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) 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 has been carried out by S Sajgo and T Badea, with assistance from Seid Ali and in collaboration with O. Popescu of the Romanian Academy of Sciences: Sajgo S, Ali S, Popescu O, Badea TC. Dynamic expression of transcription factor Brn3b during mouse cranial nerve development. J Comp Neurol. 2015 Sep 10. doi: 10.1002/cne.23890. Epub ahead of print PubMed PMID: 26356988. b) 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, and tested them in tissue culture. We have generated and tested viral vectors based on these constructs, and are analyzing the consequences of overexpressing these genes in Brn3b positive RGCs in vivo. People contributing to this project were S. Sajgo, M. Ghinia, K. Chuang, B. Wu, and T. Badea, in collaboration with Matthew Brooks in the genomics core of the N-NRL, and a manuscript is in preparation. This project will be carried forward by Vladimir Muzyka, a newly recruited postdoctoral fellow. c) We are expanding our genetic toolbox by generating mouse lines dependent on Dre recombination. We have previously generated two Dre dependent conditional knock-in Cre expressing mouse lines. In these lines, the Brn3a or Brn3b genes are flanked by Dre recombinase target sites (roxP sites), and followed by the Cre recombinase. Upon germline Dre recombination, Brn3aCre and Brn3bCre lines have been created and demonstrated to drive Cre dependent recombination and expression of viral vectors in RGCs. We are currently using these lines to analyze the biology of candidate genes described in point b. In addition, we have performed a screen for novel roxP target sites, which are mutually exclusive to the wild type roxP. These sites allowed us to design a Dre-dependent combined inversion-excision strategy (FREX) analogous to the ones developed for the Cre-loxP system (FLEX). The discovered novel sites and the FREX strategy were validated in vitro, in bacterial recombination assays and in HEK293 cells. A manuscript is currently under review. This project was carried out by K. Chuang and E. Nguyen, with support from B. Wu, and in collaboration with Y. Sergeev (NEI) and M. Cashel (NICHD). d) 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 have validated 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. We are able to objectively and quantitatively determine and compare head and eye gain during Optomotor and Optokinetic tasks, thus making determination of visual deficits in genetically modified mice, models of retinal disease or therapy far easier. Our apparatus has allowed us to perform the first direct comparison of the two image stabilization reflexes in mice, and define their relative contributions to gaze compensation. The blueprints for building the apparatus, as well as the many software innovations associated with it have been published (Kretschemr et al 2015), and made freely available on line. We have applied this new methodology to some of the knock-out models developed in the group (Kretschmer and Badea, manuscript in preparation), as well as participated in the analysis of retinal disease (Wai Wong, Anand Swaroop, NEI) and therapy (Brian Brooks, NEI) paradigms, several manuscripts from these groups being in preparation or submitted. Kretschmer F, Sajgo S, Kretschmer V, Badea TC. A system to measure the Optokinetic and Optomotor response in mice. J Neurosci Methods. 2015 Aug 14. pii: S0165-0270(15)00288-5. doi: 10.1016/j.jneumeth.2015.08.007. Epub ahead of print PubMed PMID: 26279344. We will pursue the development of visual behavior apparatus and complement it with Multielectrode Array Recordings. This project will be carried forward by Alireza Ghahari, a newly recruited postdoctoral fellow. Significance This past year we have made significant advances in understanding the mechanisms by which Brn3 transcription factors regulate RGC and indeed sensory projection neuron development. In addition, downstream transcriptional targets of Brn3, which are potentially involved in the development of RGC types.
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