Fig. 7. The effect of Numb overexpression on clonal development in newborn rat retinal explants. (A) Immunostaining of Numb in the newborn rat retina using polyclonal antibodies (Upstate Biotech). Punctate Numb staining is concentrated at the apical side of both interphase and mitotic RNECs (arrow in inset). (B) Control retinal section, stained as in A but without the primary anti-Numb antibodies. (C) The retroviral vector encoding Numb and alkaline phosphatase. The full-length open-reading frame for Numb was cloned in front of an internal ribosome entry site (IRES), which is upstream of the coding sequence for placental alkaline phosphatase (PLAP). The bicistronic mRNA encoding Numb and PLAP is transcribed from the Xenopus EF1 promoter. (D-G) Examples of clones in frozen sections of a newborn retinal explant infected with a control retrovirus encoding alkaline phosphatase (PLAP) alone and cultured for 10 days. Four clones containing a photoreceptor (D), an amacrine cell (E), a bipolar cell (F) and a Müller cell (G) are shown. (H) Composite of a z-series of confocal images showing Numb immunostaining in a frozen section of a newborn retinal explant infected with the Numb-expressing retrovirus. There is strong Numb staining throughout the two interphase RNECs shown. This contrasts with the apical concentration of endogenous Numb in nontransfected RNECs, shown in A. (I,J) Wholemounts of newborn retinal explants 10 days after infection with either the control retrovirus (I) or the Numb-expressing retrovirus (J). Note that the control explant contains a larger diversity of cell morphologies. Scale bars: 20 µm in A,B; 10 µm in D-G; 5 µm in H; 100 µm in I,J.

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