In view of the recent association of Brn-3 transcription factors with neuroblastomas, cervical, breast, and prostate cancers we examined the expression of Brn-3a(l) in normal ovaries and in different histological grades of ovarian tumors. The expression of Brn-3a(l) was also evaluated in normal ovarian and cancer cell lines and tumor cells isolated from the ascites of advanced-stage ovarian cancer patients.

In cancer cells, elevated transcription factor-related Brn-3a regulator isolated from brain cDNA (Brn-3b) transcription factor enhances proliferation in vitro and increases tumour growth in vivo whilst conferring drug resistance and migratory potential, whereas reducing Brn-3b slows growth both in vitro and in vivo. Brn-3b regulates distinct groups of key target genes that control cell growth and behaviour. Brn-3b is elevated in >65% of breast cancer biopsies, but mechanisms controlling its expression in these cells are not known.

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.

The Brn-3a and Brn-3b transcription factor have opposite and antagonistic effects in neuroblastoma cells since Brn-3a is associated with differentiation whilst Brn-3b enhances proliferation in these cells. In this study, we demonstrate that like Brn-3a, Brn-3b physically interacts with p53. However, whereas Brn-3a repressed p53 mediated Bax expression but cooperated with p53 to increase p21cip1/waf1, this study demonstrated that co-expression of Brn-3b with p53 increases trans-activation of Bax promoter but not p21cip1/waf1. Consequently co-expression of Brn-3b with p53 resulted in enhanced apoptosis, which is in contrast to the increased survival and differentiation, when Brn-3a is co-expressed with p53. For Brn-3b to cooperate with p53 on the Bax promoter, it requires binding sites that flank p53 sites on this promoter. Furthermore, neurons from Brn-3b knock-out (KO) mice were resistant to apoptosis and this correlated with reduced Bax expression upon induction of p53 in neurons lacking Brn-3b compared with controls. Thus, the ability of Brn-3b to interact with p53 and modulate Bax expression may demonstrate an important mechanism that helps to determine the fate of cells when p53 is induced.

While the transcriptional code governing retinal ganglion cell (RGC) type specification begins to be understood, its interplay with neurotrophic signaling is largely unexplored. In mice, the transcription factor Brn3a/Pou4f1 is expressed in most RGCs, and is required for the specification of RGCs with small dendritic arbors. The Glial Derived Neurotrophic Factor (GDNF) receptor Ret is expressed in a subset of RGCs, including some expressing Brn3a, but its role in RGC development is not defined.

Glaucoma is one of the leading causes of irreversible blindness and can result from abnormalities in anterior segment structures required for aqueous humor outflow, including the trabecular meshwork (TM) and Schlemm's canal (SC). Transcription factors such as AP-2β play critical roles in anterior segment development. Here, we show that the Mgp-Cre knock-in (Mgp-Cre.KI) mouse can be used to target the embryonic periocular mesenchyme giving rise to the TM and SC. Fate mapping of male and female mice indicates that AP-2β loss causes a decrease in iridocorneal angle cells derived from Mgp-Cre.KI-expressing populations compared to controls. Moreover, histological analyses revealed peripheral iridocorneal adhesions in AP-2β mutants that were accompanied by a decrease in expression of TM and SC markers, as observed using immunohistochemistry. In addition, rebound tonometry showed significantly higher intraocular pressure (IOP) that was correlated with a progressive significant loss of retinal ganglion cells, reduced retinal thickness, and reduced retinal function, as measured using an electroretinogram, in AP-2β mutants compared with controls, reflecting pathology described in late-stage glaucoma patients. Importantly, elevated IOP in AP-2β mutants was significantly reduced by treatment with latanoprost, a prostaglandin analog that increases unconventional outflow. These findings demonstrate that AP-2β is critical for TM and SC development, and that these mutant mice can serve as a model for understanding and treating progressive human primary angle-closure glaucoma.

