Dre - Cre Sequential Recombination Provides New Tools for Retinal Ganglion Cell Labeling and Manipulation in Mice

Abstract

BackgroundGenetic targeting methods have greatly advanced our understanding of many of the 20 Retinal Ganglion Cell (RGC) types conveying visual information from the eyes to the brain. However, the complexity and partial overlap of gene expression patterns in RGCs call for genetic intersectional or sparse labeling strategies. Loci carrying the Cre recombinase in conjunction with conditional knock-out, reporter or other genetic tools can be used for targeted cell type ablation and functional manipulation of specific cell populations. The three members of the Pou4f family of transcription factors, Brn3a, Brn3b and Brn3c, expressed early during RGC development and in combinatorial pattern amongst RGC types are excellent candidates for such gene manipulations.Methods and FindingsWe generated conditional Cre knock-in alleles at the Brn3a and Brn3b loci, Brn3aCKOCre and Brn3bCKOCre. When crossed to mice expressing the Dre recombinase, the endogenous Brn3 gene expressed by Brn3aCKOCre or Brn3bCKOCre is removed and replaced with a Cre recombinase, generating Brn3aCre and Brn3bCre knock-in alleles. Surprisingly both Brn3aCre and Brn3bCre knock-in alleles induce early ubiquitous recombination, consistent with germline expression. However in later stages of development, their expression is limited to the expected endogenous pattern of the Brn3a and Brn3b genes. We use the Brn3aCre and Brn3bCre alleles to target a Cre dependent Adeno Associated Virus (AAV) reporter to RGCs and demonstrate its use in morphological characterization, early postnatal gene delivery and tracing the expression of Brn3 genes in RGCs.ConclusionsDre recombinase effectively recombines the Brn3aCKOCre and Brn3bCKOCre alleles containing its roxP target sites. Sequential Dre to Cre recombination reveals Brn3a and Brn3b expression in early mouse development. The generated Brn3aCre and Brn3bCre alleles are useful tools that can target exogenously delivered Cre dependent reagents to RGCs in early postnatal development, opening up a large range of potential applications.

Identification of Retinal Ganglion Cell Types and Brain Nuclei expressing the transcription factor Brn3c/Pou4f3 using a Cre recombinase knock-in allele

“…Both Brn3c CKOCre (in CAG:Dre; Brn3c CKOCre ; Rosa26 AP mice) and Brn3c Cre in Brn3c Cre ; Rosa26 tdTomato mice) drive Cre expression almost exclusively in Brn3c + cells (~91% of AP positive cells are Brn3c + - Figure 2m1, p1, and ~ 99 % of tdTomato positive cells are Brn3c + Figure 3e, g). As previously reported (Anastassiadis et al, 2009;Sajgo et al, 2014), Dre is not inducing any recombination of the Cre reporter Rosa26 AP as both retinas (Figure 2d, j) and brains (Figure 8a-a4) of CAG:Dre; Rosa26 AP mice are completely devoid of AP staining. The Cre recombinase expression correctly reproduces the expression of Brn3c, either after inversion-excision mediated by Dre ( Figure 1h), or in constitutive knock-in configuration (Figure 1i).…”

“…The same retinorecipient areas labeling is observed in the CAG:Dre; Brn3c CKOCre/WT ; ROSA26 AP/WT brains (Figure 8 b-b4), however several other thalamic and brainstem areas are stained, presumably reflecting extraretinal expression of Brn3c (see below). No AP staining was seen in CAG:Dre; ROSA26 AP/WT brains, further confirming that there is no cross-reactivity between the Dre recombinase and the loxP target sites (Anastassiadis et al, 2009;Sajgo et al, 2014) (Figure 8a-a4). We note that significant amounts of AP staining are seen in Brn3c CKOCre/WT ; ROSA26 AP/WT brains, further suggesting that the Cre recombinase is also transcribed/translated in the inverted configuration (Fuller and Badea, data not shown).…”

Ipsilateral and Contralateral Retinal Ganglion Cells Express Distinct Genes during Decussation at the Optic Chiasm

“…The vertebrate retina, with its three cellular layers and six neuronal classes, has been a useful model for studying general principles of neurogenesis and axon guidance. Each class of retinal cells can be further divided into morphologically and functionally distinct subtypes, and recent efforts have identified the molecular programs that establish these differences within neuronal classes, such as amacrine, bipolar, and retinal ganglion cell (RGC) subtypes (Kim et al, 2008; Badea et al, 2009; Kay et al, 2011a, 2011b; Watson et al, 2012; Jiang et al, 2013; Sajgo et al, 2014; Macosko et al, 2015; Osterhout et al, 2015; Sanes and Masland, 2015; Tang et al, 2015; Jin et al, 2015; Rousso et al, 2016; Shekhar et al, 2016). RGCs, as the only projection neurons of the retina, can be additionally distinguished by the laterality of their axonal projection to targets in the thalamus and midbrain.…”

Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies

“…It is a close homolog of Cre, but has similar recombination efficiency and specificity for its own target sites, while exhibiting no cross-reactivity with the other well established recombinases, Cre and Flp. Several mouse and zebrafish models have been developed and validated demonstrating both the feasibility of Dre recombination in genetic manipulations and its use in combinatorial approaches with other recombinases ( Anastassiadis et al 2009 ; Park and Leach 2013 ; Fenno et al 2014 ; Sajgo et al 2014 ; Madisen et al 2015 ). Some of these studies have reported a moderate level of cross-talk between Dre and Cre, especially in the context of viral delivery of the recombinases and targets.…”