A Transgenic Mouse Line Expressing Cre Recombinase in Undifferentiated Postmitotic Mouse Retinal Bipolar Cell Precursors

Approaches for manipulating cell type-specific gene expression during development depend on the identification of novel genetic tools. Here, we report the generation of a transgenic mouse line that utilizes Vsx2 upstream sequences to direct Cre recombinase to developing retinal bipolar cells. In contrast to the endogenous Vsx2 expression pattern, transgene expression was not detected in proliferating retinal progenitor cells and was restricted to post-mitotic bipolar cells. Cre immunolabeling was detected in rod bipolar cells and a subset of ON and OFF cone bipolar cells. Expression was first observed at postnatal day 3 and was detectable between 24 hours and 36 hours after the last S-phase of the cell cycle. The appearance of Cre-immunolabeled cells preceded the expression of bipolar cell type-specific markers such as PKCα and Cabp5 suggesting that transgene expression is initiated prior to terminal differentiation. In the presence of a constitutive conditional reporter transgene, reporter fluorescence was detected in Cre-expressing bipolar cells in the mature retina as expected, but was also observed in Cre-negative Type 2 bipolar cells and occasionally in Cre-negative photoreceptor cells. Together these findings reveal a new transgenic tool for directing gene expression to post-mitotic retinal precursors that are mostly committed to a bipolar cell fate

<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> 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

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

Vsx1 and Chx10 paralogs sequentially secure V2 interneuron identity during spinal cord development.

Paralog factors are usually described as consolidating biological systems by displaying redundant functionality in the same cells. Here, we report that paralogs can also cooperate in distinct cell populations at successive stages of differentiation. In mouse embryonic spinal cord, motor neurons and V2 interneurons differentiate from adjacent progenitor domains that share identical developmental determinants. Therefore, additional strategies secure respective cell fate. In particular, Hb9 promotes motor neuron identity while inhibiting V2 differentiation, whereas Chx10 stimulates V2a differentiation while repressing motor neuron fate. However, Chx10 is not present at the onset of V2 differentiation and in other V2 populations. In the present study, we show that Vsx1, the single paralog of Chx10, which is produced earlier than Chx10 in V2 precursors, can inhibit motor neuron differentiation and promote V2 interneuron production. However, the single absence of Vsx1 does not impact on V2 fate consolidation, suggesting that lack of Vsx1 may be compensated by other factors. Nevertheless, Vsx1 cooperates with Chx10 to prevent motor neuron differentiation in early V2 precursors although these two paralog factors are not produced in the same cells. Hence, this study uncovers an original situation, namely labor division, wherein paralog genes cooperate at successive steps of neuronal development

<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