mTORC1 accelerates retinal development via the immunoproteasome.

The numbers and types of cells constituting vertebrate neural tissues are determined by cellular mechanisms that couple neurogenesis to the proliferation of neural progenitor cells. Here we identified a role of mammalian target of rapamycin complex 1 (mTORC1) in the development of neural tissue, showing that it accelerates progenitor cell cycle progression and neurogenesis in mTORC1-hyperactive tuberous sclerosis complex 1 (Tsc1)-deficient mouse retina. We also show that concomitant loss of immunoproteasome subunit Psmb9, which is induced by Stat1 (signal transducer and activator of transcription factor 1), decelerates cell cycle progression of Tsc1-deficient mouse retinal progenitor cells and normalizes retinal developmental schedule. Collectively, our results establish a developmental role for mTORC1, showing that it promotes neural development through activation of protein turnover via a mechanism involving the immunoproteasome.

Differential Expression of NF2 in Neuroepithelial Compartments Is Necessary for Mammalian Eye Development.

The optic neuroepithelial continuum of vertebrate eye develops into three differentially growing compartments: the retina, the ciliary margin (CM), and the retinal pigment epithelium (RPE). Neurofibromin 2 (Nf2) is strongly expressed in slowly expanding RPE and CM compartments, and the loss of mouse Nf2 causes hyperplasia in these compartments, replicating the ocular abnormalities seen in human NF2 patients. The hyperplastic ocular phenotypes were largely suppressed by heterozygous deletion of Yap and Taz, key targets of the Nf2-Hippo signaling pathway. We also found that, in addition to feedback transcriptional regulation of Nf2 by Yap/Taz in the CM, activation of Nf2 expression by Mitf in the RPE and suppression by Sox2 in retinal progenitor cells are necessary for the differential growth of the corresponding cell populations. Together, our findings reveal that Nf2 is a key player that orchestrates the differential growth of optic neuroepithelial compartments during vertebrate eye development.

Donating Otx2 to support neighboring neuron survival.

Mutations of orthodentricle homeobox 2 (OTX2) in human and mice often cause retinal dystrophy and nyctalopia, suggesting a role of OTX2 in mature retina, in addition to its functions in the development of the eye and retina. In support of this, the number of bipolar cells in Otx2＋/- post-natal mouse retina was found to be significantly lower than normal. Degeneration of the cells becomes greater as the mice age, leading to the loss of vision. Especially, the type-2 OFF-cone bipolar cells, which do not express Otx2 mRNA but carry Otx2 protein, are most sensitive to Otx2 haplodeficiency. Interestingly, this bipolar cell subpopulation imports Otx2 protein from photoreceptors to protect itself from glutamate excitotoxicity in the dark. Moreover, in the bipolar cells, the exogenous Otx2 relocates to the mitochondria to support mitochondrial ATP synthesis. This novel mitochondrial activity of exogenous Otx2 highlights the therapeutic potential of Otx2 protein transduction in retinal dystrophy. [BMB Reports 2016; 49(2): 69-70].

Mitochondrial Protection by Exogenous Otx2 in Mouse Retinal Neurons.

OTX2 (orthodenticle homeobox 2) haplodeficiency causes diverse defects in mammalian visual systems ranging from retinal dysfunction to anophthalmia. We find that the retinal dystrophy of Otx2(+/GFP) heterozygous knockin mice is mainly due to the loss of bipolar cells and consequent deficits in retinal activity. Among bipolar cell types, OFF-cone bipolar subsets, which lack autonomous Otx2 gene expression but receive Otx2 proteins from photoreceptors, degenerate most rapidly in Otx2(+/GFP) mouse retinas, suggesting a neuroprotective effect of the imported Otx2 protein. In support of this hypothesis, retinal dystrophy in Otx2(+/GFP) mice is prevented by intraocular injection of Otx2 protein, which localizes to the mitochondria of bipolar cells and facilitates ATP synthesis as a part of mitochondrial ATP synthase complex. Taken together, our findings demonstrate a mitochondrial function for Otx2 and suggest a potential therapeutic application of OTX2 protein delivery in human retinal dystrophy.

Regulation of retinal axon growth by secreted Vax1 homeodomain protein.

