The rhombic lip gives rise to neuronal populations that contribute to cerebellar, proprioceptive and interoceptive networks. Cell production depends on the expression of the basic helix-loop-helix (bHLH) transcription factor Atoh1. In rhombomere 1, Atoh1-positive cells give rise to both cerebellar neurons and extra-cerebellar nuclei in ventral hindbrain. The origin of this cellular diversity has previously been attributed to temporal signals rather than spatial patterning. Here, we show that in both chick and mouse the cerebellar Atoh1 precursor pool is partitioned into initially cryptic spatial domains that reflect the activity of two different organisers: an isthmic Atoh1 domain, which gives rise to isthmic nuclei, and the rhombic lip, which generates deep cerebellar nuclei and granule cells. We use a combination of in vitro explant culture, genetic fate mapping and gene overexpression and knockdown to explore the role of isthmic signalling in patterning these domains. We show that an FGF-dependent isthmic Atoh1 domain is the origin of distinct populations of Lhx9-positive neurons in the extra-cerebellar isthmic nuclei. In the cerebellum, ectopic FGF induces proliferation while blockade reduces the length of the cerebellar rhombic lip. FGF signalling is not required for the specification of cerebellar cell types from the rhombic lip and its upregulation inhibits their production. This suggests that although the isthmus regulates the size of the cerebellar anlage, the downregulation of isthmic FGF signals is required for induction of rhombic lip-derived cerebellar neurons.
Lhx9; Otx2; Gbx2; Isthmo-optic nucleus; Deep cerebellar nuclei; FGF receptor knockout; Cerebellum; Chick; Mouse
During the embryonic development of the cerebellum, neurons are produced from progenitor cells located along a ventricular zone within dorsal rhombomere 1 that extends caudally to the roof plate of the fourth ventricle. The apposition of the caudal neuroepithelium and roof plate results in a unique inductive region termed the cerebellar rhombic lip, which gives rise to granule cell precursors and other glutamatergic neuronal lineages. Recently, we and others have shown that, at early embryonic stages prior to the emergence of granule cell precursors (E12), waves of neurogenesis in the cerebellar rhombic lip produce specific hindbrain nuclei followed by deep cerebellar neurons. How the induction of rhombic lip-derived neurons from cerebellar progenitors is regulated during this phase of cerebellar development to produce these temporally discrete neuronal populations while maintaining a progenitor pool for subsequent neurogenesis is not known.
Employing both gain- and loss-of-function methods, we find that Notch1 signaling in the cerebellar primordium regulates the responsiveness of progenitor cells to bone morphogenetic proteins (BMPs) secreted from the roof plate that stimulate the production of rhombic lip-derived neurons. In the absence of Notch1, cerebellar progenitors are depleted during the early production of hindbrain neurons, resulting in a severe decrease in the deep cerebellar nuclei that are normally born subsequently. Mechanistically, we demonstrate that Notch1 activity prevents the induction of Math1 by antagonizing the BMP receptor-signaling pathway at the level of Msx2 expression.
Our results provide a mechanism by which a balance between neural induction and maintenance of neural progenitors is achieved in the rhombic lip throughout embryonic development.
Retinal neurons and glia arise from a common progenitor pool in a temporal order, with retinal ganglion cells (RGCs) appearing first, and Müller glia last. The transcription factors Atoh7/Math5 and Ascl1/Mash1 represent divergent bHLH clades, and exhibit distinct spatial and temporal retinal expression patterns, with little overlap during early development. Here, we tested the ability of Ascl1 to change the fate of cells in the Atoh7 lineage when misexpressed from the Atoh7 locus, using an Ascl1-IRES-DsRed2 knock-in allele. In Atoh7Ascl1KI/+ and Atoh7Ascl1KI/Ascl1KI embryos, ectopic Ascl1 delayed cell cycle exit and differentiation, even in cells coexpressing Atoh7. The heterozygous retinas recovered, and eventually produced a normal complement of RGCs, while homozygous substitution of Ascl1 for Atoh7 did not promote postnatal retinal fates precociously, nor rescue Atoh7 mutant phenotypes. However, our analyses revealed two unexpected findings. First, ectopic Ascl1 disrupted cell cycle progression within the marked Atoh7 lineage, but also nonautonomously in other retinal cells. Second, the size of the Atoh7 retinal lineage was unaffected, supporting the idea of a compensatory shift of the non-proliferative cohort to maintain lineage size. Overall, we conclude that Ascl1 acts dominantly to block cell cycle exit, but is incapable of redirecting the fates of early RPCs.
