Crucial role of TFAP2B in the nervous system for regulating NREM sleep.

The AP-2 transcription factors are crucial for regulating sleep in both vertebrate and invertebrate animals. In mice, loss of function of the transcription factor AP-2ß (TFAP2B) reduces non-rapid eye movement (NREM) sleep. When and where TFAP2B functions, however, is unclear. Here, we used the Cre-loxP system to generate mice in which Tfap2b was specifically deleted in the nervous system during development and mice in which neuronal Tfap2b was specifically deleted postnatally. Both types of mice exhibited reduced NREM sleep, but the nervous system-specific deletion of Tfap2b resulted in more severe sleep phenotypes accompanied by defective light entrainment of the circadian clock and stereotypic jumping behavior. These findings indicate that TFAP2B in postnatal neurons functions at least partly in sleep regulation and imply that TFAP2B also functions either at earlier stages or in additional cell types within the nervous system.

SIK3-HDAC4 in the suprachiasmatic nucleus regulates the timing of arousal at the dark onset and circadian period in mice.

Mammals exhibit circadian cycles of sleep and wakefulness under the control of the suprachiasmatic nucleus (SCN), such as the strong arousal phase-locked to the beginning of the dark phase in laboratory mice. Here, we demonstrate that salt-inducible kinase 3 (SIK3) deficiency in gamma-aminobutyric acid (GABA)-ergic neurons or neuromedin S (NMS)-producing neurons delayed the arousal peak phase and lengthened the behavioral circadian cycle under both 12-h light:12-h dark condition (LD) and constant dark condition (DD) without changing daily sleep amounts. In contrast, the induction of a gain-of-function mutant allele of Sik3 in GABAergic neurons exhibited advanced activity onset and a shorter circadian period. Loss of SIK3 in arginine vasopressin (AVP)-producing neurons lengthened the circadian cycle, but the arousal peak phase was similar to that in control mice. Heterozygous deficiency of histone deacetylase (HDAC) 4, a SIK3 substrate, shortened the circadian cycle, whereas mice with HDAC4 S245A, which is resistant to phosphorylation by SIK3, delayed the arousal peak phase. Phase-delayed core clock gene expressions were detected in the liver of mice lacking SIK3 in GABAergic neurons. These results suggest that the SIK3-HDAC4 pathway regulates the circadian period length and the timing of arousal through NMS-positive neurons in the SCN.

Kinase signalling in excitatory neurons regulates sleep quantity and depth.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1-SIK3-HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

Mice Lacking Cerebellar Cortex and Related Structures Show a Decrease in Slow-Wave Activity With Normal Non-REM Sleep Amount and Sleep Homeostasis.

In addition to the well-known motor control, the cerebellum has recently been implicated in memory, cognition, addiction, and social behavior. Given that the cerebellum contains more neurons than the cerebral cortex and has tight connections to the thalamus and brainstem nuclei, it is possible that the cerebellum also regulates sleep/wakefulness. However, the role of the cerebellum in sleep was unclear, since cerebellar lesion studies inevitably involved massive inflammation in the adjacent brainstem, and sleep changes in lesion studies were not consistent with each other. Here, we examine the role of the cerebellum in sleep and wakefulness using mesencephalon- and rhombomere 1-specific Ptf1a conditional knockout (Ptf1a cKO) mice, which lack the cerebellar cortex and its related structures, and exhibit ataxic gait. Ptf1a cKO mice had similar wake and non-rapid eye movement sleep (NREMS) time as control mice and showed reduced slow wave activity during wakefulness, NREMS and REMS. Ptf1a cKO mice showed a decrease in REMS time during the light phase and had increased NREMS delta power in response to 6 h of sleep deprivation, as did control mice. Ptf1a cKO mice also had similar numbers of sleep spindles and fear memories as control mice. Thus, the cerebellum does not appear to play a major role in sleep-wake control, but may be involved in the generation of slow waves.

The Role of Reproductive Hormones in Sex Differences in Sleep Homeostasis and Arousal Response in Mice.

