Electrochemical properties of a lithium-impregnated metal foam anode (LIMFA FeCrAl) for molten salt thermal batteries.

Although numerous cathode materials with excellent properties have been developed for use in molten salt thermal batteries, similar progress is yet to be made with anode materials. Herein, a high-performance lithium-impregnated metal foam anode (LIMFA) is fabricated by impregnating molten lithium into a gold-coated iron-chrome-aluminium (FeCrAl) foam at 400 °C. A test cell employing the LIMFA FeCrAl anode exhibited a specific capacity of 2627 As g-1. For comparison, a cell with a conventional Li(Si) anode was also discharged, demonstrating a specific capacity of 982 As g-1. This significant improvement in performance can be attributed to the large amount (18 wt%) of lithium incorporated into the FeCrAl foam and the ability of the FeCrAl foam to absorb and immobilize molten lithium without adopting a cup system. For thermal batteries without a cup, the LIMFA FeCrAl provides the highest-reported specific capacity and a flat discharge voltage curve of molten lithium. After cell discharge, the FeCrAl foam exhibited no lithium leakage, surface damage, or structural collapse. Given these advantageous properties, in addition to its high specific capacity, LIMFA FeCrAl is expected to aid the development of thermal batteries with enhanced performance.

Age-Related Macular Degeneration (AMD) Transmitochondrial Cybrids Protected from Cellular Damage and Death by Human Retinal Progenitor Cells (hRPCs).

PURPOSE: One of the leading causes of irreversible blindness worldwide, age-related macular degeneration (AMD) is a progressive disorder leading to retinal degeneration. While several treatment options exist for the exudative form of AMD, there are currently no FDA-approved treatments for the more common nonexudative (atrophic) form. Mounting evidence suggests that mitochondrial damage and retinal pigment epithelium (RPE) cell death are linked to the pathogenesis of AMD. Human retinal progenitor cells (hRPCs) have been studied as a potential restorative therapy for degenerative conditions of the retina; however, the effects of hRPC treatment on retinal cell survival in AMD have not been elucidated. METHODS: In this study, we used a cell coculture system consisting of hRPCs and AMD or age-matched normal cybrid cells to characterize the effects of hRPCs in protecting AMD cybrids from cellular and mitochondrial damage and death. RESULTS: AMD cybrids cocultured with hRPCs showed (1) increased cell viability; (2) decreased gene expression related to apoptosis, autophagy, endoplasmic reticulum (ER) stress, and antioxidant pathways; and (3) downregulation of mitochondrial replication genes compared to AMD cybrids without hRPC treatment. Furthermore, hRPCs cocultured with AMD cybrids showed upregulation of (1) neuronal and glial markers, as well as (2) putative neuroprotective factors, responses not found when hRPCs were cocultured with age-matched normal cybrids. CONCLUSION: The current study provides the first evidence that therapeutic benefits may be obtainable using a progenitor cell-based approach for atrophic AMD. Our results suggest that bidirectional interactions exist between hRPCs and AMD cybrids such that hRPCs release trophic factors that protect the cybrids against the cellular and mitochondrial changes involved in AMD pathogenesis while, conversely, AMD cybrids upregulate the release of these neuroprotective factors by hRPCs while promoting hRPC differentiation. These in vitro data provide evidence that hRPCs may have therapeutic potential in atrophic AMD.

Hydrothermally synthesized homogeneous Ni-Mo-S structures on Ni-foam cathodes for thermal batteries.

Hydrothermally synthesized homogeneous structures based on Ni, Mo, and S on Ni metal foam cathodes (NiMoSs) were characterized electrochemically. A NiMoS-containing cell exhibited a much higher specific capacity of 1534 A s g-1 than an FeS2 cathode, owing to its homogeneous structure, demonstrating promise for thermal battery applications.

Immunosuppressive Treatment Can Alter Visual Performance in the Royal College of Surgeons Rat.