Atoh7; Ascl1; Retinal ganglion cell; cell cycle; retinal neurogenesis; retinal cell competence
Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment?
We show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2.
Different overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip precursor pool. By contrast, a subset of coextensive nuclei is refractory to ectopic Hoxa2 and is presumably induced solely by isthmic organiser activity. Thus, graded (isthmic) and segmental mechanisms may operate exclusively of one another in the specification of different neuronal populations within rhombomere 1. The early designation of an Otx2-negative, Hoxa2-negative region, prior to the appearance of the isthmic organiser, is a key initial step in the specification of the cerebellum.
The basic helix-loop-helix (bHLH) transcription factor Math5 (Atoh7) is required for retinal ganglion cell (RGC) and optic nerve development. Using Math5-lacZ knockout mice, we have identified an additional expression domain for Math5 outside the eye, in functionally connected structures of the central auditory system. In the adult hindbrain, the cytoplasmic Math5-lacZ reporter is expressed within the ventral cochlear nucleus (VCN), in a subpopulation of neurons that project to medial nucleus of the trapezoid body (MNTB), lateral superior olive (LSO), and lateral lemniscus (LL). These cells were identified as globular and small spherical bushy cells based on their morphology, abundance, distribution within the cochlear nucleus (CN), co-expression of Kv1.1, Kv3.1b and Kcnq4 potassium channels, and projection patterns within the auditory brainstem. Math5-lacZ is also expressed by cochlear root neurons in the auditory nerve. During embryonic development, Math5-lacZ was detected in precursor cells emerging from the caudal rhombic lip from embryonic day (E)12 onwards, consistent with the time course of CN neurogenesis. These cells co-express MafB, Math1 and Math5 and are post-mitotic. Math5 expression in the CN was verified by mRNA in situ hybridization, and the identity of positive neurons was confirmed morphologically using a Math5-Cre BAC transgene with an alkaline phosphatase reporter.
The hindbrains of Math5 mutants appear grossly normal, with the exception of the CN. Although overall CN dimensions are unchanged, the lacZ positive cells are significantly smaller in Math5 −/− mice compared to Math5 +/− mice, suggesting these neurons may function abnormally. The Auditory Brainstem Response (ABR) of Math5 mutants was evaluated in a BALB/cJ congenic background. ABR thresholds of Math5 −/− mice were similar to those of wild-type and heterozygous mice, but the interpeak latencies for Peaks II-IV were significantly altered. These temporal changes are consistent with a higher level auditory processing disorder involving the CN, potentially affecting the integration of binaural sensory information.
Math5; cochlear nucleus; AVCN; PVCN; mouse; atonal; Math1; bHLH; globular bushy cells; spherical bushy cells; cochlear root neurons; auditory processing; ABR latency; binaural integration; interaural intensity difference; interaural time difference; phase locking; Calyx of Held; Kv1.1; Kv3.1; Kcnq4; mafB; Cre recombinase; fluorogold; tract-tracing; inferior colliculus; multipolar cells; rhombic lip
The cerebellum (Cb) controls movement related physiology using a diverse array of morphologically and biochemically distinct neurons. During development, the Cb is derived from rhombomere 1 (r1), an embryonic compartment patterned by a signaling center referred to as the isthmus organizer. The secreted glycoprotein WNT1 is expressed in the midbrain primordia (mesencephalon, mes) and at the posterior limit of the mes, WNT1 plays a pivotal role in maintaining the isthmus organizer. Mutations in Wnt1 produce severe Cb defects that are generally attributed to aberrant organizer activity. Interestingly, Wnt1 is also expressed at the most posterior limit of dorsal r1, in a region known as the upper rhombic lip (URL). However, the distribution and molecular identity of Wnt1 expressing progenitors have not been carefully described in r1. We used Wnt1-Venus transgenic mice to generate a molecular map of Wnt1 expressing progenitors in relation to other well characterized Cb biomarkers such as MATH1 (ATOH1), LMX1a and OTX2. Our analysis validated Wnt1 expression in the URL and revealed molecularly-defined developmental zones in r1. We then used genetic inducible fate mapping (GIFM) to link transient Wnt1 expression in r1 to terminal cell fates in the mature Cb. Wnt1 expressing progenitors primarily contributed to deep cerebellar nuclei, granule cells, and unipolar brush cells in distinct but overlapping temporal windows and sparsely contributed to inhibitory neurons and Bergmann glia. We further demonstrate that the Wnt1 lineage does not follow a competency model of progressive lineage restriction to generate the Cb or the functionally related precerebellar system. Instead, progenitors initiate Wnt1 expression de novo to give rise to each Cb cell type and precerebellar nuclei. We then used GIFM to determine how the temporal control of Wnt1 expression is related to molecular identity and cell migration in Cb development. Our findings provide new insight into how lineage and timing establish cell diversity within the Cb system.
