MAB1585 Sigma-AldrichAnti-Brn-3a Antibody, POU-domain protein, clone 5A3.2

Retinogenesis is a precisely controlled developmental process during which different types of neurons and glial cells are generated under the influence of intrinsic and extrinsic factors. Three transcription factors, Foxn4, RORβ1 and their downstream effector Ptf1a, have been shown to be indispensable intrinsic regulators for the differentiation of amacrine and horizontal cells. At present, however, it is unclear how Ptf1a specifies these two cell fates from competent retinal precursors. Here, through combined bioinformatic, molecular and genetic approaches in mouse retinas, we identify the Tfap2a and Tfap2b transcription factors as two major downstream effectors of Ptf1a. RNA-seq and immunolabeling analyses show that the expression of Tfap2a and 2b transcripts and proteins is dramatically downregulated in the Ptf1a null mutant retina. Their overexpression is capable of promoting the differentiation of glycinergic and GABAergic amacrine cells at the expense of photoreceptors much as misexpressed Ptf1a is, whereas their simultaneous knockdown has the opposite effect. Given the demonstrated requirement for Tfap2a and 2b in horizontal cell differentiation, our study thus defines a Foxn4/RORβ1-Ptf1a-Tfap2a/2b transcriptional regulatory cascade that underlies the competence, specification and differentiation of amacrine and horizontal cells during retinal development.

We investigated the progressive degeneration of retinal and superior collicular functions in a mouse model of sustained ocular hypertension.Focal laser illumination and injection of polystyrene microbeads were used to induce chronic ocular hypertension. Retinal ganglion cell (RGC) loss was characterized by in vivo optical coherence tomography (OCT) and immunohistochemistry. Retinal dysfunction was also monitored by the full-field ERG. Retinal ganglion cell light responses were recorded using a 256-channel multielectrode array (MEA), and RGC subtypes were characterized by noncentered spike-triggered covariance (STC-NC) analysis. Single-unit extracellular recordings from superficial layers of the superior colliculus (SC) were performed to examine the receptive field (RF) properties of SC neurons.The elevation of intraocular pressure (IOP) lasted 4 months in mice treated with a combination of laser photocoagulation and microbead injection. Progressive RGC loss and functional degeneration were confirmed in ocular hypertensive (OHT) mice. These mice had fewer visually responsive RGCs than controls. Using the STC-NC analysis, we classified RGCs into ON, OFF, and ON-OFF functional subtypes. We showed that ON and OFF RGCs were more susceptible to the IOP elevation than ON-OFF RGCs. Furthermore, SC neurons of OHT mice had weakened responses to visual stimulation and exhibited mismatched ON and OFF subfields and irregular RF structure.We demonstrated that the functional degeneration of RGCs is subtype-dependent and that the ON and OFF pathways from the retina to the SC were disrupted. Our study provides a foundation to investigate the mechanisms underlying the progressive vision loss in experimental glaucoma.

We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNPIGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNPTD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNPIGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma.

Humans and mice detect pain, itch, temperature, pressure, stretch and limb position via signaling from peripheral sensory neurons. These neurons are divided into three functional classes (nociceptors/pruritoceptors, mechanoreceptors and proprioceptors) that are distinguished by their selective expression of TrkA, TrkB or TrkC receptors, respectively. We found that transiently coexpressing Brn3a with either Ngn1 or Ngn2 selectively reprogrammed human and mouse fibroblasts to acquire key properties of these three classes of sensory neurons. These induced sensory neurons (iSNs) were electrically active, exhibited distinct sensory neuron morphologies and matched the characteristic gene expression patterns of endogenous sensory neurons, including selective expression of Trk receptors. In addition, we found that calcium-imaging assays could identify subsets of iSNs that selectively responded to diverse ligands known to activate itch- and pain-sensing neurons. These results offer a simple and rapid means for producing genetically diverse human sensory neurons suitable for drug screening and mechanistic studies.