PURPOSE: Immunosuppression is frequently employed to enhance survival of xenografted human cells as part of translational proof-of-concept studies. However, the potential effects of this treatment are easily overlooked. METHODS: As part of baseline testing in the dark-eyed variant of the dystrophic Royal College of Surgeons (RCS) rat, we documented the time course of retinal degenerative changes versus Long Evans controls using bright field retinal imaging, fluorescein angiography, and histology and examined the impact of immunosuppression on visual function. Rats received either no treatment or systemic immunosuppression with oral cyclosporine A and injectable dexamethasone and subsequently underwent functional evaluation by optomotor response testing and electroretinography (ERG) at multiple intervals from P45 to P180. RESULTS: Immunosuppressed RCS animals demonstrated poorer performance on functional tests than age-matched untreated rats during the earlier stages of degeneration, including significantly lower spatial acuities on optomotor threshold testing and significantly lower b-wave amplitudes on scotopic and photopic ERGs. Retinal imaging documented the progression of degenerative changes in the RCS fundus and histologic evaluation of the RCS retina confirmed progressive thinning of the outer nuclear layer. CONCLUSIONS: A standard regimen of cyclosporine A plus dexamethasone, administered to RCS rats, results in demonstrable systemic side effects and depressed scores on behavioral and electrophysiological testing at time points before P90. The source of the functional impairment was not identified. This finding has implications for the interpretation of data generated using this commonly used translational model.

Reprogramming amacrine and photoreceptor progenitors into retinal ganglion cells by replacing Neurod1 with Atoh7.

The specification of the seven retinal cell types from a common pool of retina progenitor cells (RPCs) involves complex interactions between the intrinsic program and the environment. The proneural basic helix-loop-helix (bHLH) transcriptional regulators are key components for the intrinsic programming of RPCs and are essential for the formation of the diverse retinal cell types. However, the extent to which an RPC can re-adjust its inherent program and the mechanisms through which the expression of a particular bHLH factor influences RPC fate is unclear. Previously, we have shown that Neurod1 inserted into the Atoh7 locus activates the retinal ganglion cell (RGC) program in Atoh7-expressing RPCs but not in Neurod1-expressing RPCs, suggesting that Atoh7-expressing RPCs are not able to adopt the cell fate determined by Neurod1, but rather are pre-programmed to produce RGCs. Here, we show that Neurod1-expressing RPCs, which are destined to produce amacrine and photoreceptor cells, can be re-programmed into RGCs when Atoh7 is inserted into the Neurod1 locus. These results suggest that Atoh7 acts dominantly to convert a RPC subpopulation not destined for an RGC fate to adopt that fate. Thus, Atoh7-expressing and Neurod1-expressing RPCs are intrinsically different in their behavior. Additionally, ChIP-Seq analysis identified an Atoh7-dependent enhancer within the intronic region of Nrxn3. The enhancer recognized and used Atoh7 in the developing retina to regulate expression of Nrxn3, but could be forced to use Neurod1 when placed in a different regulatory context. The results indicate that Atoh7 and Neurod1 activate distinct sets of genes in vivo, despite their common DNA-binding element.

Expression and localization of CERKL in the mammalian retina, its response to light-stress, and relationship with NeuroD1 gene.