Cerebellum; Wnt1; Genetic Inducible Fate Mapping; Lineage; Cell Fate; Mouse Genetics
Neural basic helix-loop-helix (bHLH) transcription factors are crucial in regulating the differentiation and neuronal subtype specification of neurons. Precisely how these transcription factors direct such processes is largely unknown due to the lack of bona fide targets in vivo. Genetic evidence suggests that bHLH factors have shared targets in their common differentiation role, but unique targets with respect to their distinct roles in neuronal subtype specification. However, whether neuronal subtype specific targets exist remains an unsolved question. To address this question, we focused on Atoh1 (Math1), a bHLH transcription factor that specifies distinct neuronal subtypes of the proprioceptive pathway in mammals including the dorsal interneuron 1 (dI1) population of the developing spinal cord. We identified transcripts unique to the Atoh1-derived lineage using microarray analyses of specific bHLH-sorted populations from mouse. Chromatin immunoprecipitation-sequencing (ChIP-seq) experiments followed by enhancer reporter analyses identified five direct neuronal subtype specific targets of Atoh1 in vivo along with their Atoh1-responsive enhancers. These targets, Klf7, Rab15, Rassf4, Selm, and Smad7, have diverse functions that range from transcription factors to regulators of endocytosis and signaling pathways. Only Rab15 and Selm are expressed across several different Atoh1-specified neuronal subtypes including external granule cells (EGL) in the developing cerebellum, hair cells of the inner ear, and Merkel cells. Our work establishes on a molecular level that neuronal differentiation bHLH transcription factors have distinct lineage-specific targets.
The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs). The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.
This review summarizes vertebrate rhombic lip and early cerebellar development covering classic approaches up to modern developmental genetics which identifies the relevant differential gene expression domains and their progeny. Most of this information is derived from amniotes. However, progress in anamniotes, particularly in the zebrafish, has recently been made. The current picture suggests that rhombic lip and cerebellar development in jawed vertebrates (gnathostomes) share many characteristics. Regarding cerebellar development, these include a ptf1a expressing ventral cerebellar proliferation (VCP) giving rise to Purkinje cells and other inhibitory cerebellar cell types, and an atoh1 expressing upper rhombic lip giving rise to an external granular layer (EGL, i.e., excitatory granule cells) and an early ventral migration into the anterior rhombencephalon (cholinergic nuclei). As for the lower rhombic lip (LRL), gnathostome commonalities likely include the formation of precerebellar nuclei (mossy fiber origins) and partially primary auditory nuclei (likely convergently evolved) from the atoh1 expressing dorsal zone. The fate of the ptf1a expressing ventral LRL zone which gives rise to (excitatory cells of) the inferior olive (climbing fiber origin) and (inhibitory cells of ) cochlear nuclei in amniotes, has not been determined in anamniotes. Special for the zebrafish in comparison to amniotes is the predominant origin of anamniote excitatory deep cerebellar nuclei homologs (i.e., eurydendroid cells) from ptf1a expressing VCP cells, the sequential activity of various atoh1 paralogs and the incomplete coverage of the subpial cerebellar plate with proliferative EGL cells. Nevertheless, the conclusion that a rhombic lip and its major derivatives evolved with gnathostome vertebrates only and are thus not an ancestral craniate character complex is supported by the absence of a cerebellum (and likely absence of its afferent and efferent nuclei) in jawless fishes
atoh1; cerebellum; cochlear nuclei; precerebellar systems; ptf1a; wnt1; zebrafish; rhombic lip