There are various sex differences in sleep/wake behaviors in mice. However, it is unclear whether there are sex differences in sleep homeostasis and arousal responses and whether gonadal hormones are involved in these sex differences. Here, we examined sleep/wake behaviors under baseline condition, after sleep deprivation by gentle handling, and arousal responses to repeated cage changes in male and female C57BL/6 mice that are hormonally intact, gonadectomized, or gonadectomized with hormone supplementation. Compared to males, females had longer wake time, shorter non-rapid eye movement sleep (NREMS) time, and longer rapid eye movement sleep (REMS) episodes. After sleep deprivation, males showed an increase in NREMS delta power, NREMS time, and REMS time, but females showed a smaller increase. Females and males showed similar arousal responses. Gonadectomy had only a modest effect on homeostatic sleep regulation in males but enhanced it in females. Gonadectomy weakened arousal response in males and females. With hormone replacement, baseline sleep in gonadectomized females was similar to that of intact females, and baseline sleep in gonadectomized males was close to that of intact males. Gonadal hormone supplementation restored arousal response in males but not in females. These results indicate that male and female mice differ in their baseline sleep-wake behavior, homeostatic sleep regulation, and arousal responses to external stimuli, which are differentially affected by reproductive hormones.

Efficient induction of proximity-dependent labelling by biotin feeding in BMAL1-BioID knock-in mice.

Proximity-dependent biotin identification (BioID) is a useful method to identify unknown protein-protein interactions. Few reports have described genetically engineered knock-in mouse models for in vivo BioID. Thus, little is known about the proper method for biotin administration and which tissues are applicable. Here, we established a BioID knock-in mouse model of Brain and Muscle ARNT-Like 1 (BMAL1) and the BirA biotin ligase with R118G mutation (BirA*). The BMAL1-BioID mouse model was used to investigate the effect of biotin diet feeding on protein biotinylation in several tissues. The BMAL1-BirA* fusion protein-retained proper intracellular localization of BMAL1 and binding to CLOCK protein in HEK293T cells. A biotin labelling assay in mouse embryonic fibroblasts revealed the protein biotinylation activity of BMAL1-BirA* expressed in knock-in mouse cells depending on biotin supplementation. Lastly, feeding a 0.5% biotin diet for 7 days induced protein biotinylation in the brain, heart, testis and liver of BMAL1-BioID mice without adverse effects on spermatogenesis. In the kidney, the biotin diet increased biotinylated protein levels in BMAL1-BioID and control mice, suggesting the existence of endogenous biotinylation activity. These results provide valuable information to optimize the in vivo BioID procedure.

An endothelial activin A-bone morphogenetic protein receptor type 2 link is overdriven in pulmonary hypertension.

Pulmonary arterial hypertension is a progressive fatal disease that is characterized by pathological pulmonary artery remodeling, in which endothelial cell dysfunction is critically involved. We herein describe a previously unknown role of endothelial angiocrine in pulmonary hypertension. By searching for genes highly expressed in lung microvascular endothelial cells, we identify inhibin-ß-A as an angiocrine factor produced by pulmonary capillaries. We find that excess production of inhibin-ß-A by endothelial cells impairs the endothelial function in an autocrine manner by functioning as activin-A. Mechanistically, activin-A induces bone morphogenetic protein receptor type 2 internalization and targeting to lysosomes for degradation, resulting in the signal deficiency in endothelial cells. Of note, endothelial cells isolated from the lung of patients with idiopathic pulmonary arterial hypertension show higher inhibin-ß-A expression and produce more activin-A compared to endothelial cells isolated from the lung of normal control subjects. When endothelial activin-A-bone morphogenetic protein receptor type 2 link is overdriven in mice, hypoxia-induced pulmonary hypertension was exacerbated, whereas conditional knockout of inhibin-ß-A in endothelial cells prevents the progression of pulmonary hypertension. These data collectively indicate a critical role for the dysregulated endothelial activin-A-bone morphogenetic protein receptor type 2 link in the progression of pulmonary hypertension, and thus endothelial inhibin-ß-A/activin-A might be a potential pharmacotherapeutic target for the treatment of pulmonary arterial hypertension.

