Molecular codes for cell type specification in PNAS PLUS Brn3 retinal ganglion cells Szilard Sajgoa,1, Miruna Georgiana Ghiniaa,2, Matthew Brooksb, Friedrich Kretschmera,3, Katherine Chuanga,4, Suja Hiriyannac, Zhijian Wuc, Octavian Popescud,e, and Tudor Constantin Badeaa,5 aRetinal Circuits Development and Genetics Unit, Neurobiology–Neurodegeneration and Repair Laboratory, National Eye Institute, Bethesda, MD 20892; bGenomics Core, Neurobiology–Neurodegeneration and Repair Laboratory, National Eye Institute, Bethesda, MD 20892; cOcular Gene Therapy Core, National Eye Institute, Bethesda, MD 20892; dInstitute of Biology, Romanian Academy, Bucharest 060031, Romania; and eMolecular Biology Center, Interdisciplinary Research Institute on Bio-Nano-Science, Babes-Bolyai University, Cluj-Napoca 400084, Romania Edited by Jeremy Nathans, Johns Hopkins University, Baltimore, MD, and approved April 12, 2017 (received for review November 8, 2016) Visual information is conveyed from the eye to the brain by Onecut2 (6, 13, 26–33). Together with Isl1 and Brn3b, these distinct types of retinal ganglion cells (RGCs). It is largely unknown downstream factors are expressed in partially overlapping patterns how RGCs acquire their defining morphological and physiological in RGC types, and some were shown to be required for survival features and connect to upstream and downstream synaptic and/or dendrite and axon formation in various RGC types. partners. The three Brn3/Pou4f transcription factors (TFs) participate However, many other TFs may be involved in generating the in a combinatorial code for RGC type specification, but their exact diversity of RGC types (34–38). We have previously used re- molecular roles are still unclear. We use deep sequencing to define porter knock-in alleles expressing alkaline phosphatase (AP; a i ii ( )transcriptomesofBrn3a-and/orBrn3b-positiveRGCs,( )Brn3a- glycosylphosphatidylinositol (GPI)-linked, extracellular molecule) iii and/or Brn3b-dependent RGC transcripts, and ( ) transcriptomes of at the loci of Brn3a, Brn3b, and Brn3c (Brn3CKOAP) to describe retinorecipient areas of the brain at developmental stages relevant their cell type distribution among RGCs and other sensory pro- for axon guidance, dendrite formation, and synaptogenesis. We re- jection neurons. We also identified axonal and dendrite arbor veal a combinatorial code of TFs, cell surface molecules, and deter- defects in RGCs missing Brn3a, Brn3b, or Brn3c either alone or in minants of neuronal morphology that is differentially expressed in BIOLOGY specific RGC populations and selectively regulated by Brn3a and/or combination (6, 13, 31, 39, 40). We now describe an immu- noaffinity purification strategy using anti-AP antibodies to isolate DEVELOPMENTAL Brn3b. This comprehensive molecular code provides a basis for un- AP derstanding neuronal cell type specification in RGCs. RGCs from Brn3 RGCs that are either WT or KO for Brn3a or Brn3b. Using our knowledge of partially overlapping RGC pop- retinal ganglion cells | transcription factors | neuronal cell types | Pou4f1 | ulations expressing Brn3s, we can identify molecules selectively Pou4f2 enriched in RGCs, selectively expressed in distinct Brn3 RGC subpopulations, and/or regulated by Brn3a or Brn3b in these RGC he molecular analysis of neuronal circuits benefits signifi- Tcantly from modern approaches to gene expression profiling Significance and genetic manipulation. The mechanisms of cell type specification are still poorly understood, but experiments in model organisms We report here transcriptome analysis by RNA sequencing suggest a combination of transcriptional regulation, extracellular (RNASeq) of genetically labeled and affinity-purified mouse signals, and cell–cell interactions (1–4). Retinal ganglion cells retinal ganglion cell (RGC) populations. Using a previously (RGCs) are a particularly powerful system for illustrating the established conditional knock-in reporter strategy, we label molecular and activity-dependent mechanisms of cell type speci- RGCs from which specific transcription factors have been re- fication. Based on molecular markers, dendritic arbor morphol- moved and determine the consequences on transcriptional ogies, axonal projections to retinorecipient areas of the brain, programs at different stages critical to RGC development. We – synaptic partners, physiological properties, and roles within the find that Brn3b and Brn3a control only small subsets of Brn3 – visual circuit, mouse RGCs can be cataloged in 20–30 different RGC specific transcripts. We identify extensive combinatorial types (5–10). Some of the developmental mechanisms by which sets of RGC transcription factors and cell surface molecules and RGC features are