Within the developing vertebrate retina, particular subtypes of amacrine cells (ACs) tend to arise from progenitors expressing the bHLH transcription factor, Atoh7, which is necessary for the early generation of retinal ganglion cells (RGCs). All ACs require the post-mitotic expression of the bHLH transcription factor Ptf1a, however Ptf1a alone is not sufficient to give subtype identities. Here we use functional and in vivo time-lapse studies in the zebrafish retina to investigate on the developmental programs leading to ACs specification within the subsequent divisions of Atoh7-positive progenitors. We find evidences that the homeobox transcription factor Barhl2 is an AC subtype identity-biasing factor that turns on within Atoh7-positive descendants. In vivo lineage tracing reveals that particular modes of cell division tend to generate Barhl2-positive precursors from sisters of RGCs. Additionally, Atoh7 indirectly impacts these division modes to regulate the right number of barhl2-expressing cells. We finally find that Atoh7 itself influences the subtypes of Barhl2-dependent ACs. Taken together, our study uncovers lineage-related and molecular logic of subtype specification in the vertebrate retina, by showing that specific AC subtypes arise via a particular mode of cell division and a transcriptional network cascade involving the sequential expression of first atoh7 followed by ptf1a and then barhl2.
fate determination; cell lineage; Barhl2; subtype specification; retina; zebrafish
The granule cell layer of the cerebellum comprises the largest population of neurons in the vertebrate CNS. In amniotes, its precursors undergo a unique phase of transit amplification, regulated by Sonic hedgehog. They do so within a prominent but transient, secondary proliferative epithelium, the external germinal layer, which is formed by tangential migration of precursor cells from the rhombic lip. This behaviour is a hallmark of bird and mammal cerebellum development. Despite its significance for both development and disease, it is unclear whether an external germinal layer is a requirement for granule cell production or an expedient of transit amplification. Evidence for its existence in more basal vertebrates is contradictory. We therefore examined cerebellum development in the zebrafish, specifically in relation to the expression of the bHLH gene Atonal 1, which definitively characterises granule cell precursors. The expression of Atoh1a, b and c, in combination with patterns of proliferation and fate-maps, define precursor pools at the rhombic lip and cerebellar midline, but demonstrate that an external germinal layer is absent. Sonic hedgehog signalling is correspondingly absent in the zebrafish cerebellum. Sustained roofplate derived signals suggest that, in the absence of transit amplification, primary granule cell precursor pools are maintained throughout development. To determine whether this pattern is specific to zebrafish, or reflects a more general anamniote organisation we examined the expression of similar genes in the dogfish, Scylliorhinus canicula. We show that these anamniotes share a common groundplan of granule cell production that does not include an external germinal layer.
granule cell; migration; Math1/Atonal1/Atoh1; Purkinje cell; cell proliferation; fate-map
Atonal homolog1 (Atoh1) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO) mouse line using Tg(Atoh1-cre), in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to “self-terminate” its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.
The morphogen and mitogen Sonic Hedgehog activates a Gli1-dependent transcription program that drives proliferation of granule neuron progenitors (GNPs) within the external germinal layer of the postnatally developing cerebellum. Medulloblastomas with mutations activating the Sonic Hedgehog signaling pathway preferentially arise within the external germinal layer, and the tumor cells closely resemble GNPs. Atoh1/Math1, a basic helix-loop-helix transcription factor essential for GNP histogenesis, does not induce medulloblastomas when expressed in primary mouse GNPs that are explanted from the early postnatal cerebellum and transplanted back into the brains of naïve mice. However, enforced expression of Atoh1 in primary GNPs enhances the oncogenicity of cells overexpressing Gli1 by almost three orders of magnitude. Unlike Gli1, Atoh1 cannot support GNP proliferation in the absence of Sonic Hedgehog signaling and does not govern expression of canonical cell cycle genes. Instead, Atoh1 maintains GNPs in a Sonic Hedgehog-responsive state by regulating genes that trigger neuronal differentiation, including many expressed in response to bone morphogenic protein-4. Therefore, by targeting multiple genes regulating the differentiation state of GNPs, Atoh1 collaborats with the pro-proliferative Gli1-dependent transcriptional program to influence medulloblastoma development.
