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Aberration of Purkinje cell (PC) dendrites was directly proportional to granule cell loss in Neurod1 conditional mutant cerebellum, as shown by calbindin staining (a-d " ) and dye tracing (e-e " ) in adult mice (ML molecular layer, PCL PC layer, GCL granule cell layer). aa " , e PCs were oriented in a monolayer with nicely organized dendrites in ML throughout all the lobules in control cerebellum. b-b " In Neurod1 mutant cerebellum, normal organization of PCs were observed only in lobules X and IX, where PCs were arranged in a single layer, and their dendrites projected toward the ML. c-c " , e' In the central lobules (1/2VI-1/2VIII) of mutant cerebellum, PCs were randomly organized with no specific direction of their dendrites. d-d " , e " In the anterior lobules (I-1/2VI) of mutant cerebellum, PCs almost formed a single layer, but their dendrites were either inversely polarized (arrow) toward GCL or Bifurcated and bidirectional (e " ) to both the pial surface (dotted blue line) and GCL. Bars 100 μm (a-b', c-c', d-d'), 50 μm (c " , d " ), 10 μm (e-e " )
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Neurod1 is a crucial basic helix-loop-helix gene for most cerebellar granule cells and mediates the differentiation of these cells downstream of Atoh1-mediated proliferation of the precursors. In Neurod1 null mice, granule cells die throughout the posterior two thirds of the cerebellar cortex during development. However, Neurod1 is also necessary f...
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Background
Focused ultrasound (FUS) has excellent characteristics over other non-invasive stimulation methods in terms of spatial resolution and steering capability of the target. FUS has not been tested in the cerebellar cortex and cellular effects of FUS are not fully understood.
Objective/Hypothesis
To investigate how the activity of cerebellar...
While multiple monoamines modulate cerebellar output, the mechanistic details of dopaminergic signaling in the cerebellum remain poorly understood. Here we show that Drd1 dopamine receptors are expressed in unipolar brush cells (UBCs) of the cerebellar vermis. Drd1 activation increases UBC firing rate and postsynaptic NMDA receptor-mediated current...
Citations
... While ELP1 was absent from the EGL of the anterior and central lobules of Elp1 cKO cerebella, ELP1 was present in the nodular lobules IX and X, where Atoh1-Cre transgene activity is known to be markedly reduced (Pan et al. 2009). ...
Familial Dysautonomia (FD) is an autosomal recessive disorder caused by a splice site mutation in the gene ELP1, which disproportionally affects neurons. While classically characterized by deficits in sensory and autonomic neurons, neuronal defects in the central nervous system have been described. ELP1 is highly expressed in the normal developing and adult cerebellum, but its role in cerebellum development is unknown. To investigate the cerebellar function of Elp1, we knocked out Elp1 in cerebellar granule cell progenitors (GCPs) and examined the outcome on animal behavior and cellular composition. We found that GCP-specific conditional knockout of Elp1 (Elp1cKO) resulted in ataxia by 8 weeks of age. Cellular characterization showed that the animals had smaller cerebella with fewer granule cells. This defect was already apparent 7 days after birth, when Elp1cKO animals also exhibited fewer mitotic GCPs and shorter Purkinje dendrites. Through molecular characterization, we found that loss of Elp1 was associated with an increase in apoptotic cell death and cell stress pathways in GCPs. Our study demonstrates the importance of ELP1 within the developing cerebellum, and suggests that Elp1 loss in the GC lineage may also play a role in the progressive ataxia phenotypes of FD patients.
... ATOH1 regulates early neuronal differentiation leading to the formation of CGC precursors, and its deletion results in the elimination of the granule cell population (Brown and Witman 2014;Lange et al. 2021). NEUROD1 acts subsequently to promote the differentiation of CGC precursors towards the mature cerebellar granule cell lineage (Pan et al. 2009); it is also involved in the maintenance of this differentiated population, as evidenced by the increased cell death detected in NeuroD1 knockout models (Warburton-Pitt et al. 2012). In light of this, it is tempting to speculate that the observed reduction of NEUROD1 expression may somehow relate to the massive cell loss experienced by three mutant cell lines. ...
Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability, and a peculiar cerebellar and brainstem malformation, the “molar tooth sign.” Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper, we developed an in vitro neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (AHI1, CPLANE1, TMEM67, and CC2D2A) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31 days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context.
... S1), there was evident separation between KIRREL2 positive cells that generate Purkinje cell progenitors and PAX6 positive cells that are granule cell progenitors. At D80, Calbindin D28K (CALB) was expressed in mature Purkinje cells(35) and NEUROD1 was expressed in mature granule neurons(36). CALB positive Purkinje cells were expressed at one side of the organoid in organoids from both lines (Fig. 2 and Supplementary Material, Fig. S1). ...
Neurons within the cerebellum form temporal-spatial connections through the cerebellum, and the entire brain. Organoid models provide an opportunity to model the early differentiation of the developing human cerebellum, which is difficult to study in vivo, and affords the opportunity to study neurodegenerative and neurodevelopmental diseases of the cerebellum. Previous cerebellar organoid models focused on early neuron generation and single cell activity. Here, we modify previous protocols to generate more mature cerebellar organoids that allow for the establishment of several classes of mature neurons during cerebellar differentiation and development, including the establishment of neural networks during whole organoid maturation. This will provide a means to study the generation of several more mature cerebellar cell types, including Purkinje cells, granule cells, interneurons expression as well as neuronal communication for biomedical, clinical, and pharmaceutical application.
... Of these genes, only 55/964 have previously been implicated in cerebellar development using a previously established database of genes critical for cerebellar development and function [32]. These included genes critical for cerebellar granule cell development such as Neurod1 and Pax6 [33,34] (Fig. 3B). Strikingly, the vast majority of these targets (909/964) have not yet been investigated in the context of cerebellar development. ...
... Overall, the results of the GO enrichment analysis were corroborated by the functions and expression patterns of known cerebellar genes within these clusters (Fig. 4B). In Cluster 1, we identified genes that are expressed in granule and Purkinje cells and are essential for the differentiation and maturation such as Neurod1 and Cacana1 [33,34]. Cluster 2-contained genes, such as Foxp2 and Cdk5r1, which are expressed in cells within the cerebellar parenchyma which contain developing PCs and interneurons. ...
Background
The development of the brain requires precise coordination of molecular processes across many cell-types. Underpinning these events are gene expression programs which require intricate regulation by non-coding regulatory sequences known as enhancers. In the context of the developing brain, transcribed enhancers (TEs) regulate temporally-specific expression of genes critical for cell identity and differentiation. Transcription of non-coding RNAs at active enhancer sequences, known as enhancer RNAs (eRNAs), is tightly associated with enhancer activity and has been correlated with target gene expression. TEs have been characterized in a multitude of developing tissues, however their regulatory role has yet to be described in the context of embryonic and early postnatal brain development. In this study, eRNA transcription was analyzed to identify TEs active during cerebellar development, as a proxy for the developing brain. Cap Analysis of Gene Expression followed by sequencing (CAGE-seq) was conducted at 12 stages throughout embryonic and early postnatal cerebellar development.
Results
Temporal analysis of eRNA transcription identified clusters of TEs that peak in activity during either embryonic or postnatal times, highlighting their importance for temporally specific developmental events. Functional analysis of putative target genes identified molecular mechanisms under TE regulation revealing that TEs regulate genes involved in biological processes specific to neurons. We validate enhancer activity using in situ hybridization of eRNA expression from TEs predicted to regulate Nfib, a gene critical for cerebellar granule cell differentiation.
Conclusions
The results of this analysis provide a valuable dataset for the identification of cerebellar enhancers and provide insight into the molecular mechanisms critical for brain development under TE regulation. This dataset is shared with the community through an online resource (https://goldowitzlab.shinyapps.io/trans-enh-app/).
