Essential but partially redundant roles for POU4F1/Brn-3a and POU4F2/Brn-3b transcription factors in the developing heart.

Congenital heart defects contribute to embryonic or neonatal lethality but due to the complexity of cardiac development, the molecular changes associated with such defects are not fully understood. Here, we report that transcription factors (TFs) Brn-3a (POU4F1) and Brn-3b (POU4F2) are important for normal cardiac development. Brn-3a directly represses Brn-3b promoter in cardiomyocytes and consequently Brn-3a knockout (KO) mutant hearts express increased Brn-3b mRNA during mid-gestation, which is linked to hyperplastic growth associated with elevated cyclin D1, a known Brn-3b target gene. However, during late gestation, Brn-3b can cooperate with p53 to enhance transcription of pro-apoptotic genes e.g. Bax, thereby increasing apoptosis and contribute to morphological defects such as non-compaction, ventricular wall/septal thinning and increased crypts/fissures, which may cause lethality of Brn-3a KO mutants soon after birth. Despite this, early embryonic lethality in e9.5 double KO (Brn-3a-/- : Brn-3b-/-) mutants indicate essential functions with partial redundancy during early embryogenesis. High conservation between mammals and zebrafish (ZF) Brn-3b (87%) or Brn-3a (76%) facilitated use of ZF embryos to study potential roles in developing heart. Double morphant embryos targeted with morpholino oligonucleotides to both TFs develop significant cardiac defects (looping abnormalities and valve defects) suggesting essential roles for Brn-3a and Brn-3b in developing hearts.

Pou4f2-GFP knock-in mouse line: A model for studying retinal ganglion cell development.

Pou4f2 acts as a key node in the comprehensive and step-wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2-green fluorescent protein (GFP) fusion protein expressed in RGCs. Co-localization of POU4F2 and GFP in the retina and brain of Pou4f2-GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild-type mice, the expression patterns of POU4F2 and POU4F1 and the co-expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2-GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2-GFP/GFP homozygote and wild-type mice. These results demonstrated that the development of RGCs in Pou4f2-GFP/GFP homozygote mice was the same as in wild-type mice. Thus, the present Pou4f2-GFP knock-in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.

Transient Expression of Fez Family Zinc Finger 2 Protein Regulates the Brn3b Gene in Developing Retinal Ganglion Cells.

Retinal ganglion cells (RGCs) are projection neurons in the neural retina that relay visual information from the environment to the central nervous system. The early expression of MATH5 endows the post-mitotic precursors with RGC competence and leads to the activation ofBrn3bthat marks committed RGCs. Nevertheless, this fate commitment process and, specifically, regulation ofBrn3bremain elusive. To explore the molecular mechanisms underlying RGC generation in the mouse retina, we analyzed the expression and function of Fez family zinc finger 2 (FEZF2), a transcription factor critical for the development of projection neurons in the cerebral cortex.Fezf2mRNA and protein were transiently expressed at embryonic day 16.5 in the inner neuroblast layer and the prospective ganglion cell layer of the retina, respectively. Knockout ofFezf2in the developing retina reduced BRN3B+ cells and increased apoptotic cell markers.Fezf2knockdown by retinalin uteroelectroporation diminished BRN3B but not the coexpressed ISLET1 and BRN3A, indicating that the BRN3B decrease was the cause, not the result, of the overall reduction of BRN3B+ RGCs in theFezf2knockout retina. Moreover, the mRNA and promoter activity ofBrn3bwere increasedin vitroby FEZF2, which bound to a 5' regulatory fragment in theBrn3bgenomic locus. These results indicate that transient expression ofFezf2in the retina modulates the transcription ofBrn3band the survival of RGCs. This study improves our understanding of the transcriptional cascade required for the specification of RGCs and provides novel insights into the molecular basis of retinal development.

Dynamic expression of transcription factor Brn3b during mouse cranial nerve development.