Induction of Mutant Sik3Sleepy Allele in Neurons in Late Infancy Increases Sleep Need.

Sleep is regulated in a homeostatic manner. Sleep deprivation increases sleep need, which is compensated mainly by increased EEG δ power during non-rapid eye movement sleep (NREMS) and, to a lesser extent, by increased sleep amount. Although genetic factors determine the constitutive level of sleep need and sleep amount in mice and humans, the molecular entity behind sleep need remains unknown. Recently, we found that a gain-of-function Sleepy (Slp) mutation in the salt-inducible kinase 3 (Sik3) gene, which produces the mutant SIK3(SLP) protein, leads to an increase in NREMS EEG δ power and sleep amount. Since Sik3Slp mice express SIK3(SLP) in various types of cells in the brain as well as multiple peripheral tissues from the embryonic stage, the cell type and developmental stage responsible for the sleep phenotype in Sik3Slp mice remain to be elucidated. Here, we generated two mouse lines, synapsin1CreERT2 and Sik3ex13flox mice, which enable inducible Cre-mediated, conditional expression of SIK3(SLP) in neurons on tamoxifen administration. Administration of tamoxifen to synapsin1CreERT2 mice during late infancy resulted in higher recombination efficiency than administration during adolescence. SIK3(SLP) expression after late infancy increased NREMS and NREMS δ power in male synapsin1CreERT2; Sik3ex13flox/+ mice. The expression of SIK3(SLP) after adolescence led to a higher NREMS δ power without a significant change in NREMS amounts. Thus, neuron-specific expression of SIK3(SLP) after late infancy is sufficient to increase sleep.SIGNIFICANCE STATEMENT The propensity to accumulate sleep need during wakefulness and to dissipate it during sleep underlies the homeostatic regulation of sleep. However, little is known about the developmental stage and cell types involved in determining the homeostatic regulation of sleep. Here, we show that Sik3Slp allele induction in mature neurons in late infancy is sufficient to increase non-rapid eye movement sleep amount and non-rapid eye movement sleep δ power. SIK3 signaling in neurons constitutes an intracellular mechanism to increase sleep.

Sleep Architecture in Mice Is Shaped by the Transcription Factor AP-2ß.

The molecular mechanism regulating sleep largely remains to be elucidated. In humans, families that carry mutations in TFAP2B, which encodes the transcription factor AP-2ß, self-reported sleep abnormalities such as short-sleep and parasomnia. Notably, AP-2 transcription factors play essential roles in sleep regulation in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster Thus, AP-2 transcription factors might have a conserved role in sleep regulation across the animal phyla. However, direct evidence supporting the involvement of TFAP2B in mammalian sleep was lacking. In this study, by using the CRISPR/Cas9 technology, we generated two Tfap2b mutant mouse strains, Tfap2bK144 and Tfap2bK145 , each harboring a single-nucleotide mutation within the introns of Tfap2b mimicking the mutations in two human kindreds that self-reported sleep abnormalities. The effects of these mutations were compared with those of a Tfap2b knockout allele (Tfap2b-). The protein expression level of TFAP2B in the embryonic brain was reduced to about half in Tfap2b+/- mice and was further reduced in Tfap2b-/- mice. By contrast, the protein expression level was normal in Tfap2bK145/+ mice but was reduced in Tfap2bK145/K145 mice to a similar extent as Tfap2b-/- mice. Tfap2bK144/+ and Tfap2bK144/K144 showed normal protein expression levels. Tfap2b+/- female mice showed increased wakefulness time and decreased nonrapid eye movement sleep (NREMS) time. By contrast, Tfap2bK145/+ female mice showed an apparently normal amount of sleep but instead exhibited fragmented NREMS, whereas Tfap2bK144/+ male mice showed reduced NREMS time specifically in the dark phase. Finally, in the adult brain, Tfap2b-LacZ expression was detected in the superior colliculus, locus coeruleus, cerebellum, and the nucleus of solitary tract. These findings provide direct evidence that TFAP2B influences NREMS amounts in mice and also show that different mutations in Tfap2b can lead to diverse effects on sleep architecture.

