Pou4f1 and Pou4f2 Are Dispensable for the Long-Term Survival of Adult Retinal Ganglion Cells in Mice

Purpose

To investigate the role of Pou4f1 and Pou4f2 in the survival of adult retinal ganglion cells (RGCs).

Methods

Conditional alleles of Pou4f1 and Pou4f2 were generated (Pou4f1^loxP and Pou4f2^loxP respectively) for the removal of Pou4f1 and Pou4f2 in adult retinas. A tamoxifen-inducible Cre was used to delete Pou4f1 and Pou4f2 in adult mice and retinal sections and flat mounts were subjected to immunohistochemistry to confirm the deletion of both alleles and to quantify the changes in the number of RGCs and other retinal neurons. To determine the effect of loss of Pou4f1 and Pou4f2 on RGC survival after axonal injury, controlled optic nerve crush (CONC) was performed and RGC death was assessed.

Results

Pou4f1 and Pou4f2 were ablated two weeks after tamoxifen treatment. Retinal interneurons and Müller glial cells are not affected by the ablation of Pou4f1 or Pou4f2 or both. Although the deletion of both Pou4f1 and Pou4f2 slightly delays the death of RGCs at 3 days post-CONC in adult mice, it does not affect the cell death progress afterwards. Moreoever, deletion of Pou4f1 or Pou4f2 or both has no impact on the long-term viability of RGCs at up to 6 months post-tamoxifen treatment.

Conclusion

Pou4f1 and Pou4f2 are involved in the acute response to damage to RGCs but are dispensable for the long-term survival of adult RGC in mice.     

Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B‐GFP transgenic mice

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B‐GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (?540 to +31). Using the fresh brain sections of F1B‐GFP transgenic mice, we found that the F1B‐GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B‐GFP⁺ cells existed in the brains of F1B‐GFP transgenic mice. We demonstrated that one population of F1B‐GFP⁺ cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B‐GFP⁺ cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B‐GFP⁺ cells and tyrosine hydroxylase indicated that a subpopulation of F1B‐GFP⁺‐neuronal cells was dopaminergic neurons. Importantly, these F1B‐GFP⁺/TH⁺ cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B‐GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 232–248, 2015     

Developmental wave of Brn3b expression leading to RGC fate specification is synergistically maintained by miR‐23a and miR‐374

Differential regulation of Brn3b is essential for the Retinal Ganglion Cell (RGC) development in the two phases of retinal histogenesis. This biphasic Brn3b regulation is required first, during early retinal histogenesis for RGC fate specification and secondly, during late histogenesis, where Brn3b is needed for RGC axon guidance and survival. Here, we have looked into how the regulation of Brn3b at these two stages happens. We identified two miRNAs, miR‐23a and miR‐374, as regulators of Brn3b expression, during the early stage of RGC development. Temporal expression pattern of miR‐23a during E10–19, PN1–7, and adult retina revealed an inverse relation with Brn3b expression. Though miR‐374 did not show such a pattern, its co‐expression with miR‐23a evidently inhibited Brn3b. We further substantiated these findings by ex vivo overexpression of these miRNAs in E14 mice retina and found that miR‐23a and miR‐374 together brings about a change in Brn3b expression pattern in ganglion cell layer (GCL) of the developing retina. From our results, it appears that the combined expression of these miRNAs could be regulating the timing of the wave of Brn3b expression required for early ganglion cell fate specification and later for its survival and maturation into RGCs. Taken together, here we provide convincing evidences for the existence of a co‐ordinated mechanism by miRNAs to down regulate Brn3b that will ultimately regulate the development of RGCs from their precursors. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1155–1171, 2014     

Pou3f4 deficiency causes defects in otic fibrocytes and stria vascularis by different mechanisms

DFN3, the most prevalent X-linked hearing loss, is caused by mutations in the POU3F4 gene. Previous studies in Pou3f4 knockout mice suggest that defective otic fibrocytes in the spiral ligament of the cochlear lateral wall may underlie the hearing loss in DFN3. To better understand the pathological mechanisms of the DFN3 hearing loss, we analyzed inner ears of Pou3f4-deficient mice during development. Our results indicate that compartmentalization of the spiral ligament mesenchyme setting up boundaries for specific otic fibrocytes occurs normally in Pou3f4-deficient cochlea. However, differentiation of the compartmentalized mesenchyme into specific otic fibrocytes was blocked in the absence of Pou3f4 function. In addition, we found that stria vascularis in the cochlear lateral wall was also affected in Pou3f4-deficient cochlea. Unlike the otic fibrocytes, differentiation of stria vascularis was completed in the absence of Pou3f4 function, yet expression of Kir4.1 channels in the strial intermediate cells, essential for the sound transduction, was lost afterwards. These results suggest that Pou3f4 deficiency causes defects in both otic fibrocytes and stria vascularis at different developmental stages and by different pathological mechanisms, which may account for the progressive nature of DFN3 hearing loss.     