Mutations in the Ceramide kinase like (CERKL) gene are associated with retinitis pigmentosa (RP26) and cone-rod dystrophy. CERKL is homologous to Ceramide kinase (CERK), and its function is still unknown. The purpose of this study was to test the expression and distribution of this gene and its protein in rat and in mouse tissues, in light-stressed rat retinas and in the retinas of NeuroD1 knock-out mice to understand the role of CERKL in the retina. Expression of Cerkl and Cerk mRNA was determined by quantitative RT-PCR. Expression of the protein was determined by Western blotting with anti-CERKL antibody. Localization of the protein was determined by using immunofluorescence microscopy. With qRT-PCR, we revealed that the relative mRNA expression of Cerkl was the highest in the retina among all the rat tissue tested; it was >10-fold higher than in the brain. On the other hand, Cerk has ubiquitous expression and its relative abundance is >2 fold of Cerkl in the retina. Cerkl was expressed minimally in the developing mouse eyes and reached a peak at retinal maturity at 2 months. Western blots of retinal tissues revealed two major CERKL protein bands: 59 kDa (C1) and 37 kDa (C2). However, only C2 CERKL was found in the rat retinal rod outer segment (ROS) at level of that was not changed in light vs. dark adaptation. In the light-stressed retina, expression of Cerkl mRNA increased significantly, which was reflected in only on C2 CERKL protein. The CERKL protein localized prominently to the ganglion cells, inner nuclear layers (INL), retinal pigment epithelial (RPE) cells, and photoreceptor inner segments in the retinal sections. Nuclear localization of CERKL was not affected in RPE, INL and the ganglion cell layers in the light-stressed retina; however, the perinuclear and outer segment locations appear to be altered. In the NeuroD1 knock-out mouse retina, the expression of Cerkl mRNA and protein decreased and that decrease also pertains to C2 CERKL. In conclusion, the retina had the highest level of Cerkl mRNA and protein expression, which reached its maximum in the adult retina; CERKL localized to ROS and RPE cells and the light-adaptation did not change the level of CERKL in ROS; light-stress induced Cerkl expression in the retina; and its expression decreased in NeuroD1 knock-out retina. Thus, CERKL may be important for the stress responses and protection of photoreceptor cells.

Adult mice transplanted with embryonic retinal progenitor cells: new approach for repairing damaged optic nerves.

PURPOSE: Retinal ganglion cell (RGC) death and optic nerve degeneration are complex processes whose underlying molecular mechanisms are only vaguely understood. Treatments commonly used for optic nerve degeneration have little long-term value and only prolong degeneration. Recent advances in stem cell replacement therapy offer new ways to overcome RGC loss by transferring healthy cells into eyes of afflicted individuals. However, studies on stem cell replacement for optic nerve degeneration are hampered by limitations of the available animal models, especially genetic models. We have developed a mouse model in which RGCs are genetically ablated in adult mice with subsequent degeneration of the optic nerve. In the study reported here, we used this model to determine whether embryonic retinal progenitor cells (RPCs) removed from donor retinas when RPCs are committing to an RGC fate could restore lost RGCs. METHODS: We used the RGC-depleted model as a host for transplanting donor green fluorescent protein (GFP)-labeled RPCs from embryonic retinas that are maximally expressing Atoh7, a basic helix-loop-helix gene essential for RGC specification. Dissociated GFP-labeled RPCs were characterized in situ by immunolabeling with antibodies against proteins known to be expressed in RPCs at embryonic day (E)14.5. Dissociated retinal cells were injected into the vitreous of one eye of RGC-depleted mice at two to six months of age. The injected and non-injected retinas were analyzed for gene expression using immunolabeling, and the morphology of optic nerves was assessed visually and with histological staining at different times up to four months after injection. RESULTS: We demonstrate the successful transfer of embryonic GFP-labeled RPCs into the eyes of RGC-depleted mice. Many transplanted RPCs invaded the ganglion cell layer, but the efficiency of the invasion was low. GFP-labeled cells within the ganglion cell layer expressed genes associated with early and late stages of RGC differentiation, including Pou4f1, Pou4f2, NFL, Map2, and syntaxin. Several GFP-labeled cells were detected within the injected optic nerves of RGC-depleted mice, and in most cases, we observed a significant increase in the thickness of the RPC-injected optic nerves compared with non-injected controls. We also observed more bundled axons emanating from RPC-injected retinas compared with RGC-depleted controls. CONCLUSIONS: The results offer a new approach for regenerating damaged optic nerves and indicate that a significant number of E14.5 RPCs are able to differentiate into RGCs in the foreign environment of the adult retina. However, the proportion of RPCs that populated the ganglion cell layer and contributed to the optic nerve was not sufficient to account for the increased thickness and higher number of axons. The results support the hypothesis that the injected E14.5 RPCs are contributing autonomously and non-autonomously to restoring damaged optic nerves.