cerebellar neurogenesis; Sonic Hedgehog (Shh); tumor-initiating cells; Patched1 (Ptch1); bone morphogenic protein (BMP)
Math6/atoh8, a bHLH transcription factor, is thought to be indispensable for early embryonic development and likely has important roles in vertebrate tissue-specific differentiation. However, the function of Atoh8 during early development is not clear because homozygous knockout causes embryonic lethality in mice. We have examined the effects of the atoh8 gene on the differentiation of retina and skeletal muscle during early development in zebrafish.
We isolated a Math6 homologue in zebrafish, designated as zebrafish atoh8. Whole -mount in situ hybridization analysis showed that zebrafish atoh8 is dynamically expressed mainly in developing retina and skeletal muscle. Atoh8-MO knock-down resulted in reduced eye size with disorganization of retinal lamination. The reduction of atoh8 function also affected the arrangement of paraxial cells and differentiated muscle fibers during somite morphogenesis.
Our results show that Atoh8 is an important regulator for the development of both the retina and skeletal muscles necessary for neural retinal cell and myogenic differentiation during zebrafish embryogenesis.
Atoh1 encodes a basic helix-loop-helix (bHLH) transcription factor required for the development of the inner ear sensory epithelia, the dorsal spinal cord, brainstem, cerebellum, and intestinal secretory cells. In this study to create a genetic tool for the research on gene function in the ear sensory organs, we generated an Atoh1-Cre knock-in mouse line by replacing the entire Atoh1 coding sequences with the Cre coding sequences. Atoh1Cre/+mice were viable, fertile, and displayed no visible defects whereas the Atoh1Cre/Cremice died perinatally. The spatiotemporal activities of Cre recombinase were examined by crossing Atoh1-Cre mice with the R26R-lacZ conditional reporter mice. Atoh1-Cre activities were detected in the developing inner ear, the hindbrain, the spinal cord, and the intestine. In the inner ear, Atoh1-Cre activities were confined to the sensory organs in which lacZ expression is detected in nearly all of the hair cells and in many supporting cells. Thus, Atoh1-Cre mouse line serves as a useful tool for the functional study of genes in the inner ear. In addition, our results demonstrate that Atoh1 is expressed in the common progenitors destined for both hair and supporting cells.
Math1; Atoh1; Cre recombinase; inner ear; hair cells; supporting cells
•Differential responsiveness to early panocular atoh7 expression.•Early panocular atoh7 only mildly affects dorsal retina (reduced Müller glia cells).•Early panocular atoh7 severely affects ventral retina (predominant RGC fate).•Early panocular atoh7 expression results in coloboma.
During vertebrate eye development retinal progenitor cells (RPCs) differentiate into all neural cell types of the retina. Retinal ganglion cells (RGCs) represent the first cell type to be generated. For their development, Atoh7, a basic Helix Loop Helix (bHLH) transcription factor is crucial. Atoh7 loss of function results in a massive reduction or even a total loss of RGCs. However, inconsistent results have been obtained in atoh7 gain of function experiments with respect to ganglion cell genesis, implying that the effect of Atoh7 is likely to be dependent on the competence state of the RPC.
In this study we addressed the differential susceptibilities of early RPCs to Atoh7 in vivo, using medaka. Unexpectedly, we observed a largely normal development of the dorsal retina, although atoh7 was precociously expressed. However, the development of the retina close to the optic nerve head (part of the ventral retina) was disturbed severely. Photoreceptors were largely absent and the Müller glia cell number was reduced significantly. The majority of cells in this domain were ganglion cells and the abnormal development of this area affected the closure of the optic fissure resulting in coloboma.
