Background We are interested in the development and function of neuronal circuits. Our approach consists of generating genetically modified mouse lines, in which specific genes are ablated and replaced by histochemical reporters that allow the visualization of the individual cells in which the gene manipulation has been carried out. This manipulation allows us to describe the anatomy, and developmental history of the manipulated neuronal populations, in the context of either wild type or mutant gene dosages, and thereby gain insights both about the function of the cell within the circuit and the gene within the cell. Results a) We have generated Brn3bKO/KO;Brn3aKO/AP, Brn3cKO/KO;Brn3aKO/AP and Brn3bKO/KO;Brn3cKO/AP double knock-outs, in which RGCs were labeled respectively by Brn3aAP, Brn3aAP or Brn3cAP, and studied the effects of deleting each Brn3 alone or in combination on the selected Brn3AP expressing population. The first dramatic finding is that many Brn3aAP RGCs survive in the absence of Brn3b, however combined loss of Brn3a and Brn3b results in an almost complete depletion of Brn3aAP RGCs, and RGCs in general. This finding strongly argues for distinct roles of Brn3a and Brn3b in RGC development. We also find a a reduction in Brn3cAP RGCs, in Brn3bKO retinas, coupled with a distinct and very specific deletion of the OFF morphology of Brn3cAP RGCs, and an overall increase in dendritic arbor area increase in surviving Brn3cAP RGCs. This is consistent with the fact that the OFF Brn3cAP RGC cell type is the only one also positive for Brn3b. No genetic interactions were observed between Brn3b and Brn3c or Brn3a and Brn3c with respect to RGC development, as double knock-outs of the two pairs of transcription factors do not exhibit distinct phenotypes when compared respectively to the Brn3b or Brn3a single knock-outs. In addition, we have identified Isl1 as the transcription factor that is responsible for Melanopsin expression in intrinsically photosensitive RGCs. This study, carried out by former lab members M. Shi, S.R. Kumar, O. Motajo and T. Badea, with computational support form F. Kretschmer, and in collaboration with X. Mu is published in PLOS ONe b) To understand the developmental history of our cells, we are using sparse labeling strategy to label early (Embryonic 12-15) stages in RGC development. We can thus study the trajectories of individual Brn3AP RGC axons as they progress through the various decision points inside and outside the brain (intraretinal, optic disc, optic stalk, optic chiasm and tract, diencephalon, pretectal area, optic tectum), under wild type and mutant circumstances. In addition, we have observed expression of Brn3b in many of the central targets of sensory projection neurons: - Superior Colliculus for RGCs, - nucleus of cranial nerve V for the Trigeminal Ganglion, - auditory and vestibular nuclei for the Spiral and Scarpa ganglia, and dorsal horn of the spinal chord for the DRG. We find that nuclei of the V, VII, IX, and X cranial nerves are positive for Brn3b at the ages we are investigating. These findings further strengthen the link between Brn3 transcription factors and the major sensory pathways, suggesting a marking of the ascending sensory pathway by Brn3s. We have validated the identity of the various nuclei with molecular markers, and describing the peripheral projections of these neurons. This project is being carried out by S Sajgo and T Badea. This work is a manuscript under revision. In addition, we have participated in a study describing the segregation of the ascending sensory pathways of mechano and proprioceptors in the spinal chord, carried out in the lab of Wenqin Luo at U. Penn, published in J. of Neuroscience. c) To understand the molecular requirements for RGC cell type determination, we have conducted a screen for genes specific for RGCs expressing Brn3a, Brn3b or Brn3c at ages relevant for neuronal arbor formation (E15 for axon guidance and P3 for dendrite formation and synapse formation). This is done by magnetic separation of Brn3AP RGCs, using monoclonal antibodies against AP, a GPI linked surface molecule, followed by RNA extraction and RNA sequencing in the N-NRL genomics core. Data sets for Brn3a and Brn3b RGCs at P3 and Brn3b at E15 have already been generated, and an impressive set of candidate genes with functions relevant to cell morphology development identified. We have now validated by in situ hybridization 265 candidate genes from the collection generated through our data analysis. We have generated Cre dependent AAV constructs which will allow us the expression of these genes in RGCs expressing Brn3a or Brn3b (see below), and tested them in tissue culture. We are now generating and testing viral vectors based on these constructs, in tissue culture and in vivo. This project is carried out by S. Sajgo, M. Ghinia, B. Wu, K. Chunag N. Parmhans and T. Badea, in collaboration with Matthew Brooks in the genomics core of the N-NRL. d) We are expanding our genetic toolbox by generating mouse lines dependent on Dre recombination. Dre recombinase is an equally effective homologue of Cre recombinase, but operating on distinct molecular target sites. We have generated two Dre dependent conditional knock-in Cre expressing mouse lines. In these lines, named Brn3aCKOCre and Brn3bCKOCre, the Brn3a or Brn3b genes are flanked by Dre recombinase target sites (roxP sites), and followed by the Cre recombinase. When exposed to Dre recombinase activity, the endogenous genes are removed, and replaced by the Cre recombinase, resulting in conditionally knocked-in Cre alleles. The Cre, now expressed specifically from either the Brn3a or Brn3b locus will activate any desired downstream target. Using these lines, we will be able to specifically remove Brn3a or Brn3c from RGCs that also express Brn3b using the Brn3bCKOCre in conjunction with the Brn3aCKOAP or Brn3cCKOAP alleles. At this point, we have generated and confirmed by southern blotting the Brn3aCKOCre and Brn3bCKOCre lines. We are now using the Brn3bCre and Brn3acre alleles in conjunction with the AAV vectors to describe and manipulate RGC development. The initial description of the targeting alleles and virus manipulations are now published in PLOS One M. Ghinia, S Sajgo, M. Shi, N. Parmhans and T. Badea have contributed to this project, with the collaboration of Lijin Dong in the transgenic core. e) We have developed a visual behavior setup which has the ability to detect head and eye movements of mice under scotopic and photopic light conditions, and are validating it by analyzing several lines of visually impaired mice. This setup constitutes a great progress, as it allows for automated detection of head movements, and constitutes a great improvement over the existing apparatus. It will be a great service to the NEI community and the overall community. Ongoing project carried out by F. Kretschmer, S. Sajgo and T. Badea in collaboration with Haohua Qian from the Visual function core of the NEI. f) To address the complexity of Brn3 overlap at different ages, we have generated antibodies against the three transcription factors in rabbits and chickens and have validated them by western blot on purified proteins. We have affinity purified and tested them in retinal sections. (project by B. Wu and T. Badea, with advise from T. Li). g) We have performed a screen to identify novel targets for the Dre recombinase which are able of self recombination, but do not interact with the wild type roxP site. We have identified several of these sites, confirmed them and tested them in prokaryotic and eukaryotic expression system. This project performed by Katherine Chuang, with assistance from Yuri Sergeev in the Ophthalmic Genetics core, is now complete and submitted for publication in NA
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