Loss of the conserved PKA sites of SIK1 and SIK2 increases sleep need.

Although sleep is one of the most conserved behaviors, the intracellular mechanism regulating sleep/wakefulness remains unknown. We recently identified a protein kinase, SIK3, as a sleep-regulating molecule. Mice that lack a well-conserved protein kinase A (PKA) phosphorylation site, S551, showed longer non-rapid eye movement (NREM) sleep and increased NREMS delta density. S551 of SIK3 is conserved in other members of the SIK family, such as SIK1 (S577) and SIK2 (S587). Here, we examined whether the PKA phosphorylation sites of SIK1 and SIK2 are involved in sleep regulation by generating Sik1S577A and Sik2S587A mice. The homozygous Sik1S577A mice showed a shorter wake time, longer NREMS time, and higher NREMS delta density than the wild-type mice. The heterozygous and homozygous Sik2S587A mice showed increased NREMS delta density. Both the Sik1S577A and Sik2S587A mice exhibited proper homeostatic regulation of sleep need after sleep deprivation. Despite abundant expression of Sik1 in the suprachiasmatic nucleus, the Sik1S577A mice showed normal circadian behavior. Although Sik2 is highly expressed in brown adipose tissue, the male and female Sik2S587A mice that were fed either a chow or high-fat diet showed similar weight gain as the wild-type littermates. These results suggest that PKA-SIK signaling is involved in the regulation of sleep need.

Ptf1a inactivation in adult pancreatic acinar cells causes apoptosis through activation of the endoplasmic reticulum stress pathway.

Pancreas transcription factor 1 subunit alpha (PTF1A) is one of the key regulators in pancreatogenesis. In adults, it transcribes digestive enzymes, but its other functions remain largely unknown. Recent conditional knockout studies using Ptf1aCreER/floxed heterozygous mouse models have found PTF1A contributes to the identity maintenance of acinar cells and prevents tumorigenesis caused by the oncogenic gene Kras. However, Ptf1a heterozygote is known to behave differently from homozygote. To elucidate the effects of Ptf1a homozygous loss, we prepared Elastase-CreERTM; Ptf1afloxed/floxed mice and found that homozygous Ptf1a deletion in adult acinar cells causes severe apoptosis. Electron microscopy revealed endoplasmic reticulum (ER) stress, a known cause of unfolded protein responses (UPR). We confirmed that UPR was upregulated by the activating transcription factor 6 (ATF6) and protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) pathways, but not the inositol requiring enzyme 1 (IRE1) pathway. Furthermore, we detected the expression of CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP), a pro-apoptotic factor, indicating the apoptosis was induced through UPR. Our homozygous model helps clarify the role PTF1A has on the homeostasis and pathogenesis of exocrine pancreas in mice.

A single phosphorylation site of SIK3 regulates daily sleep amounts and sleep need in mice.

Sleep is an evolutionally conserved behavior from vertebrates to invertebrates. The molecular mechanisms that determine daily sleep amounts and the neuronal substrates for homeostatic sleep need remain unknown. Through a large-scale forward genetic screen of sleep behaviors in mice, we previously demonstrated that the Sleepy mutant allele of the Sik3 protein kinase gene markedly increases daily nonrapid-eye movement sleep (NREMS) amounts and sleep need. The Sleepy mutation deletes the in-frame exon 13 encoding a peptide stretch encompassing S551, a known PKA recognition site in SIK3. Here, we demonstrate that single amino acid changes at SIK3 S551 (S551A and S551D) reproduce the hypersomnia phenotype of the Sleepy mutant mice. These mice exhibit increased NREMS amounts and inherently increased sleep need, the latter demonstrated by increased duration of individual NREMS episodes and higher EEG slow-wave activity during NREMS. At the molecular level, deletion or mutation at SIK3 S551 reduces PKA recognition and abolishes 14-3-3 binding. Our results suggest that the evolutionally conserved S551 of SIK3 mediates, together with PKA and 14-3-3, the intracellular signaling crucial for the regulation of daily sleep amounts and sleep need at the organismal level.