Atoh7 (Ath5); RPC; Retinal differentiation; Fate shift; Optic fissure; Coloboma
Atonal homolog1 (Atoh1, formerly Math1) is a crucial bHLH transcription factor for inner ear hair cell differentiation. Its absence in embryos results in complete absence of mature hair cells at birth and its misexpression can generate extra hair cells. Thus Atoh1 may be both necessary and sufficient for hair cell differentiation in the ear. Atoh1 null mice die at birth and have some undifferentiated cells in sensory epithelia carrying Atoh1 markers. The fate of these undifferentiated cells in neonates is unknown due to lethality. We use Tg(Pax2-Cre) to delete floxed Atoh1 in the inner ear. This generates viable conditional knockout (CKO) mice for studying the postnatal development of the inner ear without differentiated hair cells. Using in situ hybridization we find that Tg(Pax2-Cre) recombines the floxed Atoh1 prior to detectable Atoh1 expression. Only the posterior canal crista has Atoh1 expressing hair cells due to incomplete recombination. Most of the organ of Corti cells are lost in CKO mice via late embryonic cell deaths. Marker genes indicate that the organ of Corti is reduced to two rows of cells wedged between flanking markers of the organ of Corti (Fgf10 and Bmp4). These two rows of cells (instead of five rows of supporting cells) are positive for Prox1 in neonates. By postnatal day 14 (P14), most of the developing organ of Corti is lost through embryonic cell deaths, with the remaining cells transformed into a flat epithelium with no distinction of any specific cell type. However, some of the remaining organ of Corti cells express Myo7a at late postnatal stages and are innervated by remaining afferent fibers. Initial growth of afferents and efferents in embryos shows no difference between control mice and Tg(Pax2-Cre)::Atoh1 CKO mice. Most afferents and efferents are lost in the CKO mutant before birth, leaving only few basal and a more prominent apical innervation. Afferents focus their projections on patches that express the prosensory specifying gene, Sox2. This pattern of innervation by sensory neurons is maintained at least until P14, but fibers target the few Myo7a positive cells found in later stages.
hair cell differentiation; flat epithelium; organ of Corti; innervation of the ear; conditional deletion; mouse ear mutants
The vertebrate basic helix–loop–helix (bHLH) transcription factor ATOH7 (Math5) is specifically expressed in the embryonic neural retina and is required for the genesis of retinal ganglion cells (RGCs) and optic nerves. In Atoh7 mutant mice, the absence of trophic factors secreted by RGCs prevents the development of the intrinsic retinal vasculature and the regression of fetal blood vessels, causing persistent hyperplasia of the primary vitreous (PHPV). We therefore screened patients with hereditary PHPV, as well as bilateral optic nerve aplasia (ONA) or hypoplasia (ONH), for mutations in ATOH7. We identified a homozygous ATOH7 mutation (N46H) in a large family with an autosomal recessive PHPV disease trait linked to 10q21, and a heterozygous variant (R65G, p.Arg65Gly) in one of five sporadic ONA patients. High-density single-nucleotide polymorphism analysis also revealed a CNTN4 duplication and an OTX2 deletion in the ONA cohort. Functional analysis of ATOH7 bHLH domain substitutions, by electrophoretic mobility shift and luciferase cotransfection assays, revealed that the N46H variant cannot bind DNA or activate transcription, consistent with structural modeling. The N46H variant also failed to rescue RGC development in mouse Atoh7−/− retinal explants. The R65G variant retains all of these activities, similar to wild-type human ATOH7. Our results strongly suggest that autosomal recessive persistent hyperplastic primary vitreous is caused by N46H and is etiologically related to nonsyndromic congenital retinal nonattachment. The R65G allele, however, cannot explain the ONA phenotype. Our study firmly establishes ATOH7 as a retinal disease gene and provides a functional basis to analyze new coding variants.
To gain insight into the lineage of origin of medulloblastomas, the mRNA expression of NEUROG1, a gene encoding a proneural transcription factor transiently detected during nervous system development, was investigated in 27 human medulloblastomas characterized for mRNA expression of ATOH1, a marker of cerebellar granule precursors and corresponding medulloblastomas. Expression of Ngn1, the mouse homolog of NEUROG1, was also analyzed in the mouse cerebellar primordium. In addition, we studied mRNA expression of GLI1 as a marker of the SHH pathway activation, and nuclear β-catenin staining, β-catenin mutations, and mRNA expression of MYC as indicators of the WNT pathway status. In 15 cases, we also examined expression of OTX2, a transcription factor recently indicated as a positive marker of medulloblastomas originating from cerebellar granule precursors. The mRNA expression of NEUROG1 and Ngn1 was selectively found in medulloblastomas not expressing ATOH1 and in progenitors of the cerebellar ventricular zone, respectively. GLI1 transcript was expressed in medulloblastomas with ATOH1 transcript, whereas high levels of MYC transcript were unrelated to NEUROG1 or ATOH1 expression. No clear association between MYC overexpression and nuclear β-catenin staining was found. Finally, OTX2 mRNA was expressed in all medulloblastomas with NEUROG1 transcript, but also in a subset of these malignancies with ATOH1 transcript. These observations may help to define the lineage of origin of medulloblastomas, and support a role for ATOH1 and NEUROG1 in the classification of these malignancies.