Deregulation of CREB signaling pathway induced by chronic hyperglycemia downregulates NeuroD transcription.

CREB mediates the transcriptional effects of glucose and incretin hormones in insulin-target cells and insulin-producing ß-cells. Although the inhibition of CREB activity is known to decrease the ß-cell mass, it is still unknown what factors inversely alter the CREB signaling pathway in ß-cells. Here, we show that ß-cell dysfunctions occurring in chronic hyperglycemia are not caused by simple inhibition of CREB activity but rather by the persistent activation of CREB due to decreases in protein phophatase PP2A. When freshly isolated rat pancreatic islets were chronically exposed to 25 mM (high) glucose, the PP2A activity was reduced with a concomitant increase in active pCREB. Brief challenges with 15 mM glucose or 30 µM forskolin after 2 hour fasting further increased the level of pCREB and consequently induced the persistent expression of ICER. The excessively produced ICER was sufficient to repress the transcription of NeuroD, insulin, and SUR1 genes. In contrast, when islets were grown in 5 mM (low) glucose, CREB was transiently activated in response to glucose or forskolin stimuli. Thus, ICER expression was transient and insufficient to repress those target genes. Importantly, overexpression of PP2A reversed the adverse effects of chronic hyperglycemia and successfully restored the transient activation of CREB and ICER. Conversely, depletion of PP2A with siRNA was sufficient to disrupt the negative feedback regulation of CREB and induce hyperglycemic phenotypes even under low glucose conditions. Our findings suggest that the failure of the negative feedback regulation of CREB is the primary cause for ß-cell dysfunctions under conditions of pathogenic hyperglycemia, and PP2A can be a novel target for future therapies aiming to protect ß-cells mass in the late transitional phase of non-insulin dependent type 2 diabetes (NIDDM).

Overlapping spatiotemporal patterns of regulatory gene expression are required for neuronal progenitors to specify retinal ganglion cell fate.

Retinal progenitor cells (RPCs) are programmed early in development to acquire the competence for specifying the seven retinal cell types. Acquiring competence is a complex spatiotemporal process that is still only vaguely understood. Here, our objective was to more fully understand the mechanisms by which RPCs become competent for specifying a retinal ganglion cell (RGC) fate. RGCs are the first retinal cell type to differentiate and their abnormal development leads to apoptosis and optic nerve degeneration. Previous work demonstrated that the paired domain factor Pax6 and the bHLH factor Atoh7 are required for RPCs to specify RGCs. RGC commitment is marked by the expression of the Pou domain factor Pou4f2 and the Lim domain factor Isl1. We show that three RPC subpopulations can specify RGCs: Atoh7-expressing RPCs, Neurod1-expressing RPCs, and Atoh7-Neurod1-expressing RPCs. All three RPC subpopulations were highly interspersed throughout retinal development, although each subpopulation maintained a distinct temporal pattern. Most, but not all, RPCs from each subpopulation were postmitotic. Atoh7-Neurod1 double knockout mice were generated and double-mutant retinas revealed an unexpected role for Neurod1 in specifying RGC fate. We conclude that RPCs have a complex regulatory gene expression program in which they acquire competence using highly integrated mechanisms.

Neuronal transcriptional repressor REST suppresses an Atoh7-independent program for initiating retinal ganglion cell development.