... NEUROD1 is a basic helix-loop-helix (bHLH) transcription factor regulating the development of the cerebellum, hippocampal dentate gyrus, olfactory system, inner ear and auditory system, retina, and endocrine pancreas; it forms heterodimers with other bHLH transcription factors and binds to E box-containing promoter sequences to regulate gene expression of target genes (Naya et al., 1997;Poulin et al., 1997;Miyata et al., 1999;Liu et al., 2000;Breslin et al., 2003;Bernardo et al., 2008;Pan et al., 2009;Boutin et al., 2010;Evsen et al., 2013;Mastracci et al., 2013). NEUROD1 is located on human chromosome 2 and well-known of regulating β-cell development, insulin synthesis and secretion, as well as glucose homeostasis (Huang et al., 2002;Petersen et al., 2002;Andrali et al., 2007;Romer et al., 2019). ...
Photoreceptor development of the vertebrate visual system is controlled by a complex transcription regulatory network. OTX2 is expressed in the mitotic retinal progenitor cells (RPCs) and controls photoreceptor genesis. CRX that is activated by OTX2 is expressed in photoreceptor precursors after cell cycle exit. NEUROD1 is also present in photoreceptor precursors that are ready to specify into rod and cone photoreceptor subtypes. NRL is required for the rod fate and regulates downstream rod-specific genes including the orphan nuclear receptor NR2E3 which further activates rod-specific genes and simultaneously represses cone-specific genes. Cone subtype specification is also regulated by the interplay of several transcription factors such as THRB and RXRG. Mutations in these key transcription factors are responsible for ocular defects at birth such as microphthalmia and inherited photoreceptor diseases such as Leber congenital amaurosis (LCA), retinitis pigmentosa (RP) and allied dystrophies. In particular, many mutations are inherited in an autosomal dominant fashion, including the majority of missense mutations in CRX and NRL. In this review, we describe the spectrum of photoreceptor defects that are associated with mutations in the above-mentioned transcription factors, and summarize the current knowledge of molecular mechanisms underlying the pathogenic mutations. At last, we deliberate the outstanding gaps in our understanding of the genotype-phenotype correlations and outline avenues for future research of the treatment strategies.
... Differentiating granule cells form axons that interact with the dendrites of Purkinje cells, while the differentiating GCs migrate from the EGL to the IGL [91]. With a NeuroD1 knockout mouse model of NeuroD1 depletion in cerebellar granule cell precursors, it was demonstrated that NeuroD1 can influence a rapid transition from proliferative precursors to granule cells [92]. Similarly, in the global absence of NeuroD1, preferential posterior cerebellar defects in granule cells have been reported [93], as well as the elimination of granule cells in the central lobes and abnormalities in Purkinje cells in a GPC-selective NeuroD1 knockout model [92]. ...
... With a NeuroD1 knockout mouse model of NeuroD1 depletion in cerebellar granule cell precursors, it was demonstrated that NeuroD1 can influence a rapid transition from proliferative precursors to granule cells [92]. Similarly, in the global absence of NeuroD1, preferential posterior cerebellar defects in granule cells have been reported [93], as well as the elimination of granule cells in the central lobes and abnormalities in Purkinje cells in a GPC-selective NeuroD1 knockout model [92]. These studies with transgene mice support the importance of interaction between Purkinje and granule cells during cerebellar development, despite the variability in phenotypes when NeuroD1 is impaired. ...
