<i>Vsx2-5.3-PRE-Cre</i> is specifically localized to Vsx2-expressing neurons of the inner nuclear layer in the mature retina.

<p>(A–C) The vast majority of Vsx2-labeled nuclei (A) in the inner nuclear layer (boundaries indicated by broken lines) co-label for Cre (B). Arrowheads in (C) indicate Vsx2-positive/Cre-negative nuclei outlined in (B). (D–F) Sox9-positive Müller glial nuclei (D) do not express Cre. Arrowheads (F) show examples of Cre-negative/Sox9-positive nuclei outlined in (E). (G–I) Horizontal cells labelled with Calbindin-D28k (G) do not label for Cre (H). Sections are from >6 week old mice. Scale bar  = 10 µm.</p

Cre expression precedes expression of the mature bipolar markers PKCα and Cabp5.

<p>At postnatal day 3 (P3) Cre-positive nuclei do not co-localize to Cabp5 (A–C), PKCα (D–F), or with the early bipolar fate marker Bhlhb5 (M–O). Insets (G–I) are high magnification view of Cre-positive (solid arrowhead) and Cre-negative (open arrowhead) nuclei located apical to the Bhlhb5-positive layer (arrow for example). Scale bar (O)  = 20 µm.</p

Summary of <i>Vsx2-5.3-PRE-Cre</i> transgenic mouse lines.

<p>*“+” indicates Cre immunolabeling restricted to postmitotic presumptive bipolar cells and/or mature retinal bipolar cells</p><p>**“+” indicates reporter expression in the mature retina in bipolar cells and a subset of photoreceptor cells</p

<i>Vsx2-5.3-PRE-Cre</i> is expressed in a large subset of bipolar neurons in the adult retina.

<p>Immunolabeling for Cre-recombinase under control by the <i>Vsx2-5.3-PRE</i> transgene is localized in nearly all PKCα expressing rod bipolar cells (A–C); a large subset of CaBp5-expressing Type-III a/b, Type-V ON, or rod bipolar cells (D–F); <i>mGlur6-lacZ</i> expressing ON bipolar cells (G–I); and a large subset of PKARIIβ-expressing Type-IIb and OFF bipolar neurons (J–L). Bhlhb5 expressing Type-II OFF bipolar cells do not express Vsx2-5.3-PRE-Cre (M–O). Arrows indicate co-localized cells; solid arrowheads indicate Cre-only cells; open arrowheads indicate Cre-negative cells. Scale bar (N)  = 10 µm.</p

<i>Vsx2-5.3-PRE-Cre</i> is up-regulated postnatally in Vsx2-expressing cells.

<p>(A–D) P3 marks the earliest age at which Vsx2-5.3-PRE-Cre can be detected by immunofluorescence. Strong, Cre-immunolabeled nuclei (A) co-label with Vsx2 (B) within the inner region of the Vsx2-positive neuroblastic layer (demarcated with dashed lines in C). Inset (D–F) shows high magnification view of examples of Cre/Vsx2 double-labeled nuclei (arrowheads) within the NBL (box in (C)). (G–I) By P6, a robust up-regulation of Vsx2-5.3-PRE-Cre is evident in Vsx2-expressing cells located throughout the NBL, although the majority of these cells localize at the apical margin of the NBL. Insets (J–L) show a high magnification view of the apical NBL. The presence of Cre-negative/Vsx2-positive nuclei located at the apical NBL boundary (arrows) may represent a newly postmitotic (G1) cell arriving to the Cre-expressing layer.</p

Summary of <i>Vsx2-5.3-PRE-Cre</i> expression in the developing retina.

<p>Although Cre immunolabeling is not evident in proliferating RPCs, genetic fate mapping identifies <i>Vsx2-5.3-PRE-Cre</i> activity in bipolar and photoreceptor cells. (A) Following bipolar cell specification, Cre immunolabeling is strongly up-regulated in all bipolar neuron subtypes, with the exception of blhlb5-positive Type 2 OFF cells. TdTomato-positive photoreceptors could be derived from postmitotic bipolar cell/photoreceptor cell precursors. (B) Alternatively, TdTomato expression may result from transient low-level Cre expression in photoreceptor cells derived from either RPCs, or from uncommitted bipolar cell precursors that switch their fate to that of photoreceptor cells.</p

List of Antibodies.

<p>List of Antibodies.</p

Genetic fate mapping of the <i>Vsx2-5.3-PRE-Cre</i> transgene using tdTomato conditional reporter mice.

<p><i>Vsx2-5.3-PRE-Cre</i> mice were crossed with mice containing the <i>Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}</i> transgene. Offspring in which Cre-mediated recombination events were present continually express tdTomato in those cells. (A–C) The vast majority of tdTomato-expressing cells in <i>Vsx2-5.3-PRE-Cre/CAG:cond-tdTomato</i> mice were located in the outer portion of the inner nuclear layer. The morphology of these cells was consistent with that of a bipolar fate, including terminal lamination in the inner plexiform layer (bracketed region). Immunolabeling for Cre revealed tdTomato-expressing cells in which Cre expression does not persist in the adult (open arrowheads). A second population of tdTomato expressing cells is also evident in the outer nuclear layer, and bear morphological resemblance to photoreceptors (arrows).</p

<i>Vsx2-5.3-PRE-Cre</i> expression in Type 2 bipolar cells and photoreceptors of the mature retina indicated by tdTomato conditional reporter expression.

<p>TdTomato fluorescence in >6 weeks old <i>Vsx2-5.3-PRE-Cre</i> mice harbouring the <i>Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}</i> transgene did not co-label Type 2 bipolar cells immunolabeled for recoverin (A–D, arrow) or photoreceptors that were co-labeled for recoverin (E–H, arrows). The asterisk in (E) indicates a region of the tdT-fluorescing cell with photoreceptor outer segment morphology. The dashed lines in (B) and (H) indicate the boundaries of the inner nuclear layer.</p

<i>Vsx2-5.3-PRE-Cre</i> is up-regulated postmitotically, and corresponds to early bipolar cell differentiation.

<p>(A–C) Retinas pulsed with CldU at P4 showed examples of Cre/CldU co-localization (solid arrowheads) at 36 h. This is in contrast to only rare examples detected at 24 h (not shown). The presence of adjacent, Cre-positive/CldU-negative nuclei (open arrowheads) indicate that the timing of Cre onset of expression is tightly regulated. (D–F) By 48 h, Cre/CldU cells were clearly evident at high frequency (G–O).</p