Members of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell (RGCs) types, the main transducers of visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing alkaline phosphatase (AP) and intersectional genetics had identified three types of Brn3c positive (Brn3c+ ) RGCs. Here, we describe a novel Brn3cCre mouse allele generated by serial Dre to Cre recombination and use it to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus response properties of Brn3c+ RGC types. Furthermore, we explore brain nuclei that express Brn3c or receive input from Brn3c+ neurons. Our analysis reveals a much larger number of Brn3c+ RGCs and more diverse set of RGC types than previously reported. Most RGCs expressing Brn3c during development are still Brn3c positive in the adult, and all express Brn3a while only about half express Brn3b. Genetic Brn3c-Brn3b intersection reveals an area of increased RGC density, extending from dorsotemporal to ventrolateral across the retina and overlapping with the mouse binocular field of view. In addition, we report a Brn3c+ RGC projection to the thalamic reticular nucleus, a visual nucleus that was not previously shown to receive retinal input. Furthermore, Brn3c+ neurons highlight a previously unknown subdivision of the deep mesencephalic nucleus. Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity, and cytoarchitectonic.

Retinal ganglion cells (RGCs) are tasked with transmitting all light information from the eye to the retinal recipient areas of the brain. RGCs can be classified into many different types by morphology, gene expression, axonal projections, and functional responses to different light stimuli. Ultimately, these classification systems should be unified into an all-encompassing taxonomy. Toward that end, we show here that nearly all RGCs express either Islet-2 (Isl2), Tbr2, or a combination of Satb1 and Satb2. We present gene expression data supporting the hypothesis that Satb1 and Satb2 are expressed in ON-OFF direction-selective (DS) RGCs, complementing our previous work demonstrating that RGCs that express Isl2 and Tbr2 are non-DS and non-image-forming, respectively. Expression of these transcription factors emerges at distinct embryonic ages and only in postmitotic cells. Finally, we demonstrate that these transcription factor-defined RGC classes are born throughout RGC genesis.

The brain's primary circadian pacemaker, the suprachiasmatic nucleus (SCN), is required to translate day-length and circadian rhythms into neuronal, hormonal, and behavioral rhythms. Here, we identify the homeodomain transcription factor ventral anterior homeobox 1 (Vax1) as required for SCN development, vasoactive intestinal peptide expression, and SCN output. Previous work has shown that VAX1 is required for gonadotropin-releasing hormone (GnRH/LHRH) neuron development, a neuronal population controlling reproductive status. Surprisingly, the ectopic expression of a Gnrh-Cre allele (Gnrhcre) in the SCN confirmed the requirement of both VAX1 (Vax1flox/flox:Gnrhcre, Vax1Gnrh-cre) and sine oculis homeobox protein 6 (Six6flox/flox:Gnrhcre, Six6Gnrh-cre) in SCN function in adulthood. To dissociate the role of Vax1 and Six6 in GnRH neuron and SCN function, we used another Gnrh-cre allele that targets GnRH neurons, but not the SCN (Lhrhcre). Both Six6Lhrh-cre and Vax1Lhrh-cre were infertile, and in contrast to Vax1Gnrh-cre and Six6Gnrh-cre mice, Six6Lhrh-cre and Vax1Lhrh-cre had normal circadian behavior. Unexpectedly, ~ 1/4 of the Six6Gnrh-cre mice were unable to entrain to light, showing that ectopic expression of Gnrhcre impaired function of the retino-hypothalamic tract that relays light information to the brain. This study identifies VAX1, and confirms SIX6, as transcription factors required for SCN development and function and demonstrates the importance of understanding how ectopic CRE expression can impact the results.