As neuronal progenitors differentiate into neurons, they acquire a unique set of transcription factors. The transcriptional repressor REST prevents progenitors from undergoing differentiation. Notably, REST binding sites are often associated with retinal ganglion cell (RGC) genes whose expression in the retina is positively controlled by Atoh7, a factor essential for RGC formation. The key regulators that enable a retinal progenitor cell (RPC) to commit to an RGC fate have not been identified. We show here that REST suppresses RGC gene expression in RPCs. REST inactivation causes aberrant expression of RGC transcription factors in proliferating RPCs, independent of Atoh7, resulting in increased RGC formation. Strikingly, inactivating REST in Atoh7-null retinas restores transcription factor expression, which partially activates downstream RGC genes but is insufficient to prevent RGC loss. Our results demonstrate an Atoh7-independent program for initial activation of RGC genes and suggest a novel role for REST in preventing premature expression in RPCs.

Retinal ganglion cell death and optic nerve degeneration by genetic ablation in adult mice.

Despite the magnitude of the problem, no effective treatments exist to prevent retinal ganglion cell (RGC) death and optic nerve degeneration from occurring in diseases affecting the human eye. Animal models currently available for developing treatment strategies suffer from cumbersome procedures required to induce RGC death or rely on mutations that induce defects in developing retinas rather than in mature retinas of adults. Our objective was to develop a robust genetically engineered adult mouse model for RGC loss and optic nerve degeneration based on genetic ablation. To achieve this, we took advantage of Pou4f2 (Brn3b), a gene activated immediately as RGCs begin to differentiate and expressed throughout life. We generated adult mice whose genomes harbored a conditional Pou4f2 allele containing a floxed-lacZ-stop-diphtheria toxin A cassette and a CAGG-Cre-ER transgene. In this bigenic model, Cre recombinase is fused to a modified estrogen nuclear receptor in which the estrogen-binding domain binds preferentially to the estrogen agonist tamoxifen rather than to endogenous estradiol. Upon binding to the estrogen-binding domain, tamoxifen derepresses Cre recombinase, leading to the efficient genomic deletion of the floxed-lacZ-stop DNA sequence and expression of diphtheria toxin A. Tamoxifen administered to adult mice at different ages by intraperitoneal injection led to rapid RGC loss, reactive gliosis, progressive degradation of the optic nerve over a period of several months, and visual impairment. Perhaps more reflective of human disease, partial loss of RGCs was achieved by modulating the tamoxifen treatment. Especially relevant for RGC death and optic nerve degeneration in human retinal pathologies, RGC-ablated retinas maintained their structural integrity, and other retinal neurons and their connections in the inner and outer plexiform layers appeared unaffected by RGC ablation. These events are hallmarks of progressive optic nerve degeneration observed in human retinal pathologies and demonstrate the validity of this model for use in developing stem cell therapies for replacing dead RGCs with healthy ones.

The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis.

Vascularization is essential for tissue development and in restoration of tissue integrity after an ischemic injury. In studies of vascularization, the focus has largely been placed on vascular endothelial growth factor (VEGF), yet other factors may also orchestrate this process. Here we show that succinate accumulates in the hypoxic retina of rodents and, via its cognate receptor G protein-coupled receptor-91 (GPR91), is a potent mediator of vessel growth in the settings of both normal retinal development and proliferative ischemic retinopathy. The effects of GPR91 are mediated by retinal ganglion neurons (RGCs), which, in response to increased succinate levels, regulate the production of numerous angiogenic factors including VEGF. Accordingly, succinate did not have proangiogenic effects in RGC-deficient rats. Our observations show a pathway of metabolite signaling where succinate, acting through GPR91, governs retinal angiogenesis and show the propensity of RGCs to act as sensors of ischemic stress. These findings provide a new therapeutic target for modulating revascularization.

Myogenin regulates a distinct genetic program in adult muscle stem cells.

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.

Targeted disruption of NeuroD, a proneural basic helix-loop-helix factor, impairs distal lung formation and neuroendocrine morphology in the neonatal lung.

