and fish have the ability to recover hearing, and recent studies have focused on understanding and applying what we have learned from them for restoring hearing in humans. However, studies have been hampered by low efficiency, limited cell numbers, and subsequent death of these newly generated auditory cells. Here, we describe a combinatorial approach, which results in the generation of auditory cells in greater numbers than previously reported, with most of them surviving to adult ages in vivo. These results provide a basis for combinatorial therapeutics for hearing restoration efforts.

Vagal neural crest cell migratory behavior: a transition between the cranial and trunk crest.

Migration and differentiation of cranial neural crest cells are largely controlled by environmental cues, whereas pathfinding at the trunk level is dictated by cell-autonomous molecular changes owing to early specification of the premigratory crest. Here, we investigated the migration and patterning of vagal neural crest cells. We show that (1) vagal neural crest cells exhibit some developmental bias, and (2) they take separate pathways to the heart and to the gut. Together these observations suggest that prior specification dictates initial pathway choice. However, when we challenged the vagal neural crest cells with different migratory environments, we observed that the behavior of the anterior vagal neural crest cells (somite-level 1-3) exhibit considerable migratory plasticity, whereas the posterior vagal neural crest cells (somite-level 5-7) are more restricted in their behavior. We conclude that the vagal neural crest is a transitional population that has evolved between the head and the trunk.

Regional differences in neural crest morphogenesis.

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively, and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: 1) what establishes the pathways of migration and 2) what controls the final destination and differentiation of various neural crest subpopulations. These questions will be addressed in this review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube, and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted, or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk. The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: 1) the vagal-level neural crest cells exhibit modest developmental bias; 2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively; 3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.