combined to determine RGC types are begin- show that several RGC-specific genes can induce neurite-like ning to be uncovered. Mouse RGCs become postmitotic and start processes cell autonomously in a heterologous system. exhibiting specific molecular markers and morphological features Author contributions: S.S., M.G.G., F.K., K.C., O.P., and T.C.B. designed research; S.S., M.G.G., around embryonic day 11 (E11). As soon as E12, RGC axons cross M.B., F.K., K.C., and T.C.B. performed research; S.H. and Z.W. contributed new reagents/ the midline at the optic chiasm, and by E15, the first axons have analytic tools; S.S., M.G.G., M.B., and T.C.B. analyzed data; and S.S., M.G.G., O.P., and T.C.B. reached the superior colliculus (SC), the most remote retinor- wrote the paper. ecipient area of the brain (11, 12). RGC axons invade their target The authors declare no conflict of interest. nuclei only around birth, and the first 10 postnatal days are the This article is a PNAS Direct Submission. most active period for synapse formation. RGC dendritic arbors Data deposition: The next generation sequencing data reported in this paper have been develop mostly postnatally, with lamination within the inner deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih. gov/geo (accession no. GSE87647). plexyform layer clearly visible at postnatal days 3–4(P3–P4) and – 1Present address: Yonehara Laboratory, Danish Research Institute of Translational Neu- reaching a nearly mature distribution by P7 (13 16). Combinato- roscience, Aarhus University, 8000 Aarhus, Denmark. rial transcriptional regulation may play a major role in RGC type 2Present address: Emerson Laboratory, Biology Department, The City College of New specification. Previous work suggests the following transcriptional York, New York, NY 10031. – – cascade: the basic helix loop helix (bHLH) transcription factor 3Present address: Scientific Computing Core, Max Planck Institute for Brain Research, (TF) Atoh7 is expressed in RGC precursors and controls the ex- Frankfurt am Main 60438, Germany. pression of the POU4 family TF Brn3b and the Lim domain TF 4Present address: School of Medicine, Yale University, New Haven, CT 06510. Isl1, which are both required for the initiation of the RGC tran- 5To whom correspondence should be addressed. Email: [email protected]. – scriptional program (17 25). Further downstream TFs include This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Brn3a, Brn3c, Eomesodesmin (Tbr2), Ebf1, Ebf3, Onecut1, and 1073/pnas.1618551114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1618551114 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 KO). When paired with a WT allele, recombination results in AP- A C i) Genes enriched in RGCs: tagged heterozygote cells that are phenotypically WT (Brn3aAP/WT or Brn3bAP/WT RGCs; WT). Although recombination happens E15 Brn3AP/WT RGCs vs. Other (supernatant) throughout the retina, other retinal cell types do not express Axon Guidance either Brn3a or Brn3b and therefore, appear AP-negative (6, 13, ii) Brn3-dependent RGC Genes: 18, 21, 25, 31, 41). Using this genetic labeling strategy, we can B compare several cell populations (Fig. 1C). (i) Comparing the Brn3AP/WT RGCs vs. Brn3AP/KO RGCs expression profiles of Brn3AP/WT RGCs and retinal supernatants, we can identify genes specific for or enriched in RGCs. (ii) RGC P3 iii) Genes Specific for distinct RGC populations genes regulated by a Brn3 TF should be differentially expressed Dendrite Formation in Brn3AP/WT vs. Brn3AP/KO RGCs. (iii) Genes specific for Axon - target interaction + − − + + + Synapse formation Brn3aAP/WT RGCsvs. Brn3bAP/WT RGCs Brn3a Brn3b , Brn3a Brn3b , or Brn3a Brn3b RGC pop- ulations can be identified by comparing expression profiles of D F iii AP/WT AP/WT Supernat Brn3a with Brn3b RGCs. We dissociated retinas + from Pax6α:Cre;Brn3aCKOAP/WT,Pax6α:Cre;Brn3aCKOAP/KO, RGC Pax6α:Cre;Brn3bCKOAP/WT, and Pax6α:Cre;Brn3bCKOAP/KO mice Brn3AP αAP AP Other cells and isolated the AP-expressing Brn3 RGCs using magnetic RGCs Beads iii iv beads coupled to anti-AP mouse mAbs (Materials and Methods E ChTB P 3.5 and Fig. 1 D and F). We also have labeled the lateral geniculate P 0.5 nucleus (LGN), SC, and pretectal area (PTA) of P3 WT mice by anterograde tracing and dissected and processed them for deep sequencing (Materials and Methods and Fig. 1E). In the following, we will present gene expression data that are Fig. 1. Experimental goal and design. (A) E15 retina containing heteroge- restricted to the RefSeq (https://www.ncbi.nlm.nih.gov/refseq/) neous undifferentiated cells (gray) and RGCs (purple), which are mostly post- subset of mouse transcripts given its highest level of quality and mitotic and extend axons. (B) P3 retina