Postnatal developmental dynamics of cell type specification genes in Brn3a/Pou4f1 Retinal Ganglion Cells

Abstract

BackgroundAbout 20–30 distinct Retinal Ganglion Cell (RGC) types transmit visual information from the retina to the brain. The developmental mechanisms by which RGCs are specified are still largely unknown. Brn3a is a member of the Brn3/Pou4f transcription factor family, which contains key regulators of RGC postmitotic specification. In particular, Brn3a ablation results in the loss of RGCs with small, thick and dense dendritic arbors (‘midget-like’ RGCs), and morphological changes in other RGC subpopulations. To identify downstream molecular mechanisms underlying Brn3a effects on RGC numbers and morphology, our group recently performed a RNA deep sequencing screen for Brn3a transcriptional targets in mouse RGCs and identified 180 candidate transcripts.MethodsWe now focus on a subset of 28 candidate genes encoding potential cell type determinant proteins. We validate and further define their retinal expression profile at five postnatal developmental time points between birth and adult stage, using in situ hybridization (ISH), RT-PCR and fluorescent immunodetection (IIF).ResultsWe find that a majority of candidate genes are enriched in the ganglion cell layer during early stages of postnatal development, but dynamically change their expression profile. We also document transcript-specific expression differences for two example candidates, using RT-PCR and ISH. Brn3a dependency could be confirmed by ISH and IIF only for a fraction of our candidates.ConclusionsAmongst our candidate Brn3a target genes, a majority demonstrated ganglion cell layer specificity, however only around two thirds showed Brn3a dependency. Some were previously implicated in RGC type specification, while others have known physiological functions in RGCs. Only three genes were found to be consistently regulated by Brn3a throughout postnatal retina development – Mapk10, Tusc5 and Cdh4.Electronic supplementary materialThe online version of this article (10.1186/s13064-018-0110-0) contains supplementary material, which is available to authorized users.

Differential expression and subcellular localization of Copines in mouse retina

“…While expression profiling by Deep Sequencing is a powerful discovery tool, the huge PCR amplification involved at several stages in the process of library generation and probe detection can induce a good number of false positives. This is a well‐known fact in the field, and in fact, at least for our dataset, both Sajgo et al, and Muzyka et al, describe ISH validation rates of about 60% when compared to RNASeq results. This not uncommon for such comparisons, making the point that careful validation work is required when drawing conclusions from deep sequencing data.…”

“…But what are the Brn3‐dependent molecules that mediate the development of cell‐specific morphologies? We had previously reported combinatorial codes of transcription factors, cell surface, and intracellular molecules that are enriched in distinct RGC populations or regulated by different Brn3 transcription factors (Muzyka, Brooks, & Badea, ; Sajgo et al, ). Among these putative targets, we now focus on a protein family—Copines comprising nine members, of which six are expressed in the retina, five exhibit some level of enrichment in RGCs at early postnatal ages, and one (Cpne4) appears to be RGC specific.…”

“…At least in one subtype of these RGCs‐J‐RGCs, Tbr1 mediated regulation of cell adhesion molecules Cdh8 and Sorcs3 in these cells (Liu, Reggiani, et al, ). Brn3a similarly has been shown to regulate several types of molecules including cell adhesion molecules, cytoskeletal proteins, and others (Muzyka et al, ), some of which could potentially be important for specific morphologies of the Brn3a‐positive RGCs. If Brn3b regulated Cpne4 can play a role in determining the morphologies of the RGCs during development remains to be studied.…”

Genetic Interplay Between Transcription Factor Pou4f1/Brn3a and Neurotrophin Receptor Ret In Retinal Ganglion Cell Type Specification

“…Four of the five cell types which are unique to the Ret CreERt2/WT ; Brn3a AP/WT RGC population induced at E15 (ONS, M5, AT1, AT2), were never previously observed in sparsely labelled Brn3a AP/WT RGCs 2,14,17,51 . In addition, the ON dendritic arbors of these 4 cell types are laminated within the IPL in close proximity to the GCL, a sublamina that is typically not labelled when dendritic arbors of the entire Brn3a AP/WT RGC population are labelled by E9.5 recombination ( 14,17,23 and Figure 7a). The fifth, ON-DS, is, based on dendritic arbor areas and lamination, most likely the ON-DS RGC, which is known to occasionally have smaller branches co-stratifying with the OFF ChAT band, and has been recorded in early embryonic induced samples ( 2,14,17 , Figure 4 e-e3, Figure 5 left side).…”

“…This reduction is mostly explained by a loss of RGCs with small dendritic arbor areas and dense multistratified lamination pattern -ON and OFF  RGCs. RNA deep sequencing of affinity-purified early postnatal Brn3a AP/KO (effectively Brn3a null = Brn3a KO/KO ) RGCs revealed potential transcriptional targets for Brn3a regulation of cell type development 9,23 . However, at what developmental stage Brn3a is necessary for specification of ON and OFF  RGCs still remains unexplored.…”