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Explant cultures reveal early specification of the paleocortex. Explants of E12.5 lateral telencephalon, which include the entire lateral face (pallium and subpallium) of the hemisphere, display Dlx2 in the subpallium after 3 DIV (asterisk; a). Lmo3 expression arises in an arc in the dorsal portion of the Dlx2 expression (arrow; b). A similar pattern of Lmo3 expression is up-regulated in E10.5 explants after 5 DIV (arrow; c). In an E10.5 intact telencephalic hemisphere (d), dotted lines illustrate the “intact lateral face” explants prepared for c; similar preparations from E12.5 telencephalic hemispheres were made for the explants in a,b. When a more restricted portion of the E10.5 lateral telencephalon, which includes the presumptive paleocortex (dashed area; d), is explanted and maintained for 5 DIV, Dlx2 expression continues to identify the subpallium (asterisk; e). Lmo3 expression arises in a restricted band, consistently with its pattern in intact lateral face explants (arrows; f,c). Wnt8b and Nkx2.1, normally absent from the paleocortex, are not expressed in the lateral explants, serving as negative controls (g,h). Scale bar = 500 μm for a,b, 250 μm for c–f.
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The patterning of the telencephalon is regulated by the concerted action of distinct mechanisms operating in different portions of this structure. Although much progress has been made in understanding the mechanisms underlying the specification of dorsal and ventral structures, little is known about the specification of the paleocortex, the olfacto...
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... with re- spect to control markers, such as Dlx2, identifying the LGE and MGE (Bulfone et al., 1993; Fig. 2). With refer- ence to this marker, the paleocortical marker Lmo3 arises in an arc extending from the rostral to the caudal extent of the explant, in a position that appears to be at the dorsal extreme of the Dlx2-expressing territory (Fig. 7a,b). To test the timing of this phenomenon further, we examined younger telencephalic explants for their ability to develop a paleocortex-specific pattern of Lmo3. E10.5 explants also develop a remarkably specific band of Lmo3 expression after 5 DIV (Fig. ...
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... in a position that appears to be at the dorsal extreme of the Dlx2-expressing territory (Fig. 7a,b). To test the timing of this phenomenon further, we examined younger telencephalic explants for their ability to develop a paleocortex-specific pattern of Lmo3. E10.5 explants also develop a remarkably specific band of Lmo3 expression after 5 DIV (Fig. ...
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... that presump- tive paleocortex was well isolated from any potential sig- nals from the dorsomedial and ventromedial telencephalic tissue. In addition, to maximize isolation from adjacent neocortical and LGE tissue, the explants were made as small as possible without compromising their ability to maintain tissue integrity during the culture period (Fig. 7d). These explants were maintained in isolation for 5 DIV and were probed for the expression of Dlx2 and Lmo3. These explants show a striking recapitulation of the data seen in intact lateral face explants (Fig. 7e,f). Markers restricted to medial structures, such as Wnt8b, normally localized to the dorsomedial telencephalon, and Nkx2.1, ...
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... the explants were made as small as possible without compromising their ability to maintain tissue integrity during the culture period (Fig. 7d). These explants were maintained in isolation for 5 DIV and were probed for the expression of Dlx2 and Lmo3. These explants show a striking recapitulation of the data seen in intact lateral face explants (Fig. 7e,f). Markers restricted to medial structures, such as Wnt8b, normally localized to the dorsomedial telencephalon, and Nkx2.1, restricted to the MGE, are not expressed in these subdis- sected E10.5 explants, serving as negative controls (Fig. 7g,h). In summary, these experiments demonstrate that paleocortical specification is intrinsic to ...
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... and Lmo3. These explants show a striking recapitulation of the data seen in intact lateral face explants (Fig. 7e,f). Markers restricted to medial structures, such as Wnt8b, normally localized to the dorsomedial telencephalon, and Nkx2.1, restricted to the MGE, are not expressed in these subdis- sected E10.5 explants, serving as negative controls (Fig. 7g,h). In summary, these experiments demonstrate that paleocortical specification is intrinsic to isolated portions of lateral telencephalic tissue and can proceed normally in vitro under serum-free conditions, from as early as E10.5, without cues from more distant regions of the telenceph- alon or surrounding nontelencephalic ...
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We used ultrasound image-guided injections of high-titer retroviral vectors to obtain widespread introduction of genes into the mouse nervous system in utero as early as embryonic day 8.5 (E8.5). The vectors used included internal promoters that substantially improved proviral gene expression in the ventricular zone of the brain. To demonstrate the...
Citations
... Interestingly, although the hem, septum and ThE have been studied as independent structures, their molecular identities share some fundamental features which lead to the proposal that they rather represent a continuum referred to as the forebrain-hem system (Roy et al., 2014). Progenitors of the forebrain-hem system express the transcription factors Id3, Otx2, Zic2 (Guo and Li, 2019;La Manno et al., 2021;Roy et al., 2014) and their identity is repressed by the pallial transcription factors Lhx2 and Foxg1 (Godbole et al., 2017;Godbole et al., 2018;Mangale et al., 2008;Muzio and Mallamaci, 2005;Roy et al., 2014;Vyas et al., 2003). Therefore, the very distinctive transcriptomic signature of CRs, illustrated by their systematic divergence from other cortical excitatory neurons in scRNAseq datasets , likely stems from the equally unique identity of their progenitors. ...