During development, transcription factor combinatorial codes define a large variety of morphologically and physiologically distinct neurons. Such a combinatorial code has been proposed for the differentiation of projection neurons of the somatic and visceral components of cranial nerves. It is possible that individual neuronal cell types are not specified by unique transcription factors but rather emerge through the intersection of their expression domains. Brn3a, Brn3b, and Brn3c, in combination with each other and/or transcription factors of other families, can define subgroups of retinal ganglion cells (RGC), spiral and vestibular ganglia, inner ear and vestibular hair cell neurons in the vestibuloacoustic system, and groups of somatosensory neurons in the dorsal root ganglia. The present study investigates the expression and potential role of the Brn3b transcription factor in cranial nerves and associated nuclei of the brainstem. We report the dynamic expression of Brn3b in the somatosensory component of cranial nerves II, V, VII, and VIII and visceromotor nuclei of nerves VII, IX, and X as well as other brainstem nuclei during different stages of development into adult stage. We find that genetically identified Brn3b(KO) RGC axons show correct but delayed pathfinding during the early stages of embryonic development. However, loss of Brn3b does not affect the anatomy of the other cranial nerves normally expressing this transcription factor.

Generation of retinal ganglion cells with functional axons from human induced pluripotent stem cells.

We generated self-induced retinal ganglion cells (RGCs) with functional axons from human induced pluripotent stem cells. After development of the optic vesicle from the induced stem cell embryoid body in three-dimensional culture, conversion to two-dimensional culture, achieved by supplementation with BDNF, resulted in differentiation of RGCs at a rate of nearly 90% as indicated by a marginal subregion of an extruded clump of cells, suggesting the formation of an optic vesicle. Axons extended radially from the margin of the clump. Induced RGCs expressed specific markers, such as Brn3b and Math5, as assessed using by quantitative PCR and immunohistochemistry. The long, prominent axons contained neurofilaments and tau and exhibited anterograde axonal transport and sodium-dependent action potentials. The ability to generate RGCs with functional axons uniformly and at a high rate may contribute to both basic and clinical science, including embryology, neurology, pathognomy, and treatment of various optic nerve diseases that threaten vision.

Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

PURPOSE: Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. METHODS: Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. RESULTS: Adeno-associated virus-mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b-injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP- injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. CONCLUSIONS: Adeno-associated virus-mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma.

The transcriptional regulator BCL6 participates in the secondary gene regulatory response to vitamin D.

The vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the high affinity ligand of the transcription factor vitamin D receptor (VDR) and therefore a direct regulator of transcription. Transcriptome-wide analysis of THP-1 human monocytes had indicated more than 600 genes to be significantly (p<0.05) stimulated after 4h incubation with 1,25(OH)2D3, but only 67 of them where more than 1.5-fold up-regulated. These include the genes encoding for the transcription factors BCL6, NFE2, POU4F2 and ELF4, which are controlled by one or two VDR binding sites within their chromosomal domains. The latter are defined via DNA loop formation mediated by the transcription factor CTCF that is highly conserved in its genome-wide loci. We found BCL6 being most responsive to 1,25(OH)2D3 and selected it for further analysis. An incubation of THP-1 cells with 1,25(OH)2D3 for 24 h resulted in a significant (p<0.001) change in the mRNA expression of more than 1600 genes, of which 132 were at least 2-fold up-regulated. About half of the latter genes are secondary 1,25(OH)2D3 targets, since they do not carry any VDR binding site within their chromosomal domain. Chromatin immunoprecipitation sequencing datasets indicated that the majority of these domains contain a BCL6 binding site. We followed the secondary transcriptional response to 1,25(OH)2D3 for eight representative gene examples and confirmed the binding of CTCF and BCL6 to their respective chromosomal domains. In conclusion, our study indicated that in monocytes most of the physiological responses to 1,25(OH)2D3 involve the action of the transcription factor BCL6.

Dynamic expression of ganglion cell markers in retinal progenitors during the terminal cell cycle.

The vertebrate neural retina contains seven major cell types, which arise from a common multipotent progenitor pool. During neurogenesis, these cells stop cycling, commit to a single fate, and differentiate. The mechanism and order of these steps remain unclear. The first-born type of retinal neurons, ganglion cells (RGCs), develop through the actions of Math5 (Atoh7), Brn3b (Pou4f2) and Islet1 (Isl1) factors, whereas inhibitory amacrine and horizontal precursors require Ptf1a for differentiation. We have examined the link between these markers, and the timing of their expression during the terminal cell cycle, by nucleoside pulse-chase analysis in the mouse retina. We show that G2 phase lasts 1-2 h at embryonic (E) 13.5 and E15.5 stages. Surprisingly, we found that cells expressing Brn3b and/or Isl1 were frequently co-labeled with EdU after a short chase (<1 h) in early embryos (E15), Brn3b and Isl1 were exclusively expressed in post-mitotic cells, even as new RGCs are still generated. In contrast, Ptf1a and amacrine marker AP2α were detected only after terminal mitosis, at all developmental stages. Using a retroviral tracer in embryonic retinal explants (E12-E13), we identified two-cell clones containing paired ganglion cells, consistent with RGC fate commitment prior to terminal mitosis. Thus, although cell cycle exit and fate determination are temporally correlated during retinal neurogenesis, the order of these events varies according to developmental stage and final cell type.

