Visual information is conveyed from the eye to the brain by distinct types of retinal ganglion cells (RGCs). It is largely unknown how RGCs acquire their defining morphological and physiological features and connect to upstream and downstream synaptic partners. The three Brn3/Pou4f transcription factors (TFs) participate in a combinatorial code for RGC type specification, but their exact molecular roles are still unclear. We use deep sequencing to define () transcriptomes of Brn3a- and/or Brn3b-positive RGCs, () Brn3a- and/or Brn3b-dependent RGC transcripts, and () transcriptomes of retinorecipient areas of the brain at developmental stages relevant for axon guidance, dendrite formation, and synaptogenesis. We reveal a combinatorial code of TFs, cell surface molecules, and determinants of neuronal morphology that is differentially expressed in specific RGC populations and selectively regulated by Brn3a and/or Brn3b. This comprehensive molecular code provides a basis for understanding neuronal cell type specification in RGCs.

Background Visually evoked compensatory head movements (Optomotor responses) or eye movements (Optokinetic responses) are extensively used in experimental mouse models for developmental defects, pathological conditions, and testing the efficacy of therapeutic manipulations. New Method We present an automated system to measure Optomotor and Optokinetic responses under identical stimulation conditions, enabling a direct comparison of the two reflexes. A semi-automated calibration procedure and a commercial eye tracker are used to record angular eye velocity in the restrained animal. Novel video tracking algorithms determine the location of the mouse head in real time and allow repositioning of the stimulus relative to the mouse head. Results Optomotor and Optokinetic responses yield comparable results with respect to determining visual acuity in mice. Our new head tracking algorithms enable a far more accurate analysis of head angle determination, and reveal individual head retractions, analogous to saccadic eye movements observed during Optokinetic Nystagmus. Comparison with Existing Methods To our knowledge this is the first apparatus allowing the direct comparison of Optomotor and Optokinetic responses in mice. Our tracking algorithms, which allow an objective determination of head movements are a significant increment over existing systems which rely on subjective human observation. The increased accuracy of the novel algorithms increases the robustness of automated Optomotor response determinations and reveals novel aspects of this reflex. Conclusions We provide the blueprints for inexpensive hardware, and release open source software for our system, and describe an accurate and accessible method for Optomotor and Optokinetic response determination in mice.

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.