Molecular codes for cell type specification in Brn3 retinal ganglion cells.

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 (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 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.

Blister-inducing antibodies target multiple epitopes on collagen VII in mice.

Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA.

Brn3a and Brn3b knockout mice display unvaried retinal fine structure despite major morphological and numerical alterations of ganglion cells.

Ganglion cells (GCs), the retinal output neurons, receive synaptic inputs from bipolar and amacrine cells in the inner plexiform layer (IPL) and send information to the brain nuclei via the optic nerve. Although GCs constitute less than 1% of the total retinal cells, they occur in numerous types and are the first neurons formed during retinal development. Using Brn3a and Brn3b mutant mice in which the alkaline phosphatase gene was knocked-in (Badea et al. [Neuron] 2009;61:852-864; Badea and Nathans [Vision Res] 2011;51:269-279), we studied the general effects after gene removal on the retinal neuropil together with the consequences of lack of development of large numbers of GCs onto the remaining retinal neurons of the same class. We analyzed the morphology, number, and general architecture of various neuronal types presynaptic to GCs, searching for changes secondary to the decrement in the number of their postsynaptic partners, as well as the morphology and distribution of retinal astrocytes, for their strong topographical relation to GCs. We found that, despite GC losses, retinal organization in Brn3 null mice is remarkably similar to that of wild-type controls. J. Comp. Neurol. 527:187-211, 2019. © 2016 Wiley Periodicals, Inc.

Dre - Cre sequential recombination provides new tools for retinal ganglion cell labeling and manipulation in mice.

BACKGROUND: Genetic 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 FINDINGS: We generated conditional Cre knock-in alleles at the Brn3a and Brn3b loci, Brn3a(CKOCre) and Brn3b(CKOCre). When crossed to mice expressing the Dre recombinase, the endogenous Brn3 gene expressed by Brn3a(CKOCre) or Brn3b(CKOCre) is removed and replaced with a Cre recombinase, generating Brn3a(Cre) and Brn3b(Cre) knock-in alleles. Surprisingly both Brn3a(Cre) and Brn3b(Cre) 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 Brn3a(Cre) and Brn3b(Cre) 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. CONCLUSIONS: Dre recombinase effectively recombines the Brn3a(CKOCre) and Brn3b(CKOCre) alleles containing its roxP target sites. Sequential Dre to Cre recombination reveals Brn3a and Brn3b expression in early mouse development. The generated Brn3a(Cre) and Brn3b(Cre) 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.