Cajal-Retzius (CR) neurons are key players of cortical development that display a very unique transcriptomic identity. However, little is known about the mechanisms involved in their fate specification. Here we use scRNAseq to reconstruct the differentiation trajectory of hem-derived CR cells (CRs) and unravel the transient expression of a complete gene module previously known to control the cellular process of multiciliogenesis. However, we find that CRs do not undergo centriole amplification or multiciliation. We show that upon genetic disruption of Gmnc, the master regulator of the multiciliation cascade, CRs are initially produced but fail to reach their normal identity and lean towards an aberrant fate resulting in their massive apoptosis. We further dissect the contribution of multiciliation effector genes and identify Trp73 as a key determinant. Finally, we use in utero electroporation to demonstrate that the intrinsic competence of hem progenitors as well as the heterochronic expression of Gmnc prevent centriole amplification in the CR lineage. Our work exemplifies how the co-option of a complete gene module, repurposed to control a completely distinct process, may contribute to the emergence of novel cell identities.
... However, the said suppressive capacity of the BAF complex is likely exerted at early cortical development stages, perhaps until E12.5, as loss of BAF complex from E13.5 achieved in dcKO_hGFAP-Cre mutants did not lead to an observable hem or medial cortex expansion, albeit hippocampal formation was perturbed in such mutants (Nguyen et al., 2018). The effect of BAF complex deletion on hem expansion is consistent with outcome of other studies in which genetic manipulation of factors such as Foxg1, Lhx2, Lhx2/Pax6 (double deletion), and Nf2 in early development caused abnormal expansion of the mouse dTel midline structures and associated cortical patterning deviations (Bulchand et al., 2001;Monuki et al., 2001;Vyas et al., 2003;Muzio and Mallamaci 2005;Mangale et al., 2008;Lavado et al., 2013;Godbole et al., 2017). It would be interesting to find out the impact on cortical patterning following focal silencing of the BAF complex in the nascent hem using Lmx1a-Cre driver (Chizhikov et al., 2010;Fregoso et al., 2019). ...
Early forebrain patterning entails the correct regional designation of the neuroepithelium, and appropriate specification, generation, and distribution of neural cells during brain development. Specific signaling and transcription factors are known to tightly regulate patterning of the dorsal telencephalon to afford proper structural/functional cortical arealization and morphogenesis. Nevertheless, whether and how changes of the chromatin structure link to the transcriptional program(s) that control cortical patterning remains elusive. Here, we report that the BAF chromatin remodeling complex regulates the spatiotemporal patterning of the mouse dorsal telencephalon. To determine whether and how the BAF complex regulates cortical patterning, we conditionally deleted the BAF complex scaffolding subunits BAF155 and BAF170 in the mouse dorsal telencephalic neuroepithelium. Morphological and cellular changes in the BAF mutant forebrain were examined using immunohistochemistry and in situ hybridization. RNA sequencing, Co-immunoprecipitation, and mass spectrometry were used to investigate the molecular basis of BAF complex involvement in forebrain patterning. We found that conditional ablation of BAF complex in the dorsal telencephalon neuroepithelium caused expansion of the cortical hem and medial cortex beyond their developmental boundaries. Consequently, the hippocampal primordium is not specified, the mediolateral cortical patterning is compromised, and the cortical identity is disturbed in the absence of BAF complex. The BAF complex was found to interact with the cortical hem suppressor LHX2. The BAF complex suppresses cortical hem fate to permit proper forebrain patterning. We provide evidence that BAF complex modulates mediolateral cortical patterning possibly by interacting with the transcription factor LHX2 to drive the LHX2-dependent transcriptional program essential for dorsal telencephalon patterning. Our data suggest a putative mechanistic synergy between BAF chromatin remodeling complex and LHX2 in regulating forebrain patterning and ontogeny.
... We observed abundant SATB2 signal in upper layers of the control lateral cortex as expected, but only few SATB2 + neurons in the mutant lateral cortex (Fig 3C, 3E and 3J). Next, we analyzed the pattern of Lmo3, a paleocortex marker highly expressed in the E16.5 piriform cortex [29,35,36]. The Lmo3 + domain was shifted dorsally in Emx1-cKOs, thus replacing large parts of the lateral neocortex (Fig 3K). ...
... Furthermore, the olfactory tract (the main piriform afference) is shifted dorsally along with the ventral boundary of the piriform cortex as defined by Tbr1 and Emx1-Cre-labeling. This phenotype is reminiscent of the Gli3 -/brain, where an Lmo3 + paleocortex is specified next to a rudimentary dorsomedial pallium [36]. It also corresponds to Emx1-Cre; Lhx2 LoxP/LoxP mice, where a piriform-like cortex develops at the position of the lateral neocortex [52]. ...