Differential expression of Brn3 transcription factors in intrinsically photosensitive retinal ganglion cells in mouse.

Several subtypes of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) have been reported. The M1 type of ipRGCs exhibit distinct properties compared with the remaining (non-M1) cells. They differ not only in their soma size and dendritic arbor, but also in their physiological properties, projection patterns, and functions. However, it is not known how these differences arise. We tested the hypothesis that M1 and non-M1 cells express Brn3 transcription factors differentially. The Brn3 family of class IV POU-domain transcription factors (Brn3a, Brn3b, and Brn3c) is involved in the regulation of differentiation, dendritic stratification, and axonal projection of retinal ganglion cells during development. By using double immunofluorescence for Brn3 transcription factors and melanopsin, and with elaborate morphometric analyses, we show in mouse retina that neither Brn3a nor Brn3c are expressed in ipRGCs. However, Brn3b is expressed in a subset of ipRGCs, particularly those with larger somas and lower melanopsin levels, suggesting that Brn3b is expressed preferentially in the non-M1 cells. By using dendritic stratification to distinguish M1 from non-M1 cells, we found that whereas nearly all non-M1 cells expressed Brn3b, a vast majority of the M1 cells were negative for Brn3b. Interestingly, in the small proportion of the M1 cells that did express Brn3b, the expression level of Brn3b was significantly lower than in the non-M1 cells. These results provide insights about how expression of specific molecules in a ganglion cell could be linked to its role in visual function.

Proliferation-associated Brn-3b transcription factor can activate cyclin D1 expression in neuroblastoma and breast cancer cells.

Brn-3b transcription factor enhances proliferation of neuroblastoma (NB) and breast cancer cell lines in vitro and increases the rate and size of in vivo tumour growth, whereas reducing Brn-3b slows growth, both in vitro and in vivo. Brn-3b is elevated in >65% of breast cancer biopsies, and here we demonstrate that Brn-3b is also elevated in NB tumours. We show a significant correlation between Brn-3b and cyclin D1 (CD1) in breast cancers and NB tumours and cell lines. Brn-3b directly transactivates the CD1 promoter in co-transfection experiments, whereas electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrate that Brn-3b protein binds to an octamer sequence located in the proximal CD1 promoter. Site-directed mutagenesis of this sequence resulted in loss of transactivation of the CD1 promoter by Brn-3b. Thus, Brn-3b may act to alter growth properties of breast cancer and NB cells by enhancing CD1 expression in these cells.

SWI/SNF chromatin remodeling ATPase Brm regulates the differentiation of early retinal stem cells/progenitors by influencing Brn3b expression and Notch signaling.

Based on a variety of approaches, evidence suggests that different cell types in the vertebrate retina are generated by multipotential progenitors in response to interactions between cell intrinsic and cell extrinsic factors. The identity of some of the cellular determinants that mediate such interactions has emerged, shedding light on mechanisms underlying cell differentiation. For example, we know now that Notch signaling mediates the influence of the microenvironment on states of commitment of the progenitors by activating transcriptional repressors. Cell intrinsic factors such as the proneural basic helix-loop-helix and homeodomain transcription factors regulate a network of genes necessary for cell differentiation and maturation. What is missing from this picture is the role of developmental chromatin remodeling in coordinating the expression of disparate classes of genes for the differentiation of retinal progenitors. Here we describe the role of Brm, an ATPase in the SWI/SNF chromatin remodeling complex, in the differentiation of retinal progenitors into retinal ganglion cells. Using the perturbation of expression and function analyses, we demonstrate that Brm promotes retinal ganglion cell differentiation by facilitating the expression and function of a key regulator of retinal ganglion cells, Brn3b, and the inhibition of Notch signaling. In addition, we demonstrate that Brm promotes cell cycle exit during retinal ganglion cell differentiation. Together, our results suggest that Brm represents one of the nexus where diverse information of cell differentiation is integrated during cell differentiation.