bProneural NEUROG2 (neurogenin 2 [Ngn2]) is essential for neuronal commitment, cell cycle withdrawal, and neuronal differentiation. Although NEUROG2's influence on neuronal commitment and differentiation is beginning to be clarified, its role in cell cycle withdrawal remains unknown. We therefore set out to investigate the molecular mechanisms by which NEUROG2 induces cell cycle arrest during spinal neurogenesis. We developed a large-scale chicken embryo strategy, designed to find gene networks modified at the onset of NEUROG2 expression, and thereby we identified those involved in controlling the cell cycle. NEUROG2 activation leads to a rapid decrease of a subset of cell cycle regulators acting at G 1 and S phases, including CCND1, CCNE1/2, and CCNA2 but not CCND2. The use of NEUROG2VP16 and NEUROG2EnR, acting as the constitutive activator and repressor, respectively, indicates that NEUROG2 indirectly represses CCND1 and CCNE2 but opens the possibility that CCNE2 is also repressed by a direct mechanism. We demonstrated by phenotypic analysis that this rapid repression of cyclins prevents S phase entry of neuronal precursors, thus favoring cell cycle exit. We also showed that cell cycle exit can be uncoupled from neuronal differentiation and that during normal development NEUROG2 is in charge of tightly coordinating these two processes.O ne important challenge in neurobiology is to understand how different types of postmitotic neurons, with distinct cellular and physiological properties, are generated in the developing central nervous system (CNS) from a pool of dividing neural progenitors. The embryonic spinal cord is a good model to tackle these issues, because the role of extracellular signals and transcription factors in neuron specification and differentiation is relatively well defined. This structure is derived from the neural tube, a single pseudoepithelium that will sequentially give rise to a large variety of neurons and glial cells dedicated to serve specific functions in the adult. Neurogenesis is achieved via a succession of steps that follow a stereotypic temporal order. A neural progenitor is committed to a neuronal fate at the expense of a glial fate and becomes a neuronal precursor. Concomitantly, this neural progenitor is destined to differentiate into a specific neuronal subtype. Soon after, neuronal precursors stop cycling and initiate their differentiation to give rise to postmitotic differentiated neurons.The main positive regulators of vertebrate neurogenesis are proneural transcription factors of the neural basic helix-loop-helix (bHLH) family, including neurogenins (NeuroG1/2/3) (5, 35). They control different steps of neurogenesis, such as neuronal commitment, cell cycle exit, subtype specification, and neuronal differentiation (5, 35, 42). In the spinal cord, loss-of-function studies have shown that NEUROG2 is involved in the acquisition of motoneuron and interneuron fates (46). Together with NEUROG1, NEUROG2 also controls neuronal differentiation as shown by the loss of neurons in Ne...

Control of cell-division orientation is integral to epithelial morphogenesis and asymmetric cell division. Proper spatiotemporal localization of the evolutionarily conserved Gαi-LGN-NuMA protein complex is critical for mitotic spindle orientation, but how this is achieved remains unclear. Here we identify Suppressor APC domain containing 2 (SAPCD2) as a previously unreported LGN-interacting protein. We show that SAPCD2 is essential to instruct planar mitotic spindle orientation in both epithelial cell cultures and mouse retinal progenitor cells in vivo. Loss of SAPCD2 randomizes spindle orientation, which in turn disrupts cyst morphogenesis in three-dimensional cultures, and triples the number of terminal asymmetric cell divisions in the developing retina. Mechanistically, we show that SAPCD2 negatively regulates the localization of LGN at the cell cortex, likely by competing with NuMA for its binding. These results uncover SAPCD2 as a key regulator of the ternary complex controlling spindle orientation during morphogenesis and asymmetric cell divisions.

Cell division orientation is crucial to control segregation of polarized fate determinants in the daughter cells to produce symmetric or asymmetric fate outcomes. Most studies in vertebrates have focused on the role of mitotic spindle orientation in proliferative asymmetric divisions and it remains unclear whether altering spindle orientation is required for the production of asymmetric fates in differentiative terminal divisions. Here, we show that the GoLoco motif protein LGN, which interacts with Gαi to control apicobasal division orientation in Drosophila neuroblasts, is excluded from the apical domain of retinal progenitors undergoing planar divisions, but not in those undergoing apicobasal divisions. Inactivation of LGN reduces the number of apicobasal divisions in mouse retinal progenitors, whereas it conversely increases these divisions in cortical progenitors. Although LGN inactivation increases the number of progenitors outside the ventricular zone in the developing neocortex, it has no effect on the position or number of progenitors in the retina. Retinal progenitor cell lineage analysis in LGN mutant mice, however, shows an increase in symmetric terminal divisions producing two photoreceptors, at the expense of asymmetric terminal divisions producing a photoreceptor and a bipolar or amacrine cell. Similarly, inactivating Gαi decreases asymmetric terminal divisions, suggesting that LGN function with Gαi to control division orientation in retinal progenitors. Together, these results show a context-dependent function for LGN and indicate that apicobasal divisions are not involved in proliferative asymmetric divisions in the mouse retina, but are instead essential to generate binary fates at terminal divisions.

Proper embryonic development relies on a tight control of spatial and temporal gene expression profiles in a highly regulated manner. One good example is the ON/OFF switching of the transcription factor PAX6 that governs important steps of neurogenesis. In the neural tube PAX6 expression is initiated in neural progenitors through the positive action of retinoic acid signaling and downregulated in neuronal precursors by the bHLH transcription factor NEUROG2. How these two regulatory inputs are integrated at the molecular level to properly fine tune temporal PAX6 expression is not known. In this study we identified and characterized a 940-bp long distal cis-regulatory module (CRM), located far away from the PAX6 transcription unit and which conveys positive input from RA signaling pathway and indirect repressive signal(s) from NEUROG2. These opposing regulatory signals are integrated through HOMZ, a 94 bp core region within E940 which is evolutionarily conserved in distant organisms such as the zebrafish. We show that within HOMZ, NEUROG2 and RA exert their opposite temporal activities through a short 60 bp region containing a functional RA-responsive element (RARE). We propose a model in which retinoic acid receptors (RARs) and NEUROG2 repressive target(s) compete on the same DNA motif to fine tune temporal PAX6 expression during the course of spinal neurogenesis.