Retinal ganglion cell (RGC) axons of binocular animals cross the midline at the optic chiasm (OC) to grow toward their synaptic targets in the contralateral brain. Ventral anterior homeobox 1 (Vax1) plays an essential role in the development of the OC by regulating RGC axon growth in a non-cell autonomous manner. In this study, we identify an unexpected function of Vax1 that is secreted from ventral hypothalamic cells and diffuses to RGC axons, where it promotes axonal growth independent of its transcription factor activity. We demonstrate that Vax1 binds to extracellular sugar groups of the heparan sulfate proteoglycans (HSPGs) located in RGC axons. Both Vax1 binding to HSPGs and subsequent penetration into the axoplasm, where Vax1 activates local protein synthesis, are required for RGC axonal growth. Together, our findings demonstrate that Vax1 possesses a novel RGC axon growth factor activity that is critical for the development of the mammalian binocular visual system.

Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes.

In the human, mutations of OTX2 (Orthodenticle homeobox 2 transcription factor) translate into eye malformations of variable expressivity (even between the two eyes of the same individual) and incomplete penetrance, suggesting the existence of subtle thresholds in OTX2 activity. We have addressed this issue by analyzing retinal structure and function in six mutant mice with graded Otx2 activity: Otx2(+/+), Otx2(+/AA), Otx2(+/GFP), Otx2(AA/AA), Otx2(AA/GFP) and Otx2(GFP/GFP). Null mice (Otx2(GFP/GFP)) fail to develop the head and are embryonic lethal, and compound heterozygous Otx2(AA/GFP) mice show a truncated head and die at birth. All other genotypes develop until adulthood. We analyzed eye structure and visual physiology in the genotypes that develop until adulthood and report that phenotype severity parallels Otx2 activity. Otx2(+/AA) are only mildly affected whereas Otx2(+/GFP) are more affected than Otx2(+/AA) but less than Otx2(AA/AA) mice. Otx2(AA/AA) mice later manifest the most severe defects, with variable expressivity. Electrophysiological and histological analyses of the mouse retina revealed progressive death of bipolar cells and cone photoreceptors that is both Otx2 activity- and age-dependent with the same ranking of phenotypic severity. This study demonstrates the importance of gene dosage in the development of age-dependent pathologies and underscores the fact that small gene dosage differences can cause significant pathological states.

Compartmentalization of vertebrate optic neuroephithelium: external cues and transcription factors.

The vertebrate eye is a laterally extended structure of the forebrain. It develops through a series of events, including specification and regionalization of the anterior neural plate, evagination of the optic vesicle (OV), and development of three distinct optic structures: the neural retina (NR), optic stalk (OS), and retinal pigment epithelium (RPE). Various external signals that act on the optic neuroepithelium in a spatial- and temporal-specific manner control the fates of OV subdomains by inducing localized expression of key transcription factors. Investigating the mechanisms underlying compartmentalization of these distinct optic neuroepithelium-derived tissues is therefore not only important from the standpoint of accounting for vertebrate eye morphogenesis, it is also helpful for understanding the fundamental basis of fate determination of other neuroectoderm- derived tissues. This review focuses on the molecular signatures of OV subdomains and the external factors that direct the development of tissues originating from the OV.

Survival and differentiation of mammary epithelial cells in mammary gland development require nuclear retention of Id2 due to RANK signaling.

RANKL plays an essential role in mammary gland development during pregnancy. However, the molecular mechanism by which RANK signaling leads to mammary gland development is largely unknown. We report here that RANKL stimulation induces phosphorylation of Id2 at serine 5, which leads to nuclear retention of Id2. In lactating Id2Tg; RANKL(-/-) mice, Id2 was not phosphorylated and was localized in the cytoplasm. In addition, in lactating Id2(S5A)Tg mice, Id2(S5A) (with serine 5 mutated to alanine) was exclusively localized in the cytoplasm of mammary epithelial cells (MECs), while endogenous Id2 was localized in the nucleus. Intriguingly, nuclear expression of Id2(S5A) rescued increased apoptosis and defective differentiation of MECs in RANKL(-/-) mice. Our results demonstrate that nuclear retention of Id2 due to RANK signaling plays a decisive role in the survival and differentiation of MECs during mammary gland development.