Forebrain Ptf1a Is Required for Sexual Differentiation of the Brain.

The mammalian brain undergoes sexual differentiation by gonadal hormones during the perinatal critical period. However, the machinery at earlier stages has not been well studied. We found that Ptf1a is expressed in certain neuroepithelial cells and immature neurons around the third ventricle that give rise to various neurons in several hypothalamic nuclei. We show that conditional Ptf1a-deficient mice (Ptf1a cKO) exhibit abnormalities in sex-biased behaviors and reproductive organs in both sexes. Gonadal hormone administration to gonadectomized animals revealed that the abnormal behavior is caused by disorganized sexual development of the knockout brain. Accordingly, expression of sex-biased genes was severely altered in the cKO hypothalamus. In particular, Kiss1, important for sexual differentiation of the brain, was drastically reduced in the cKO hypothalamus, which may contribute to the observed phenotypes in the Ptf1a cKO. These findings suggest that forebrain Ptf1a is one of the earliest regulators for sexual differentiation of the brain.

Quantitative phosphoproteomic analysis of the molecular substrates of sleep need.

Sleep and wake have global effects on brain physiology, from molecular changes1-4 and neuronal activities to synaptic plasticity3-7. Sleep-wake homeostasis is maintained by the generation of a sleep need that accumulates during waking and dissipates during sleep8-11. Here we investigate the molecular basis of sleep need using quantitative phosphoproteomic analysis of the sleep-deprived and Sleepy mouse models of increased sleep need. Sleep deprivation induces cumulative phosphorylation of the brain proteome, which dissipates during sleep. Sleepy mice, owing to a gain-of-function mutation in the Sik3 gene 12 , have a constitutively high sleep need despite increased sleep amount. The brain proteome of these mice exhibits hyperphosphorylation, similar to that seen in the brain of sleep-deprived mice. Comparison of the two models identifies 80 mostly synaptic sleep-need-index phosphoproteins (SNIPPs), in which phosphorylation states closely parallel changes of sleep need. SLEEPY, the mutant SIK3 protein, preferentially associates with and phosphorylates SNIPPs. Inhibition of SIK3 activity reduces phosphorylation of SNIPPs and slow wave activity during non-rapid-eye-movement sleep, the best known measurable index of sleep need, in both Sleepy mice and sleep-deprived wild-type mice. Our results suggest that phosphorylation of SNIPPs accumulates and dissipates in relation to sleep need, and therefore SNIPP phosphorylation is a molecular signature of sleep need. Whereas waking encodes memories by potentiating synapses, sleep consolidates memories and restores synaptic homeostasis by globally downscaling excitatory synapses4-6. Thus, the phosphorylation-dephosphorylation cycle of SNIPPs may represent a major regulatory mechanism that underlies both synaptic homeostasis and sleep-wake homeostasis.

Forward-genetics analysis of sleep in randomly mutagenized mice.

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

Identification of mutations through dominant screening for obesity using C57BL/6 substrains.

The discovery of leptin substantiated the usefulness of a forward genetic approach in elucidating the molecular network regulating energy metabolism. However, no successful dominant screening for obesity has been reported, which may be due to the influence of quantitative trait loci between the screening and counter strains and the low fertility of obese mice. Here, we performed a dominant screening for obesity using C57BL/6 substrains, C57BL/6J and C57BL/6N, with the routine use of in vitro fertilization. The screening of more than 5000 mutagenized mice established two obese pedigrees in which single nucleotide substitutions in Mc4r and Sim1 genes were identified through whole-exome sequencing. The mutation in the Mc4r gene produces a premature stop codon, and the mutant SIM1 protein lacks transcriptional activity, showing that the haploinsufficiency of SIM1 and MC4R results in obesity. We further examined the hypothalamic neuropeptide expressions in the mutant pedigrees and mice with diet-induced obesity, which showed that each obesity mouse model has distinct neuropeptide expression profiles. This forward genetic screening scheme is useful and applicable to any research field in which mouse models work.

