Let-7 regulates cell cycle dynamics in the developing cerebral cortex and retina.

In the neural progenitors of the developing central nervous system (CNS), cell proliferation is tightly controlled and coordinated with cell fate decisions. Progenitors divide rapidly during early development and their cell cycle lengthens progressively as development advances to eventually give rise to a tissue of the correct size and cellular composition. However, our understanding of the molecules linking cell cycle progression to developmental time is incomplete. Here, we show that the microRNA (miRNA) let-7 accumulates in neural progenitors over time throughout the developing CNS. Intriguingly, we find that the level and activity of let-7 oscillate as neural progenitors progress through the cell cycle by in situ hybridization and fluorescent miRNA sensor analyses. We also show that let-7 mediates cell cycle dynamics: increasing the level of let-7 promotes cell cycle exit and lengthens the S/G2 phase of the cell cycle, while let-7 knock down shortens the cell cycle in neural progenitors. Together, our findings suggest that let-7 may link cell proliferation to developmental time and regulate the progressive cell cycle lengthening that occurs during development.

RBX2 maintains final retinal cell position in a DAB1-dependent and -independent fashion.

The laminated structure of the retina is fundamental for the organization of the synaptic circuitry that translates light input into patterns of action potentials. However, the molecular mechanisms underlying cell migration and layering of the retina are poorly understood. Here, we show that RBX2, a core component of the E3 ubiquitin ligase CRL5, is essential for retinal layering and function. RBX2 regulates the final cell position of rod bipolar cells, cone photoreceptors and Muller glia. Our data indicate that sustained RELN/DAB1 signaling, triggered by depletion of RBX2 or SOCS7 - a CRL5 substrate adaptor known to recruit DAB1 - causes rod bipolar cell misposition. Moreover, whereas SOCS7 also controls Muller glia cell lamination, it is not responsible for cone photoreceptor positioning, suggesting that RBX2, most likely through CRL5 activity, controls other signaling pathways required for proper cone localization. Furthermore, RBX2 depletion reduces the number of ribbon synapses and disrupts cone photoreceptor function. Together, these results uncover RBX2 as a crucial molecular regulator of retina morphogenesis and cone photoreceptor function.

Specialized filopodia: at the 'tip' of morphogen transport and vertebrate tissue patterning.

For over a century, biologists have strived to unravel the mechanisms that establish how cells are informed of their position in the embryo and differentiate to give rise to complex organs and structures. However, the historical idea that one predominant mode of ligand transport, largely accounted for by free diffusion, can explain how all signaling molecules, known as morphogens, control tissue patterning has greatly hindered our ability to fully appreciate the complexities driving the delivery and reception of signaling molecules at a distance. In reality, a cell's shape, morphology, and location change continuously as development progresses. Thus, cellular context poses distinct challenges for morphogen transport in each unique cellular environment. Emerging studies reveal that some cells overcome such obstacles in an unexpected manner: via long, cellular projections, or specialized filopodia, that link distant cells and traffic signaling components. Here, we will review recent findings describing specialized filopodia and discuss the potential mechanisms and implications for filopodia-based long-range cell signaling and communication, particularly within the developing vertebrate embryo.

FoxD3 regulates cranial neural crest EMT via downregulation of tetraspanin18 independent of its functions during neural crest formation.

The scaffolding protein tetraspanin18 (Tspan18) maintains epithelial cadherin-6B (Cad6B) to antagonize chick cranial neural crest epithelial-to-mesenchymal transition (EMT). For migration to take place, Tspan18 must be downregulated. Here, we characterize the role of the winged-helix transcription factor FoxD3 in the control of Tspan18 expression. Although we previously found that Tspan18 mRNA persists several hours past the stage it would normally be downregulated in FoxD3-deficient neural folds, we now show that Tspan18 expression eventually declines. This indicates that while FoxD3 is crucial for initial downregulation of Tspan18, other factors subsequently impact Tspan18 expression. Remarkably, the classical EMT transcription factor Snail2 is not one of these factors. As in other vertebrates, FoxD3 is required for chick cranial neural crest specification and migration, however, FoxD3 has surprisingly little impact on chick cranial neural crest cell survival. Strikingly, Tspan18 knockdown rescues FoxD3-dependent neural crest migration defects, although neural crest specification is still deficient. This indicates that FoxD3 promotes cranial neural crest EMT by eliciting Tspan18 downregulation separable from its Tspan18-independent activity during neural crest specification and survival.

Tetraspanin18 is a FoxD3-responsive antagonist of cranial neural crest epithelial-to-mesenchymal transition that maintains cadherin-6B protein.

During epithelial-to-mesenchymal transition (EMT), tightly associated, polarized epithelial cells become individual mesenchymal cells capable of migrating. Here, we investigate the role of the transmembrane protein tetraspanin18 (Tspan18) in chick cranial neural crest EMT. Tspan18 mRNA is expressed in premigratory cranial neural crest cells, but is absent from actively migrating neural crest cells. Tspan18 knockdown leads to a concomitant loss of cadherin-6B (Cad6B) protein, whereas Cad6B protein persists when Tspan18 expression is extended. The temporal profile of Cad6B mRNA downregulation is unaffected in these embryos, which indicates that Tspan18 maintains Cad6B protein levels and reveals that Cad6B is regulated by post-translational mechanisms. Although downregulation of Tspan18 is necessary, it is not sufficient for neural crest migration: the timing of neural crest emigration, basal lamina breakdown and Cad7 upregulation proceed normally in Tspan18-deficient cells. This emphasizes the need for coordinated transcriptional and post-translational regulation of Cad6B during EMT and illustrates that Tspan18-antagonized remodeling of cell-cell adhesions is only one step in preparation for cranial neural crest migration. Unlike Cad6B, which is transcriptionally repressed by Snail2, Tspan18 expression is downstream of the winged-helix transcription factor FoxD3, providing a new transcriptional input into cranial neural crest EMT. Together, our data reveal post-translational regulation of Cad6B protein levels by Tspan18 that must be relieved by a FoxD3-dependent mechanism in order for cranial neural crest cells to migrate. These results offer new insight into the molecular mechanisms of cranial neural crest EMT and expand our understanding of tetraspanin function relevant to metastasis.

Developmental cues and persistent neurogenic potential within an in vitro neural niche.

BACKGROUND: Neurogenesis, the production of neural cell-types from neural stem cells (NSCs), occurs during development as well as within select regions of the adult brain. NSCs in the adult subependymal zone (SEZ) exist in a well-categorized niche microenvironment established by surrounding cells and their molecular products. The components of this niche maintain the NSCs and their definitive properties, including the ability to self-renew and multipotency (neuronal and glial differentiation). RESULTS: We describe a model in vitro NSC niche, derived from embryonic stem cells, that produces many of the cells and products of the developing subventricular zone (SVZ) and adult SEZ NSC niche. We demonstrate a possible role for apoptosis and for components of the extracellular matrix in the maintenance of the NSC population within our niche cultures. We characterize expression of genes relevant to NSC self-renewal and the process of neurogenesis and compare these findings to gene expression produced by an established neural-induction protocol employing retinoic acid. CONCLUSIONS: The in vitro NSC niche shows an identity that is distinct from the neurally induced embryonic cells that were used to derive it. Molecular and cellular components found in our in vitro NSC niche include NSCs, neural progeny, and ECM components and their receptors. Establishment of the in vitro NSC niche occurs in conjunction with apoptosis. Applications of this culture system range from studies of signaling events fundamental to niche formation and maintenance as well as development of unique NSC transplant platforms to treat disease or injury.