SummaryStem and progenitor cells utilize asymmetric cell divisions to balance proliferation and differentiation. Evidence from lower eukaryotes shows that this process is regulated by proteins asymmetrically distributed at the cell cortex during mitosis: (1) Par3-Par6-aPKC, conferring polarity; (2) Gαi-LGN/AGS3-NuMA-p150glued, governing spindle positioning. Here, we focus on developing mouse skin, where progenitors execute a switch from predominantly symmetric to asymmetric divisions concomitant with stratification. Using in vivo skin-specific lentiviral RNAi, we investigate spindle orientation regulation and provide direct evidence that LGN, Numa1 and Dctn1 are involved. In compromising asymmetric cell divisions, we uncover profound defects in stratification, differentiation and barrier formation, and implicate Notch signalling as an important effector. Our study demonstrates the efficacy of applying RNAi in vivo to mammalian systems, and the ease of uncovering complex genetic interactions, here to gain insights into how changes in spindle orientation are coupled to establishing proper tissue architecture during skin development.

In animals with binocular vision, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. Here, we show that ephrin-Bs in the chiasm region direct the divergence of retinal axons through the selective repulsion of a subset of RGCs that express EphB1. Ephrin-B2 is expressed at the mouse chiasm midline as the ipsilateral projection is generated and is selectively inhibitory to axons from ventrotemporal (VT) retina, where ipsilaterally projecting RGCs reside. Moreover, blocking ephrin-B2 function in vitro rescues the inhibitory effect of chiasm cells and eliminates the ipsilateral projection in the semiintact mouse visual system. A receptor for ephrin-B2, EphB1, is found exclusively in regions of retina that give rise to the ipsilateral projection. EphB1 null mice exhibit a dramatically reduced ipsilateral projection, suggesting that this receptor contributes to the formation of the ipsilateral retinal projection, most likely through its repulsive interaction with ephrin-B2.

SUMMARY Ciliogenesis precedes lineage-determining signaling in skin development. To understand why, we performed shRNA-mediated knockdown of seven intraflagellar-transport-proteins (IFTs), and conditional ablation of Ift-88 and Kif3a during embryogenesis. Both in cultured keratinocytes and embryonic epidermis, all eliminated cilia, and many (not Kif3a) caused hyperproliferation. Surprisingly and independent of proliferation, ciliary-mutants displayed defects in Notch-signaling and in commitment of progenitors to differentiate. Notch-receptors and Notch-processing-enzymes colocalized specifically with cilia in wild-type epidermal cells. Moreover, differentiation defects in ciliary-mutants were cell-autonomous and rescued by activated Notch (NICD). By contrast, Shh-signaling was neither operative nor required for epidermal ciliogenesis, Notch-signaling or differentiation. Rather Shh-signaling defects in ciliary-mutants occurred later, arresting HF morphogenesis in the skin. These findings unveil temporally and spatially distinct functions for primary cilia at the nexus of signaling, proliferation and differentiation: a novel, early role in epidermis, whose morphogenesis relies upon Notch-signaling; and a later role in HFs, reliant upon Shh-signaling.