Transcription factors control cell identity by regulating diverse developmental steps such as differentiation and axon guidance. The mammalian binocular visual circuit is comprised of projections of retinal ganglion cells (RGCs) to ipsilateral and contralateral targets in the brain. A transcriptional code for ipsilateral RGC identity has been identified, but less is known about the transcriptional regulation of contralateral RGC development. Here we demonstrate that SoxC genes (Sox4, 11, and 12) act on the progenitor-to-postmitotic transition to implement contralateral, but not ipsilateral, RGC differentiation, by binding to Hes5 and thus repressing Notch signaling. When SoxC genes are deleted in postmitotic RGCs, contralateral RGC axons grow poorly on chiasm cells in vitro and project ipsilaterally at the chiasm midline in vivo, and Plexin-A1 and Nr-CAM expression in RGCs is downregulated. These data implicate SoxC transcription factors in the regulation of contralateral RGC differentiation and axon guidance.

Toxic nitric oxide (NO) levels can regulate gene expression. Using a novel protein/DNA array, we show that toxic NO levels regulate the binding of trans-factors to various cis-elements in neuroblastoma cells, including CRE and those recognized by the transcription factors AP1, AP2, Brn-3a, EGR, E2F1 and SP1. Functionality of some of the cis-elements was confirmed by electro mobility shift and reporter assays. Interestingly, CREB, AP-1, Brn-3a, EGR and E2F1 can control mammalian cell viability. NO induced the anti-apoptotic Bcl-2 protein and its mRNA prior to the onset of death of 30-60% of the cells. Promoter analysis of the bcl-2 gene confirmed the involvement of a CRE in NO-dependent bcl-2 transcription. Neuroblastoma cells over-expressing bcl-2 became much more resistant to NO-induced apoptosis; conversely, Bcl-2 knockdown cells were rendered markedly more sensitive to NO. Together these results suggest that Bcl-2 counteracts NO-induced apoptosis in a fraction of the cell population. Thus, NO stimulates the binding of many trans-factors to their cognate cis-elements, some of which can regulate cell viability through transcriptional activation of target genes. Our results emphasize that a DNA/protein array approach can reveal novel, global transcription factor activities stimulated by cell death-regulating molecules.

Previous studies demonstrated that retinal damage correlates with a massive remodeling of extracellular matrix (ECM) molecules and reactive gliosis. However, the functional significance of the ECM in retinal neurodegeneration is still unknown. In the present study, we used an intraocular pressure (IOP) independent experimental autoimmune glaucoma (EAG) mouse model to examine the role of the ECM glycoprotein tenascin-C (Tnc). Wild type (WT ONA) and Tnc knockout (KO ONA) mice were immunized with an optic nerve antigen (ONA) homogenate and control groups (CO) obtained sodium chloride (WT CO, KO CO). IOP was measured weekly and electroretinographies were recorded at the end of the study. Ten weeks after immunization, we analyzed retinal ganglion cells (RGCs), glial cells, and the expression of different cytokines in retina and optic nerve tissue in all four groups. IOP and retinal function were comparable in all groups. Although RGC loss was less severe in KO ONA, WT as well as KO mice displayed a significant cell loss after immunization. Compared to KO ONA, less βIII-tubulin+ axons, and downregulated oligodendrocyte markers were noted in WT ONA optic nerves. In retina and optic nerve, we found an enhanced GFAP+ staining area of astrocytes in immunized WT. A significantly higher number of retinal Iba1+ microglia was found in WT ONA, while a lower number of Iba1+ cells was observed in KO ONA. Furthermore, an increased expression of the glial markers Gfap, Iba1, Nos2, and Cd68 was detected in retinal and optic nerve tissue of WT ONA, whereas comparable levels were observed in KO ONA. In addition, pro-inflammatory Tnfa expression was upregulated in WT ONA, but downregulated in KO ONA. Vice versa, a significantly increased anti-inflammatory Tgfb1 expression was measured in KO ONA animals. We conclude that Tnc plays an important role in glial and inflammatory response during retinal neurodegeneration. Our results provide evidence that Tnc is involved in glaucomatous damage by regulating retinal glial activation and cytokine release. Thus, this transgenic EAG mouse model for the first time offers the possibility to investigate IOP-independent glaucomatous damage in direct relation to ECM remodeling.