Rac1 is a critical regulator of cytoskeletal dynamics in multiple cell types. In the nervous system, it has been implicated in the control of cell proliferation, neuronal migration, and axon development.To systematically investigate the role of Rac1 in axon growth and guidance in the developing nervous system, we have examined the phenotypes associated with deleting Rac1 in the embryonic mouse forebrain, in cranial and spinal motor neurons, in cranial sensory and dorsal root ganglion neurons, and in the retina. We observe a widespread requirement for Rac1 in axon growth and guidance and a cell-autonomous defect in axon growth in Rac1 (-/-) motor neurons in culture. Neuronal death, presumably a secondary consequence of the axon growth and/or guidance defects, was observed in multiple locations. Following deletion of Rac1 in the forebrain, thalamocortical axons were misrouted inferiorly, with the majority projecting to the contralateral thalamus and a minority projecting ipsilaterally to the ventral cortex, a pattern of misrouting that is indistinguishable from the pattern previously observed in Frizzled3 (-/-) and Celsr3 (-/-) forebrains. In the limbs, motor-neuron-specific deletion of Rac1 produced a distinctive stalling of axons within the dorsal nerve of the hindlimb but a much milder loss of axons in the ventral hindlimb and forelimb nerves, a pattern that is virtually identical to the one previously observed in Frizzled3 (-/-) limbs.The similarities in axon growth and guidance phenotypes caused by Rac1, Frizzled3, and Celsr3 loss-of-function mutations suggest a mechanistic connection between tissue polarity/planar cell polarity signaling and Rac1-dependent cytoskeletal regulation.

Topoisomerase IIbeta (Top2b) is an enzyme that modulates DNA supercoiling by catalyzing the passage of DNA duplexes through one another. It is ubiquitously expressed in postmitotic cells and known to function during the development of neuromuscular junctions in the diaphragm and the proper formation of laminar structure in the cerebral cortex. However, due to the perinatal death phenotype of the traditional constitutive and brain-specific Top2b knockout mice, the precise in vivo function of Top2b, especially during postnatal neural development, remains to be determined. Using both the constitutive and retina-specific knockout mouse models, we showed that Top2b deficiency resulted in delayed neuronal differentiation, degeneration of the plexiform layers and outer segment of photoreceptors, as well as dramatic reduction in cell number in the retina. Genome-wide transcriptome analysis by RNA sequencing revealed that genes involved in neuronal survival and neural system development were preferentially affected in Top2b-deficient retinas. Collectively, our findings have indicated an important function of Top2b in proper development and the maintenance/survival of postmitotic neurons in the retina.

Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations.

The Rgcs1 quantitative trait locus, on mouse chromosome 5, influences susceptibility of retinal ganglion cells to acute damage of the optic nerve. Normally resistant mice (DBA/2J) congenic for the susceptible allele from BALB/cByJ mice exhibit susceptibility to ganglion cells, not only in acute optic nerve crush, but also to chronic inherited glaucoma that is characteristic of the DBA/2J strain as they age. SNP mapping of this QTL has narrowed the region of interest to 1 Mb. In this region, a single gene (Spink2) is the most likely candidate for this effect. Spink2 is expressed in retinal ganglion cells and is increased after optic nerve damage. This gene is also polymorphic between resistant and susceptible strains, containing a single conserved amino acid change (threonine to serine) and a 220 bp deletion in intron 1 that may quantitatively alter endogenous expression levels between strains. Overexpression of the different variants of Spink2 in D407 tissue culture cells also increases their susceptibility to the apoptosis-inducing agent staurosporine in a manner consistent with the differential susceptibility between the DBA/2J and BALB/cByJ strains.

Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (ANs) using stem cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection. As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction protocol to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC; H9) toward a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyze the expression of key markers involved in AN development at defined time points of differentiation. The hiPSC- and hESC-derived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), proneurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160), and sensory AN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC- and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Furthermore, the neurons generated from this assay were found to be electrically active. While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.

The TRK-fused gene (TFG) was originally identified in chromosome translocation events, creating a pair of oncogenes in some cancers, and was recently demonstrated as the causal gene of hereditary motor and sensory neuropathy with proximal dominant involvement. Recently, we cloned an alternative splicing variant of Tfg from a cDNA library of the rat retina, tentatively naming it retinal Tfg (rTfg). Although the common form of Tfg is ubiquitously expressed in most rat tissues, rTfg expression is localized to the central nervous system. In this study, we produced an antibody against an rTFG-specific amino acid sequence and used it to examine the localization of rTFG-like protein in the rat retina by immunohistochemistry and Western blots. Western blot analysis showed that the antibody detected a single band of 24 kDa in the rat retina. When we examined rTFG recombinant protein, the antibody detected two bands of about 42 kDa and 24 kDa. The results suggest that the 24 kDa rTFG-like protein is a fragment of rTFG. In our immunohistochemical studies of the rat retina, rTFG-like immunoreactivity was observed in all calbindin D-28K-positive horizontal cells and in some syntaxin 1-positive amacrine cells (ACs). In addition, the rTFG-like immunopositive ACs were actually glycine transporter 1-positive glycinergic or glutamate decarboxylase-positive GABAergic ACs. Our findings indicate that this novel 24 kDa rTFG-like protein may play a specific role in retinal inhibitory interneurons.