Whole Number, Distribution and Co-Expression of Brn3 Transcription Factors in Retinal Ganglion Cells of Adult Albino and Pigmented Rats

Francisco M Nadal-Nicolás,Manuel Jiménez-López,Paloma Sobrado-Calvo,Manuel Salinas-Navarro,Manuel Vidal-Sanz,Juan J Alburquerque-Béjar,Marta Agudo-Barriuso,Alan R Harvey

doi:10.1371/journal.pone.0049830

Abstract

The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Brn3b and Brn3c, respectively) play, during development, essential roles in the differentiation and survival of sensory neurons. The purpose of this work is to study the expression of the three Brn3 factors in the albino and pigmented adult rat. Animals were divided into these groups: i) untouched; ii) fluorogold (FG) tracing from both superior colliculli; iii) FG-tracing from one superior colliculus; iv) intraorbital optic nerve transection or crush. All retinas were dissected as flat-mounts and subjected to single, double or triple immunohistofluorescence The total number of FG-traced, Brn3a, Brn3b, Brn3c or Brn3 expressing RGCs was automatically quantified and their spatial distribution assessed using specific routines. Brn3 factors were studied in the general RGC population, and in the intrinsically photosensitive (ip-RGCs) and ipsilateral RGC sub-populations. Our results show that: i) 70% of RGCs co- express two or three Brn3s and the remaining 30% express only Brn3a (26%) or Brn3b; ii) the most abundant Brn3 member is Brn3a followed by Brn3b and finally Brn3c; iii) Brn3 a-, b- or c- expressing RGCs are similarly distributed in the retina; iv) The vast majority of ip-RGCs do not express Brn3; v) The main difference between both rat strains was found in the population of ipsilateral-RGCs, which accounts for 4.2% and 2.5% of the total RGC population in the pigmented and albino strain, respectively. However, more ipsilateral-RGCs express Brn3 factors in the albino than in the pigmented rat; vi) RGCs that express only Brn3b and RGCs that co-express the three Brn3 members have the biggest nuclei; vii) After axonal injury the level of Brn3a expression in the surviving RGCs decreases compared to control retinas. Finally, this work strengthens the validity of Brn3a as a marker to identify and quantify rat RGCs.

Highlights

The Brn3 family of transcription factors (Brn3a/Pou4f1, Brn3b/ Pou4f2, Brn3c/Pou4f3) [1,2,3] are expressed by different sets of neurons of the trigeminal nuclei, dorsal root ganglia, inner ear and retina [4]
In the albino strain the percentage of FG+retinal ganglion cells (RGCs) that express Brn3a is 92.2% [39], while in the pigmented one this percentage goes up to 96.4%
Melanopsin and each of the Brn3 members were immunodetected in retinas from albino and pigmented animals to assess their level of co-expression (Figure 3). These results show that the vast majority of intrinsically photosensitive RGCs (ip-RGCs) do not express these transcription factors

Summary

Introduction

The Brn family of transcription factors (Brn3a/Pou4f1, Brn3b/ Pou4f2, Brn3c/Pou4f3) [1,2,3] are expressed by different sets of neurons of the trigeminal nuclei, dorsal root ganglia, inner ear and retina [4]. They are not needed for neuronal commitment but are crucial for neuronal differentiation and survival [5,6,7,8,9,10]. It has been shown that Brn3a and Brn3b are functionally equivalent, during development Brn3a can replace Brn3b function if it is expressed during the Brn3b spatiotemporal window [15,16]

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