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.     

Neuroprotective effects of Argon are mediated via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1 in retinal ganglion cells

Retinal ischemia and reperfusion injuries (R‐IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti‐apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK‐1/2 dependent regulation of heat‐shock proteins. Inhalation of Argon (75 Vol%) was performed after R‐IRI on the rats′ left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat‐shock proteins ?70, ?90 and heme‐oxygenase‐1, mitogen‐activated protein kinases (p38, JNK, ERK‐1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova . Argon significantly reduced the R‐IRI‐affected heat‐shock protein expression (p < 0.05). While Argon significantly induced ERK‐1/2 expression (p < 0.001), inhibition of ERK‐1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme‐oxygenase‐1 (p < 0.05). R‐IRI‐induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK‐1/2 activation in Müller cells. We conclude, that Argon treatment protects R‐IRI‐induced apoptotic loss of RGC via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1.

     

Matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase co‐regulate axonal outgrowth of mouse retinal ganglion cells

Restoration of correct neural activity following central nervous system (CNS) damage requires the replacement of degenerated axons with newly outgrowing, functional axons. Unfortunately, spontaneous regeneration is largely lacking in the adult mammalian CNS. In order to establish successful regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc‐dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model, we were able to show that broad‐spectrum MMP inhibition reduces axon outgrowth of mouse retinal ganglion cells (RGCs), implicating MMPs as beneficial factors in axonal regeneration. Additional studies, using more specific MMP inhibitors and MMP‐deficient mice, disclosed that both MMP‐2 and MT1‐MMP, but not MMP‐9, are involved in this process. Furthermore, administration of a novel antibody to MT1‐MMP that selectively blocks pro‐MMP‐2 activation revealed a functional co‐involvement of these proteinases in determining RGC axon outgrowth. Subsequent immunostainings showed expression of both MMP‐2 and MT1‐MMP in RGC axons and glial cells. Finally, results from combined inhibition of MMP‐2 and β1‐integrin were suggestive for a functional interaction between these molecules. Overall, our data indicate MMP‐2 and MT1‐MMP as promising axonal outgrowth‐promoting molecules.

     

Expression of Pou3f3/Brn‐1 and its genomic methylation in developing auditory epithelium

In the mammalian cochlea, both the sensory cells—called hair cells (HCs)—and nonsensory cells such as supporting cells (SCs) and mesenchymal cells participate in proper auditory function through the expression of various functional molecules. During development, expression of certain genes is repressed through genomic methylation, one of the major epigenetic regulatory mechanisms. We explored the genomic regions that were differentially methylated in rat auditory epithelium at postnatal day 1 (P1) and P14 using amplification of intermethylated sites (AIMS). An AIMS fragment was mapped to the 3′‐flanking region of Pou3f3/Brn‐1. Bisulfite‐converted PCR and quantitative methylation‐specific PCR showed that the methylation frequency of the AIMS region and the adjacent CpG island was increased at P14, when the expression of Pou3f3 and the noncoding RNAs nearby decreased. Expression of de novo DNA methyltransferases 3a and 3b also suggests a role of epigenetic regulation during postnatal inner ear development. Immunohistochemical analysis showed that Pou3f3 was expressed specifically in the SCs and mesenchymal cells in the cochlea and established that Pou3f3 is a new cell‐type marker for studying inner ear development. Mice deficient in Pou3f3 or Pou3f2 plus Pou3f3 did not exhibit any abnormality in the embryonic cochlea. Absence of Pou3f3 affected neither the proliferation nor the differentiation activities of HC progenitor cells. Pou3f3 may, however, be important for the maintenance or functional development of the postnatal cochlea. This is the first report to study involvement of an epigenetic regulatory mechanism in the developing mammalian auditory epithelium. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009