Despite the importance of airspace integrity in vertebrate gas exchange, the molecular pathways that instruct distal lung formation are poorly understood. Recently, we found that fibrillin-1 deficiency in mice impairs alveolar formation and recapitulates the pulmonary features of human Marfan syndrome. To further elucidate effectors involved in distal lung formation, we performed expression profiling analysis comparing the fibrillin-1-deficient and wild-type developing lung. NeuroD, a basic helix-loop-helix transcription factor, fulfilled the expression criteria for a candidate mediator of distal lung development. We investigated its role in murine lung development using genetically targeted NeuroD-deficient mice. We found that NeuroD deficiency results in both impaired alveolar septation and altered morphology of the pulmonary neuroendocrine cells. NeuroD-deficient mice had enlarged alveoli associated with reduced epithelial proliferation in the airway and airspace compartments during development. Additionally, the neuroendocrine compartment in these mice manifested an increased number of neuroepithelial bodies but a reduced number of solitary pulmonary neuroendocrine cells in the neonatal lung. Overexpression of NeuroD in a murine lung epithelial cell line conferred a neuroendocrine phenotype characterized by the induction of neuroendocrine markers as well as increased proliferation. These results support an unanticipated role for NeuroD in the regulation of pulmonary neuroendocrine and alveolar morphogenesis and suggest an intimate connection between the neuroendocrine compartment and distal lung development.

Target gene selectivity of the myogenic basic helix-loop-helix transcription factor myogenin in embryonic muscle.

The myogenic regulatory factors MyoD and myogenin are crucial for skeletal muscle development. Despite their importance, the mechanisms by which these factors selectively regulate different target genes are unclear. The purpose of the present investigation was to compare embryonic skeletal muscle from myogenin(+/+) and myogenin(-/-) mice to identify genes whose expression was dependent on the presence of myogenin but not MyoD and to determine whether myogenin-binding sites could be found within regulatory regions of myogenin-dependent genes independent of MyoD. We identified a set of 140 muscle-expressed genes whose expression in embryonic tongue muscle of myogenin(-/-) mice was downregulated in the absence of myogenin, but in the presence of MyoD. Myogenin bound within conserved regulatory regions of several of the downregulated genes, but MyoD bound only to a subset of these same regions, suggesting that many downregulated genes were selective targets of myogenin. The regulatory regions activated gene expression in cultured myoblasts and fibroblasts overexpressing myogenin or MyoD, indicating that expression from exogenously introduced DNA could not recapitulate the selectivity for myogenin observed in vivo. The results identify new target genes for myogenin and show that myogenin's target gene selectivity is not based solely on binding site sequences.

Altered traveling wave propagation and reduced endocochlear potential associated with cochlear dysplasia in the BETA2/NeuroD1 null mouse.

The BETA2/NeuroD1 null mouse has cochlear dysplasia. Its cochlear duct is shorter than normal, there is a lack of spiral ganglion neurons, and there is hair cell disorganization. We measured vertical movements of the tectorial membrane at acoustic frequencies in excised cochleae in response to mechanical stimulation of the stapes using laser doppler vibrometry. While tuning curve sharpness was similar between wild-type, heterozygotes, and null mice in the base, null mutants had broader tuning in the apex. At both the base and the apex, null mice had less phase lag accumulation with increasing stimulus frequency than wild-type or heterozygote mice. In vivo studies demonstrated that the null mouse lacked distortion product otoacoustic emissions, and the cochlear microphonic and endocochlear potential were found to be severely reduced. Electrically evoked otoacoustic emissions could be elicited, although the amplitudes were lower than those of wild-type mice. Cochlear cross-sections revealed an incomplete partition malformation, with fenestrations within the modiolus that connected the cochlear turns. Outer hair cells from null mice demonstrated the normal pattern of prestin expression within their lateral walls and normal FM 1-43 dye entry. Overall, these data demonstrate that while tonotopicity can exist with cochlear dysplasia, traveling wave propagation is abnormally fast. Additionally, the presence of electrically evoked otoacoustic emissions suggests that outer hair cell reverse transduction is present, although the acoustic response is shaped by the alterations in cochlear mechanics.

