We report the retinal expression pattern of Ret, a receptor tyrosine kinase for the glial derived neurotrophic factor (GDNF) family ligands (GFLs), during development and in the adult mouse. Ret is initially expressed in retinal ganglion cells (RGCs), followed by horizontal cells (HCs) and amacrine cells (ACs), beginning with the early stages of postmitotic development. Ret expression persists in all three classes of neurons in the adult. Using RNA sequencing, immunostaining and random sparse recombination, we show that Ret is expressed in at least three distinct types of ACs, and ten types of RGCs. Using intersectional genetics, we describe the dendritic arbor morphologies of RGC types expressing Ret in combination with each of the three members of the POU4f/Brn3 family of transcription factors. Ret expression overlaps with Brn3a in 4 RGC types, with Brn3b in 5 RGC types, and with Brn3c in one RGC type, respectively. Ret RGCs project to the lateral geniculate nucleus (LGN), pretectal area (PTA) and superior colliculus (SC), and avoid the suprachiasmatic nucleus and accessory optic system. Brn3a Ret and Brn3c Ret RGCs project preferentially to contralateral retinorecipient areas, while Brn3b Ret RGCs shows minor ipsilateral projections to the olivary pretectal nucleus and the LGN. Our findings establish intersectional genetic approaches for the anatomic and developmental characterization of individual Ret RGC types. In addition, they provide necessary information for addressing the potential interplay between GDNF neurotrophic signaling and transcriptional regulation in RGC type specification.

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.

Members of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell types (RGCs), the projection sensory neuron conveying visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing Alkaline Phosphatase (AP) and intersectional genetics had identified three types of Pou4f3/Brn3c positive (Brn3c + ) RGCs. Here, we describe a novel Brn3c Cre mouse allele generated by serial Dre to Cre recombination. We use this allele to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus properties of Brn3c + RGC types. Furthermore, we explore Brn3c-expressing brain nuclei. Our analysis reveals a much larger number of Brn3c + RGCs and more diverse set of RGC types than previously reported. The majority of RGCs having expressed Brn3c during development are still Brn3c positive in the adult, and all of them express Brn3a while only about half express Brn3b. Intersection of Brn3b and Brn3c expression highlights an area of increased RGC density, similar to an area centralis, corresponding to part of the binocular field of view of the mouse. Brn3c + neurons and projections are present in multiple brain nuclei. Brn3c + RGC projections can be detected in the Lateral Geniculate Nucleus (LGN), Pretectal Area (PTA) and Superior Colliculus (SC) but also in the thalamic reticular nucleus (TRN), a visual circuit station that was not previously described to receive retinal input. Most Brn3c + neurons of the brain are confined to the pretectum and the dorsal midbrain. Amongst theses we identify a previously unknown Brn3c + subdivision of the deep mesencephalic nucleus (DpMe). Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity and midbrain cytoarchitectonic, and opens the avenue for specific characterization and manipulation of these structures.Retinal Ganglion Cells (RGCs) are projection sensory neurons that convey multiple aspects of visual information from the eye to retinorecipient areas of the brain (Ramón y Cajal, 1972). Work in many species has documented the abundant diversity among RGC types, by characterizing distinctions in dendritic arbors, responses to visual stimuli, projections to a variety of brain nuclei and circuit

Members of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell (RGCs) types, the main transducers of visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing alkaline phosphatase (AP) and intersectional genetics had identified three types of Brn3c positive (Brn3c +) RGCs. Here, we describe a novel Brn3c Cre mouse allele generated by serial Dre to Cre recombination and use it to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus response properties of Brn3c + RGC types. Furthermore, we explore brain nuclei that express Brn3c or receive input from Brn3c + neurons. Our analysis reveals a much larger number of Brn3c + RGCs and more diverse set of RGC types than previously reported. Most RGCs expressing Brn3c during development are still Brn3c positive in the adult, and all express Brn3a while only about half express Brn3b. Genetic Brn3c-Brn3b intersection reveals an area of increased RGC density, extending from dorsotemporal to ventrolateral across the retina and overlapping with the mouse binocular field of view. In addition, we report a Brn3c + RGC projection to the thalamic reticular nucleus, a visual nucleus that was not previously shown to receive retinal input. Furthermore, Brn3c + neurons highlight a previously unknown subdivision of the deep mesencephalic nucleus. Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity, and cytoarchitectonic.

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