Impaired cerebellar development of premature infants and the associated impairment of cerebellar functions in cognitive development could be crucial factors for neurodevelopmental disorders. Anesthetic- and hyperoxia-induced neurotoxicity of the immature brain can lead to learning and behavioral disorders. Dexmedetomidine (DEX), which is associated with neuroprotective properties, is increasingly being studied for off-label use in the NICU. For this purpose, six-day-old Wistar rats (P6) were exposed to hyperoxia (80% O2) or normoxia (21% O2) for 24 h after DEX (5 µg/kg, i.p.) or vehicle (0.9% NaCl) application. An initial detection in the immature rat cerebellum was performed after the termination of hyperoxia at P7 and then after recovery in room air at P9, P11, and P14. Hyperoxia reduced the proportion of Calb1+-Purkinje cells and affected the dendrite length at P7 and/or P9/P11. Proliferating Pax6+-granule progenitors remained reduced after hyperoxia and until P14. The expression of neurotrophins and neuronal transcription factors/markers of proliferation, migration, and survival were also reduced by oxidative stress in different manners. DEX demonstrated protective effects on hyperoxia-injured Purkinje cells, and DEX without hyperoxia modulated neuronal transcription in the short term without any effects at the cellular level. DEX protects hyperoxia-damaged Purkinje cells and appears to differentially affect cerebral granular cell neurogenesis following oxidative stress.
... For instance, Atoh1 expression extends along the roof plate to the cerebellum, parallel to the slightly more ventral expression of Neurog1/2, and the loss of dorsal neurons in Atoh1 null mice [57,58] results in a reduced cerebellum and auditory nuclei [52]. Neurod1 negatively regulates Atoh1 expression during cerebellum, and gut proliferation and manipulating Neurod1 expression that may help to counteract Medulloblastoma [67][68][69][70]. Atoh1 shows a much higher level of expression in the auditory nuclei and counteracts with Neurod1, indicating a differential regulation of expression in auditory nuclei [71]. ...
Sensorineural hearing loss is the most prevalent sensory deficit in humans. Most cases of hearing loss are due to the degeneration of key structures of the sensory pathway in the cochlea, such as the sensory hair cells, the primary auditory neurons, and their synaptic connection to the hair cells. Different cell-based strategies to replace damaged inner ear neurosensory tissue aiming at the restoration of regeneration or functional recovery are currently the subject of intensive research. Most of these cell-based treatment approaches require experimental in vitro models that rely on a fine understanding of the earliest morphogenetic steps that underlie the in vivo development of the inner ear since its initial induction from a common otic–epibranchial territory. This knowledge will be applied to various proposed experimental cell replacement strategies to either address the feasibility or identify novel therapeutic options for sensorineural hearing loss. In this review, we describe how ear and epibranchial placode development can be recapitulated by focusing on the cellular transformations that occur as the inner ear is converted from a thickening of the surface ectoderm next to the hindbrain known as the otic placode to an otocyst embedded in the head mesenchyme. Finally, we will highlight otic and epibranchial placode development and morphogenetic events towards progenitors of the inner ear and their neurosensory cell derivatives.
... How these progenitors generate such a vast array of neuron types is currently being investigated and appears to depend on the temporal expression of transcription factors that act as selector genes. In this context, the co-expression of Atoh1 with Olig3 is critical for deep cerebellar neuron development, whereas the coexpression of Atoh1 with Neurod1 is essential for granule cell progenitor specification and cerebellar and cochlear unipolar brush cell development (Ben-Arie et al., 1997;Chizhikov and Millen, 2003;Gazit et al., 2004;Pan et al., 2009;Machold et al., 2011;Lowenstein et al., 2021). The selector gene for the specification of parabrachial/Kölliker-Fuse complex is presently unknown. ...
Breathing (or respiration) is an unconscious and complex motor behavior which neuronal drive emerges from the brainstem. In simplistic terms, respiratory motor activity comprises two phases, inspiration (uptake of oxygen, O 2) and expiration (release of carbon dioxide, CO 2). Breathing is not rigid, but instead highly adaptable to external and internal physiological demands of the organism. The neurons that generate, monitor, and adjust breathing patterns locate to two major brainstem structures, the pons and medulla oblongata. Extensive research over the last three decades has begun to identify the developmental origins of most brainstem neurons that control different aspects of breathing. This research has also elucidated the transcriptional control that secures the specification of brainstem respiratory neurons. In this review, we aim to summarize our current knowledge on the transcriptional regulation that operates during the specification of respiratory neurons, and we will highlight the cell lineages that contribute to the central respiratory circuit. Lastly, we will discuss on genetic disturbances altering transcription factor regulation and their impact in hypoventilation disorders in humans.