The Wnt receptor Ryk controls specification of GABAergic neurons versus oligodendrocytes during telencephalon development.

GABAergic neurons and oligodendrocytes originate from progenitors within the ventral telencephalon. However, the molecular mechanisms that control neuron-glial cell-fate segregation, especially how extrinsic factors regulate cell-fate changes, are poorly understood. We have discovered that the Wnt receptor Ryk promotes GABAergic neuron production while repressing oligodendrocyte formation in the ventral telencephalon. We demonstrate that Ryk controls the cell-fate switch by negatively regulating expression of the intrinsic oligodendrogenic factor Olig2 while inducing expression of the interneuron fate determinant Dlx2. In addition, we demonstrate that Ryk is required for GABAergic neuron induction and oligodendrogenesis inhibition caused by Wnt3a stimulation. Furthermore, we showed that the cleaved intracellular domain of Ryk is sufficient to regulate the cell-fate switch by regulating the expression of intrinsic cell-fate determinants. These results identify Ryk as a multi-functional receptor that is able to transduce extrinsic cues into progenitor cells, promote GABAergic neuron formation, and inhibit oligodendrogenesis during ventral embryonic brain development.

Human neurospheres derived from the fetal central nervous system are regionally and temporally specified but are not committed.

Proliferating single cells were isolated from various CNS regions (telencephalon, diencephalon, midbrain, cerebellum, pons and medulla, and spinal cord) of human fetal cadavers at 13 weeks of gestation and grown as neurospheres in long-term cultures. We investigated whether neural stem cells (NSCs) or progenitors within spheres have specific regional or temporal characteristics with regard to growth, differentiation, and region-specific gene expression, and whether these molecular specifications are reversible. Regardless of regional origin, all of the neurospheres were found to contain cells of different subtypes, which suggests that multipotent NSCs, progenitors or radial glial cells co-exist with restricted neuronal or glial progenitors within the neurospheres. Neurospheres from the forebrain grew faster and gave rise to significantly more neurons than did those from either the midbrain or hindbrain, and regional differences in neuronal differentiation appeared to be sustained during long-term passage of neurospheres in culture. There was also a trend towards a reduction in neuronal emergence from the respective neurospheres over time in culture, although the percentages of neurons generated from cerebellum-derived neurospheres increased dramatically. These results suggest that differences in neuronal differentiation for the various neurospheres are spatially and temporally determined. In addition, the properties of glial fibrillary acidic protein (GFAP)-, glutamate-, and gamma-aminobutyric acid (GABA)-expressing cells derived from neurospheres of the respective CNS regions appear to be regionally and temporally different. Isolated human neurospheres from different CNS compartments expressed distinctive molecular markers of regional identity and maintained these patterns of region-specific gene expression during long-term passage in vitro. To determine the potential of human neurospheres for regional fate plasticity, single spheres from the respective regions were co-cultured with embryonic day 16.5 (E16.5 d) mouse brain slices. Specific cues from the developing mouse brain tissues induced the human neurospheres to express different marker genes of regional identity and to suppress the expression of their original marker genes. Thus, even the early regional identities of human neurospheres may not be irreversible and may be altered by local inductive cues. These findings have important implications for understanding the characteristics of growth, differentiation, and molecular specification of human neurospheres derived from the developing CNS, as well as the therapeutic potential for neural repair.

Gene and cell replacement via neural stem cells.

Neural stem cells (NSCs) are operationally defined by their ability to self-renew, to differentiate into cells of all glial and neuronal lineages throughout the neuraxis, and to populate developing or degenerating central nervous system (CNS) regions. Thus their use as graft material can be considered analogous to hematopoietic stem cell-mediated reconstitution and gene transfer. The recognition that NSCs propagated in culture could be reimplanted into mammalian brain, where they might integrate appropriately throughout the mammalian CNS and stably express foreign genes, has unveiled a new role for neural transplantation and gene therapy and a possible strategy for addressing the CNS manifestations of diseases that heretofore had been refractory to intervention. NSCs additionally have the appealing ability to home in on pathology, even over great distances. Such observations help to advance the idea that NSCs--as a prototype for stem cells from other solid organs--might aid in reconstructing the molecular and cellular milieu of maldeveloped or damaged CNS.