ATOH1 (MATH-1); cerebellum; GLI1; medulloblastoma; MYC; NEUROG1 (neurogenin-1); OTX2
The Organ of Corti, located within the mammalian cochlea, contains a precise mosaic of hair cells (HC) and supporting cells (SC), the patterning of which is critical for auditory function. Progenitors of HCs and SCs are found in the same post-mitotic region of the cochlear duct during early stages of cochlear development, and both HCs and SCs are absent in mice lacking the transcription factor Atoh1. Based on existing data, Atoh1 is thought to be the earliest determinant of HC fate, and to have a cell-autonomous role in HC differentiation, but the lineage of Atoh1-positive cells within the cochlear duct remains unclear. To address this issue, we used an inducible Atoh1Cre*PR allele to permanently mark Atoh1-expressing cells at different developmental time points. We found that up to 30% of cells from the Atoh1-lineage develop as SCs, and that the number of Atoh1-positive SCs decreases both spatially and temporally in a pattern consistent with ongoing commitment. Modulation of Notch signaling, necessary for formation of the HC-SC mosaic, changes the percentage of cells from the Atoh1-lineage that develop as either HCs or SCs. The HC-SC ratio is also affected by morphogenesis of the cochlea, as inhibiting the outgrowth of the cochlear duct increases the number of Atoh1-lineage cells that develop as SCs. Our results demonstrate that the Atoh1-lineage is established early in cochlear development, but also show that expression of Atoh1 does not absolutely result in commitment to a HC fate.
inner ear; development; cell fate; Organ of Corti
Background & Aims
The Atonal Homolog 1 (Atoh1 or Math1) transcription factor is required for intestinal secretory (goblet, Paneth, enteroendocrine) cell differentiation. Notch/γ-secretase inhibitors (GSIs) block proliferation and induce secretory cell differentiation in the intestine. We used genetic analyses of mice to determine whether Atoh1 mediates the effects of GSIs in normal and cancerous intestinal epithelia.
We studied mice with intestine-specific disruption of Atoh1 (Atoh1Δintestine), the APCmin mutation, both mutations [Atoh1Δintestine; APCmin], or littermate controls; mice were given GSI or vehicle. Colorectal cancer (CRC) cell lines were treated with GSI or vehicle and with small hairpin (sh) RNAs to reduce ATOH1. Differentiation and homeostasis was assessed by protein, RNA, and histologic analyses.
GSIs failed to induce secretory cell differentiation or apoptosis or decrease proliferation of Atoh1-null progenitor cells, compared with wild-type cells. Exposure of APCmin adenomas to GSIs decreased proliferation and increased secretory cell numbers in an Atoh1-dependent manner. In CRC cells treated with GSI, ATOH1 levels were inversely correlated with proliferation. ATOH1 was required for secretory cell gene expression in cell lines and in mice.
ATOH1 is required for all effects of GSIs in intestinal crypts and adenomas; Notch has no unique function in intestinal progenitors and cancer cells other than to regulate ATOH1 expression. Reducing ATOH1 activity might mitigate intestinal toxicity from systemic GSI therapy for non-intestinal diseases. Among gastrointestinal malignancies, ATOH1 mediates the effects of GSIs, so ATOH1 expression levels might predict responses to these inhibitors. We propose that only the subset of CRCs that retain ATOH1 expression will respond to GSIs.
biomarkers; cell fate specification; basic helix-loop-helix transcription factor; notch intracellular domain
Medulloblastoma encompasses a collection of clinically and molecularly diverse tumor subtypes that together comprise the most common malignant childhood brain tumor1–4. These tumors are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) following aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH-subtype)3–8. The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here, we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT-subtype)1,3,4, arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumors infiltrate the dorsal brainstem, while SHH-subtype tumors are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem that included aberrantly proliferating Zic1+ precursor cells. These lesions persisted in all mutant adult mice and in 15% of cases in which Tp53 was concurrently deleted, progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer.