The onset of retinal degenerative disease is often associated with neuronal loss. Therefore, how to regenerate new neurons to restore vision is an important issue. NeuroD1 is a neural transcription factor with the ability to reprogram brain astrocytes into neurons in vivo. Here, we demonstrate that in adult mice, NeuroD1 can reprogram Müller cells, the principal glial cell type in the retina, to become retinal neurons. Most strikingly, ectopic expression of NeuroD1 using two different viral vectors converted Müller cells into different cell types. Specifically, AAV7m8 GFAP681::GFP-ND1 converted Müller cells into inner retinal neurons, including amacrine cells and ganglion cells. In contrast, AAV9 GFAP104::ND1-GFP converted Müller cells into outer retinal neurons such as photoreceptors and horizontal cells, with higher conversion efficiency. Furthermore, we demonstrate that Müller cell conversion induced by AAV9 GFAP104::ND1-GFP displayed clear dose- and time-dependence. These results indicate that Müller cells in adult mice are highly plastic and can be reprogrammed into various subtypes of retinal neurons.

The mouse eye is used to model central nervous system development, pathology, angiogenesis, tumorigenesis, and regenerative therapies. To facilitate the analysis of these processes, we developed an optimized tissue clearing and depigmentation protocol, termed InVision, that permits whole-eye fluorescent marker tissue imaging. We validated this method for the analysis of normal and degenerative retinal architecture, transgenic fluorescent reporter expression, immunostaining and three-dimensional volumetric (3DV) analysis of retinoblastoma and angiogenesis. We also used this method to characterize material transfer (MT), a recently described phenomenon of horizontal protein exchange that occurs between transplanted and recipient photoreceptors. 3D spatial distribution analysis of MT in transplanted retinas suggests that MT of cytoplasmic GFP between photoreceptors is mediated by short-range, proximity-dependent cellular interactions. The InVision protocol will allow investigators working across multiple cell biological disciplines to generate novel insights into the local cellular networks involved in cell biological processes in the eye.

Daily adaptation of metabolic activity to light-dark cycles to maintain homeostasis is controlled by hypothalamic nuclei receiving information from the retina and from nutritional inputs that vary according to feeding cycles. We show that selective hypomorphic expression of the transcription factor gene Pitx3 prevents light-dependent entrainment of the central pacemaker in the suprachiasmatic nucleus. This translates into altered behavioral and metabolic outputs affecting locomotor activity, feeding patterns, energy expenditure, and corticosterone secretion that correlate with dysfunctional expression of clock genes in the ventromedial hypothalamus, liver, and brown adipose tissue. Metabolic entrainment by time-restricted feeding restores clock function in the liver and brown adipose tissue but not in the ventromedial hypothalamus and, remarkably, fails to synchronize energy expenditure and locomotor and hormonal outputs. Thus, our study reveals a central role of the priming of the suprachiasmatic nucleus with retinal innervation in the hypothalamic regulation of cyclic metabolic homeostasis.

Loss-of-function mutations in NaV1.7 cause congenital insensitivity to pain (CIP); this voltage-gated sodium channel is therefore a key target for analgesic drug development. Utilizing a multi-modal approach, we investigated how NaV1.7 mutations lead to human pain insensitivity. Skin biopsy and microneurography revealed an absence of C-fiber nociceptors in CIP patients, reflected in a reduced cortical response to capsaicin on fMRI. Epitope tagging of endogenous NaV1.7 revealed the channel to be localized at the soma membrane, axon, axon terminals, and the nodes of Ranvier of induced pluripotent stem cell (iPSC) nociceptors. CIP patient-derived iPSC nociceptors exhibited an inability to properly respond to depolarizing stimuli, demonstrating that NaV1.7 is a key regulator of excitability. Using this iPSC nociceptor platform, we found that some NaV1.7 blockers undergoing clinical trials lack specificity. CIP, therefore, arises due to a profound loss of functional nociceptors, which is more pronounced than that reported in rodent models, or likely achievable following acute pharmacological blockade. VIDEO ABSTRACT.

Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy.

We report the retinal expression pattern of Ret, a receptor tyrosine kinase for the glial derived neurotrophic factor (GDNF) family ligands (GFLs), during development and in the adult mouse. Ret is initially expressed in retinal ganglion cells (RGCs), followed by horizontal cells (HCs) and amacrine cells (ACs), beginning with the early stages of postmitotic development. Ret expression persists in all three classes of neurons in the adult. Using RNA sequencing, immunostaining and random sparse recombination, we show that Ret is expressed in at least three distinct types of ACs, and ten types of RGCs. Using intersectional genetics, we describe the dendritic arbor morphologies of RGC types expressing Ret in combination with each of the three members of the POU4f/Brn3 family of transcription factors. Ret expression overlaps with Brn3a in 4 RGC types, with Brn3b in 5 RGC types, and with Brn3c in one RGC type, respectively. Ret+ RGCs project to the lateral geniculate nucleus (LGN), pretectal area (PTA) and superior colliculus (SC), and avoid the suprachiasmatic nucleus and accessory optic system. Brn3a+ Ret+ and Brn3c+ Ret+ RGCs project preferentially to contralateral retinorecipient areas, while Brn3b+ Ret+ RGCs shows minor ipsilateral projections to the olivary pretectal nucleus and the LGN. Our findings establish intersectional genetic approaches for the anatomic and developmental characterization of individual Ret+ RGC types. In addition, they provide necessary information for addressing the potential interplay between GDNF neurotrophic signaling and transcriptional regulation in RGC type specification.

During vertebrate retinal development, subsets of progenitor cells generate progeny in a non-stochastic manner, suggesting that these decisions are tightly regulated. However, the gene-regulatory network components that are functionally important in these progenitor cells are largely unknown. Here we identify a functional role for the OTX2 transcription factor in this process. CRISPR/Cas9 gene editing was used to produce somatic mutations of OTX2 in the chick retina and identified similar phenotypes to those observed in human patients. Single cell RNA sequencing was used to determine the functional consequences OTX2 gene editing on the population of cells derived from OTX2-expressing retinal progenitor cells. This confirmed that OTX2 is required for the generation of photoreceptors, but also for repression of specific retinal fates and alternative gene regulatory networks. These include specific subtypes of retinal ganglion and horizontal cells, suggesting that in this context, OTX2 functions to repress sister cell fate choices.

The mammalian cochlea undergoes a highly dynamic process of growth and innervation during development. This process includes spiral ganglion neuron (SGN) branch refinement, a process whereby Type I SGNs undergo a phase of "debranching" before forming unramified synaptic contacts with inner hair cells. Using Sox2CreERT2 and R26RtdTomato as a strategy to genetically label individual SGNs in mice of both sexes, we report on both a time course of SGN branch refinement and a role for P2rx3 in this process. P2rx3 is an ionotropic ATP receptor that was recently implicated in outer hair cell spontaneous activity and Type II SGN synapse development (Ceriani et al., 2019), but its function in Type I SGN development is unknown. Here, we demonstrate that P2rx3 is expressed by Type I SGNs and hair cells during developmental periods that coincide with SGN branching refinement. P2rx3 null mice show SGNs with more complex branching patterns on their peripheral synaptic terminals and near their cell bodies around the time of birth. Loss of P2rx3 does not appear to confer general changes in axon outgrowth or hair cell formation, and alterations in branching complexity appear to mostly recover by postnatal day (P)6. However, when we examined the distribution of Type I SGN subtypes using antibodies that bind Calb2, Calb1, and Pou4f1, we found that P2rx3 null mice showed an increased proportion of SGNs that express Calb2. These data suggest P2rx3 may be necessary for normal Type I SGN differentiation in addition to serving a role in branch refinement.