The retrotrapezoid nucleus neurons expressing Atoh1 and Phox2b are essential for the respiratory response to CO2.

Maintaining constant CO2 and H(+) concentrations in the arterial blood is critical for life. The principal mechanism through which this is achieved in mammals is the respiratory chemoreflex whose circuitry is still elusive. A candidate element of this circuitry is the retrotrapezoid nucleus (RTN), a collection of neurons at the ventral medullary surface that are activated by increased CO2 or low pH and project to the respiratory rhythm generator. Here, we use intersectional genetic strategies to lesion the RTN neurons defined by Atoh1 and Phox2b expression and to block or activate their synaptic output. Photostimulation of these neurons entrains the respiratory rhythm. Conversely, abrogating expression of Atoh1 or Phox2b or glutamatergic transmission in these cells curtails the phrenic nerve response to low pH in embryonic preparations and abolishes the respiratory chemoreflex in behaving animals. Thus, the RTN neurons expressing Atoh1 and Phox2b are a necessary component of the chemoreflex circuitry.

Unipotent, Atoh1+ progenitors maintain the Merkel cell population in embryonic and adult mice.

Resident progenitor cells in mammalian skin generate new cells as a part of tissue homeostasis. We sought to identify the progenitors of Merkel cells, a unique skin cell type that plays critical roles in mechanosensation. We found that some Atoh1-expressing cells in the hairy skin and whisker follicles are mitotically active at embryonic and postnatal ages. Genetic fate-mapping revealed that these Atoh1-expressing cells give rise solely to Merkel cells. Furthermore, selective ablation of Atoh1(+) skin cells in adult mice led to a permanent reduction in Merkel cell numbers, demonstrating that other stem cell populations are incapable of producing Merkel cells. These data identify a novel, unipotent progenitor population in the skin that gives rise to Merkel cells both during development and adulthood.

Temporal identity transition from Purkinje cell progenitors to GABAergic interneuron progenitors in the cerebellum.

In the cerebellum, all GABAergic neurons are generated from the Ptf1a-expressing ventricular zone (Ptf1a domain). However, the machinery to produce different types of GABAergic neurons remains elusive. Here we show temporal regulation of distinct GABAergic neuron progenitors in the cerebellum. Within the Ptf1a domain at early stages, we find two subpopulations; dorsally and ventrally located progenitors that express Olig2 and Gsx1, respectively. Lineage tracing reveals the former are exclusively Purkinje cell progenitors (PCPs) and the latter Pax2-positive interneuron progenitors (PIPs). As development proceeds, PCPs gradually become PIPs starting from ventral to dorsal. In gain- and loss-of-function mutants for Gsx1 and Olig1/2, we observe abnormal transitioning from PCPs to PIPs at inappropriate developmental stages. Our findings suggest that the temporal identity transition of cerebellar GABAergic neuron progenitors from PCPs to PIPs is negatively regulated by Olig2 and positively by Gsx1, and contributes to understanding temporal control of neuronal progenitor identities.

Atoh1 directs hair cell differentiation and survival in the late embryonic mouse inner ear.

Atoh1 function is required for the earliest stages of inner ear hair cell development, which begins during the second week of gestation. Atoh1 expression in developing hair cells continues until early postnatal ages, but the function of this late expression is unknown. To test the role of continued Atoh1 expression in hair cell maturation we conditionally deleted the gene in the inner ear at various embryonic and postnatal ages. In the organ of Corti, deletion of Atoh1 at E15.5 led to the death of all hair cells. In contrast, deletion at E16.5 caused death only in apical regions, but abnormalities of stereocilia formation were present throughout the cochlea. In the utricle, deletion at E14.5 or E16.5 did not cause cell death but led to decreased expression of myosin VIIa and failure of stereocilia formation. Furthermore, we show that maintained expression of Barhl1 and Gfi1, two transcription factors implicated in cochlear hair cell survival, depends upon continued Atoh1 expression. However, maintained expression of Pou4f3 and several hair cell-specific markers is independent of Atoh1 expression. These data reveal novel late roles for Atoh1 that are separable from its initial role in hair cell development.