The ability to precisely identify separate neuronal populations is essential to the understanding of the development and function of different brain structures. This necessity is particularly evident in regions such as the brainstem, where the anatomy is quite complex and little is known about the identity, origin, and function of a number of distinct nuclei due to the lack of specific cellular markers. In this regard, the gene encoding the transcription factor Runx1 has emerged as a specific marker of restricted neuronal populations in the murine central and peripheral nervous systems. The aim of this study was to precisely characterize the expression of Runx1 in the developing and postnatal mouse brainstem.
Methods and Principal Findings
Anatomical and immunohistochemical studies were used to characterize mouse Runx1 expression in the brainstem. It is shown here that Runx1 is expressed in a restricted population of neurons located in the dorsolateral rostral hindbrain. These neurons define a structure that is ventromedial to the dorsal nucleus of the lateral lemniscus, dorsocaudal to the medial paralemniscal nucleus and rostral to the cerebellum. Runx1 expression in these cells is first observed at approximately gestational day 12.5, persists into the adult brain, and is lost in knockout mice lacking the transcription factor Atoh1, an important regulator of the development of neuronal lineages of the rhombic lip. Runx1-expressing neurons in the rostral hindbrain produce cholecystokinin and also co-express members of the Groucho/Transducin-like Enhancer of split protein family.
Based on the anatomical and molecular characteristics of the Runx1-expressing cells in the rostral hindbrain, we propose that Runx1 expression in this region of the mouse brain defines the superior lateral parabrachial nucleus.
Atoh1 is a basic helix-loop-helix transcription factor necessary for the specification of inner ear hair cells and central auditory system neurons derived from the rhombic lip. We used the Cre-loxP system and two Cre-driver lines (Egr2Cre and Hoxb1Cre) to delete Atoh1 from different regions of the cochlear nucleus (CN) and accessory auditory nuclei (AAN). Adult Atoh1-conditional knockout mice (Atoh1CKO) are behaviorally deaf, have diminished auditory brainstem evoked responses and disrupted CN and AAN morphology and connectivity. In addition, Egr2; Atoh1CKO mice lose spiral ganglion neurons in the cochlea and AAN neurons during the first 3 days of life, revealing a novel critical period in the development of these neurons. These new mouse models of predominantly central deafness illuminate the importance of the CN for support of a subset of peripheral and central auditory neurons.
The basic helix-loop-helix (bHLH) transcription factor Math5 (Atoh7) is transiently expressed during early retinal histogenesis and is necessary for retinal ganglion cell (RGC) development. Using nucleoside pulse-chase experiments and clonal analysis, we determined that progenitor cells activate Math5 during or after the terminal division, with progressively later onset as histogenesis proceeds. We have traced the lineage of Math5+ cells using mouse BAC transgenes that express Cre recombinase under strict regulatory control. Quantitative analysis showed that Math5+ progenitors express equivalent levels of Math5 and contribute to every major cell type in the adult retina, but are heavily skewed toward early fates. The Math5>Cre transgene labels 3% of cells in adult retina, including 55% of RGCs. Only 11% of Math5+ progenitors develop into RGCs; the majority become photoreceptors. The fate bias of the Math5 cohort, inferred from the ratio of cone and rod births, changes over time, in parallel with the remaining neurogenic population. Comparable results were obtained using Math5 mutant mice, except that ganglion cells were essentially absent, and late fates were overrepresented within the lineage. We identified Math5-independent RGC precursors in the earliest-born (embryonic day 11) retinal cohort, but these precursors require Math5-expressing cells for differentiation. Math5 thus acts permissively to establish RGC competence within a subset of progenitors, but is not sufficient for fate specification. It does not autonomously promote or suppress the determination of non-RGC fates. These data are consistent with progressive and temporal restriction models for retinal neurogenesis, in which environmental factors influence the final histotypic choice.
mouse; genetics; Cre recombinase; BAC transgenic; lineage; expression fate mapping; cell fate determination; retina; optic nerve; atonal; bHLH