Abstract
BackgroundWhile the transcriptional code governing retinal ganglion cell (RGC) type specification begins to be understood, its interplay with neurotrophic signaling is largely unexplored. In mice, the transcription factor Brn3a/Pou4f1 is expressed in most RGCs, and is required for the specification of RGCs with small dendritic arbors. The Glial Derived Neurotrophic Factor (GDNF) receptor Ret is expressed in a subset of RGCs, including some expressing Brn3a, but its role in RGC development is not defined.MethodsHere we use combinatorial genetic experiments using conditional knock-in reporter alleles at the Brn3a and Ret loci, in combination with retina- or Ret specific Cre drivers, to generate complete or mosaic genetic ablations of either Brn3a or Ret in RGCs. We then use sparse labelling to investigate Brn3a and Ret gene dosage effects on RGC dendritic arbor morphology. In addition, we use immunostaining and/or gene expression profiling by RNASeq to identify transcriptional targets relevant for the potential Brn3a-Ret interaction in RGC development.ResultsWe find that mosaic gene dosage manipulation of the transcription factor Brn3a/Pou4f1 in neurotrophic receptor Ret heterozygote RGCs results in altered cell fate decisions and/or morphological dendritic defects. Specific RGC types are lost if Brn3a is ablated during embryogenesis and only mildly affected by postnatal Brn3a ablation. Sparse but not complete Brn3a heterozygosity combined with complete Ret heterozygosity has striking effects on RGC type distribution. Brn3a only mildly modulates Ret transcription, while Ret knockouts exhibit slightly skewed Brn3a and Brn3b expression during development that is corrected by adult age. Brn3a loss of function modestly but significantly affects distribution of Ret co-receptors GFRα1-3, and neurotrophin receptors TrkA and TrkC in RGCs.ConclusionsBased on these observations, we propose that Brn3a and Ret converge onto developmental pathways that control RGC type specification, potentially through a competitive mechanism requiring signaling from the surrounding tissue.
Highlights
Retinal Ganglion Cells (RGCs) – the output neurons of the vertebrate retina – relay visual information to distinct projection areas in the brain
Sparse random recombination in mice carrying RetCreERt2and Brn3aCKOAPalleles induces mosaic gene dosage manipulations in RGCs We had previously demonstrated that ablation of Brn3a before the onset of its expression (E12), results in essentially complete loss of RGC types with small, dense dendritic arbors (ON and OFF β or “midget-like” RGCs), while other Brn3a+ RGC types are only modestly affected [5, 7, 93]
Ret can function as a competitive coreceptor for ligands involved in neuronal arbor formation and axon guidance, such as ephrin and p75-NTR [14] and Plexin / NCAMs [20], some of which are under transcriptional control of Brn3a [78, 92]
Summary
Retinal Ganglion Cells (RGCs) – the output neurons of the vertebrate retina – relay visual information to distinct projection areas in the brain. The developmental mechanisms orchestrating the differentiation of RGC types involve transcription factors. Downstream of Atoh, postmitotic TFs determine general traits of neuronal morphology and functional characteristics – in RGCs this group includes the three members of the Pou4f family, namely Pou4f1/Brn3a, Pou4f2/Brn3b, and Pou4f3/Brn3c [5, 7, 35, 38, 93, 105,106,107]. While the transcriptional code governing retinal ganglion cell (RGC) type specification begins to be understood, its interplay with neurotrophic signaling is largely unexplored. The transcription factor Brn3a/ Pou4f1 is expressed in most RGCs, and is required for the specification of RGCs with small dendritic arbors. The Glial Derived Neurotrophic Factor (GDNF) receptor Ret is expressed in a subset of RGCs, including some expressing Brn3a, but its role in RGC development is not defined
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