... Specifically, there is a loss of Neurog1/2 in r1-r6 and a partial loss of Ascl1 in r1-r3 (Hernandez-Miranda et al., 2017). A unique combinatorial interaction of Atoh1, Neurog1/2, Olig3 and Ptf1a, among other genes (Lowenstein et al., 2021;Pan et al., 2009), defines the cerebellum (Fig. 4). A delayed expression of Neurod1 adds to the interaction by providing a cerebellum-specific negative feedback of Atoh1 . ...
... A delayed expression of Neurod1 adds to the interaction by providing a cerebellum-specific negative feedback of Atoh1 . This helps to establish the rostral limit of the auditory nuclei, whose development depends on a higher level of Atoh1 expression (Pan et al., 2009). Lmx1a/b, Fgf8 and Wnt1 are also Fig. 3. ...
... Atoh1 and Olig3 are expressed in the spinal cord, the hindbrain and the cerebellum (Bermingham et al., 2001;Farago et al., 2006;Hernandez-Miranda et al., 2017;Pan et al., 2009). Complete knockout of Atoh1 expression using Wnt1-cre upstream of Atoh1 (Wang et al., 2005) leads to the loss of all hindbrain neurons that depend on Atoh1/Olig3, leaving only the choroid plexus . ...
Studies by His from 1868-1904 delineated the critical role of the dorsal roof plate in the development of the hindbrain choroid plexus, and of the rhombic lips in the development of hindbrain auditory centers. Modern molecular studies have confirmed these observations and placed them in a mechanistic context. Expression of the transcription factor Lmx1a/b is crucial to the development of the hindbrain choroid plexus, and also regulates the expression of Atoh1, a transcription factor that is essential for the formation of the cochlear hair cells and auditory nuclei. By contrast, development of the vestibular hair cells, vestibular ganglion and vestibular nuclei does not depend on Lmx1a/b. These findings demonstrate a common dependence on a specific gene for the hindbrain choroid plexus and the primary auditory projection from hair cells to sensory neurons to hindbrain nuclei. Thus, His’ conclusions regarding the origins of specific hindbrain structures are borne out by molecular genetic experiments conducted more than a hundred years later.
... The molecular hand-off between Pou3f1 and Atoh1 may be mediated by negative feedback regulation. Analogous to the regulatory model that governs cerebellar granule cell development, we suspect that, as cells leave the RL, Pou3f1 acts as a negative regulator of Atoh1 expression, much like how NeuroD1 suppresses Atoh1 expression in the granule cells (Pan et al., 2009). As Atoh1 is involved in cell proliferation, by thwarting Atoh1 expression, Pou3f1 then initiates glutamatergic CN neurons to differentiate as they exit the SPS. ...
The cerebellar nuclear (CN) neurons are a molecularly heterogeneous population whose specification into the different cerebellar nuclei is defined by the expression of varying sets of transcription factors. Here, we present a novel molecular marker, Pou3f1, that delineates specific sets of glutamatergic CN neurons. The glutamatergic identity of Pou3f1+ cells was confirmed by: (1) the co-expression of vGluT2, a cell marker of glutamatergic neurons; (2) the lack of co-expression between Pou3f1 and GAD67, a marker of GABAergic neurons; (3) the co-expression of Atoh1, the master regulator required for the production of all cerebellar glutamatergic lineages; and (4) the absence of Pou3f1-expressing cells in the Atoh1-null cerebellum. Furthermore, the lack of Pax6 and Tbr1 expression in Pou3f1+ cells reveals that Pou3f1-expressing CN neurons specifically settle in the interposed and dentate nuclei. In addition, the Pou3f1-labeled glutamatergic CN neurons can be further classified by the expression of Brn2 and Irx3. The results of the present study align with previous findings highlighting that the survival of the interposed and dentate CN neurons is largely independent of Pax6. More importantly, the present study extends the field’s collective knowledge of the molecular diversity of cerebellar nuclei.