Compensational regulation of bHLH transcription factors in the postnatal development of BETA2/NeuroD1-null retina.

The bHLH transcriptional factor BETA2/NeuroD1 is essential for the survival of photoreceptor cells in the retina. Although this gene is expressed throughout the retina, BETA2/NeuroD1 knockout mice show photoreceptor cell degeneration only in the outer nuclear layer of the retina; other retinal neurons are not affected. Previous studies on retina explants lacking three bHLH genes revealed that retinal neurons in the inner nuclear layer require multiple bHLH genes for their differentiation and survival. However, single- or double-gene mutations show no or a lesser degree of abnormalities during eye development, likely because of compensation or cooperative regulation among those genes. Because not all null mice survive until the retina is fully organized, no direct evidence of this concept has been reported. To understand the regulatory mechanisms between bHLH factors in retinal development, we performed a detailed analysis of BETA2/NeuroD1 knockout mice. BETA2/NeuroD1 was expressed in all 3 layers of the mouse retina, including all major types of neurons. In addition, a null mutation of BETA2/NeuroD1 resulted in up-regulation of other bHLH genes, Mash1, Neurogenin2, and Math3, in the inner nuclear layer. Our data suggest that compensatory and cross regulatory mechanisms exist among the bHLH factors during retinal development.

Mutant neurogenin-3 in congenital malabsorptive diarrhea.

BACKGROUND: Neurogenin-3 (NEUROG3) is expressed in endocrine progenitor cells and is required for endocrine-cell development in the pancreas and intestine. The NEUROG3 gene (NEUROG3) is therefore a candidate for the cause of a newly discovered autosomal recessive disorder characterized by generalized malabsorption and a paucity of enteroendocrine cells. METHODS: We screened genomic DNA from three unrelated patients with sparse enteroendocrine cells for mutations of NEUROG3. We then tested the ability of the observed mutations to alter NEUROG3 function, using in vitro and in vivo assays. RESULTS: The patients had few intestinal enteroendocrine cells positive for chromogranin A, but they had normal numbers of Paneth's, goblet, and absorptive cells. We identified two homozygous mutations in NEUROG3, both of which rendered the NEUROG3 protein unable to activate NEUROD1, a downstream target of NEUROG3, and compromised the ability of NEUROG3 to bind to an E-box element in the NEUROD1 promoter. The injection of wild-type but not mutant NEUROG3 messenger RNA into xenopus embryos induced NEUROD1 expression. CONCLUSIONS: A newly discovered disorder characterized by malabsorptive diarrhea and a lack of intestinal enteroendocrine cells is caused by loss-of-function mutations in NEUROG3.

Preferential posterior cerebellum defect in BETA2/NeuroD1 knockout mice is the result of differential expression of BETA2/NeuroD1 along anterior-posterior axis.

BETA2/NeuroD1 has been shown to play a major role in terminal differentiation of the pancreatic and enteroendocrine cells, as well as for the survival of photoreceptors. Here, we report that the loss of BETA2/NeuroD1 affected the cerebellar development with a major reduction of granule cell number. However, there is a differential reduction of granule cells along the anterior and posterior axis of the cerebellum; while the reduction of granule cells in the anterior lobes is substantial, there is an almost complete loss of granule cells in the posterior compartment. To understand the mechanism for this anterior-posterior difference, we carried out detailed analyses. We found that both BETA2/NeuroD1 and its direct target TrkC, expression commence earlier in the posterior part than those in the anterior part during cerebellum development. Consequently, loss of BETA2/NeuroD1 enhances granule cell death in the posterior 2 days earlier than the anterior. Furthermore, the higher rate of cell death in the posterior of the cerebellum is concomitant with the reduction of TrkC expression in knockout mice. Thus, our data indicate that preferential expression of BETA2/NeuroD1 and TrkC in posterior lobes explains the earlier start of cell apoptosis and preferential loss of granule cells in the posterior lobes.