Molecular codes for cell type specification in Brn3 Retinal Ganglion Cells. Mus musculus

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 the combinatorial code for RGC type specification but their exact molecular roles are still unclear. We use deep sequencing to define (i) transcriptomes of Brn3a and/or Brn3b positive RGCs, (ii) Brn3a and/or Brn3b dependent RGC transcripts and (iii) transcriptomes of retinorecipient areas of the brain at developmental stages relevant for axon guidance, dendrite formation and synaptogenesis. We reveal a combinatorial code of transcription factors, adhesion molecules and determinants of neuronal morphology that are 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. Overall design: For retinal ganglion cells (RGCs) alkaline phosphatase (AP) positive and negative cells from Brn3aAP/WT, Brn3aAP/KO, Brn3bAP/WT, and Brn3bAP/KO from dissociated mouse retina at embryonic day 15 and post-natal day 3 were isolated by magnetic beads coupled to anti-AP mouse monoclonal antibodies. Lateral geniculate nucleus (LGN), superior colliculus (SC) and pretectal area (PTA) retinorecipient nuclei from wild type post-natal day 3 mice were visualized by anterograde tracing, microdissected, and processed for RNA isolation. Total RNA was extracted by Qiagen RNeasy using on-column DNase treatment (Qiagen) and analyzed by 2100 Bioanalyzer (Agilent Technologies Genomics). High quality of total RNA (RIN: >8.0) was subjected to sequencing library construction using 20 ng of total RNA as input. Libraries were constructed using a stranded modification of the Illumina TruSeq mRNA (Brooks, et al. Meth Mol Biol 2012). Each RGC library was single-end sequenced in an independent lane of a GAIIx at a length of 76 bases. Brain samples were paired-end sequenced to 125 bases on an Illumina HiSeq2500. Fastq files were generated from reads passing chastity filter.