Biallelic mutations in DONSON , an essential gene encoding for a replication fork protection factor, were linked to skeletal abnormalities and microcephaly. To better understand DONSON function in corticogenesis, we characterized Donson expression and consequences of conditional Donson deletion in the mouse telencephalon. Donson was widely expressed in the proliferation and differentiation zones of the embryonic dorsal and ventral telencephalon, which was followed by a postnatal expression decrease. Emx1 -Cre-mediated Donson deletion in progenitors of cortical glutamatergic neurons caused extensive apoptosis in the early dorsomedial neuroepithelium, thus preventing formation of the neocortex and hippocampus. At the place of the missing lateral neocortex, these mutants exhibited a dorsal extension of an early-generated paleocortex. Targeting cortical neurons at the intermediate progenitor stage using Tbr2 -Cre evoked no apparent malformations, whereas Nkx2 . 1 -Cre-mediated Donson deletion in subpallial progenitors ablated 75% of Nkx2 . 1 -derived cortical GABAergic neurons. Thus, the early telencephalic neuroepithelium depends critically on Donson function. Our findings help explain why the neocortex is most severely affected in individuals with DONSON mutations and suggest that DONSON-dependent microcephaly might be associated with so far unrecognized defects in cortical GABAergic neurons. Targeting Donson using an appropriate recombinase is proposed as a feasible strategy to ablate proliferating and nascent cells in experimental research.
... Foxg1 is involved in the development of various organs such as the forebrain, cerebral cortex, etc. Lack of Foxg1 in the forebrain leads to defects in ventral telencephalon and dorsal structures due to the abnormal proliferation and differentiation of neuroepithelial cells [22,28,29,[46][47][48][49]. In addition, Foxg1 also contributes to the maintenance of hippocampal dentate gyrus progenitor cells and to the process of neurogenesis and glialogenesis [30]. ...
Foxg1 plays important roles in regeneration of hair cell (HC) in the cochlea of neonatal mouse. Here, we used Sox9-CreER to knock down Foxg1 in supporting cells (SCs) in the utricle in order to investigate the role of Foxg1 in HC regeneration in the utricle. We found Sox9 an ideal marker of utricle SCs and bred Sox9CreER/+Foxg1loxp/loxp mice to conditionally knock down Foxg1 in utricular SCs. Conditional knockdown (cKD) of Foxg1 in SCs at postnatal day one (P01) led to increased number of HCs at P08. These regenerated HCs had normal characteristics, and could survive to at least P30. Lineage tracing showed that a significant portion of newly regenerated HCs originated from SCs in Foxg1 cKD mice compared to the mice subjected to the same treatment, which suggested SCs trans-differentiate into HCs in the Foxg1 cKD mouse utricle. After neomycin treatment in vitro, more HCs were observed in Foxg1 cKD mice utricle compared to the control group. Together, these results suggest that Foxg1 cKD in utricular SCs may promote HC regeneration by inducing trans-differentiation of SCs. This research therefore provides theoretical basis for the effects of Foxg1 in trans-differentiation of SCs and regeneration of HCs in the mouse utricle.
... Foxg1 knock-out mice, which die at the perinatal period, show hypoplasia of the telencephalon and abnormal eye and ear development [45,55,57]. In forebrain development, Foxg1 maintains the progenitor pools and inhibits neuronal differentiation, and it is down-regulated when progenitors undergo neuronal differentiation [55,[58][59][60][61][62][63][64]. In postnatal mice, Foxg1 also plays an important role in maintaining the hippocampal dentate gyrus progenitor pool, and the lack of Foxg1 promotes both gliogenesis and neurogenesis [24]. ...
... The role of Foxg1 has been characterized mainly in forebrain development [58][59][60][61], and the absence of Foxg1 leads to structural defects of both the dorsal and ventral telencephalon due to reduced proliferation and premature differentiation of neuroepithelial cells [55]. In cortical progenitor cells, Foxg1 promotes self-renewal of neural precursors and inhibits neuronal differentiation [55,59,62,63]. ...
Foxg1 is one of the forkhead box genes that are involved in morphogenesis, cell fate determination, and proliferation, and Foxg1 was previously reported to be required for morphogenesis of the mammalian inner ear. However, Foxg1 knock-out mice die at birth, and thus the role of Foxg1 in regulating hair cell (HC) regeneration after birth remains unclear. Here we used Sox2CreER/+ Foxg1loxp/loxp mice and Lgr5-EGFPCreER/+ Foxg1loxp/loxp mice to conditionally knock down Foxg1 specifically in Sox2+ SCs and Lgr5+ progenitors, respectively, in neonatal mice. We found that Foxg1 conditional knockdown (cKD) in Sox2+ SCs and Lgr5+ progenitors at postnatal day (P)1 both led to large numbers of extra HCs, especially extra inner HCs (IHCs) at P7, and these extra IHCs with normal hair bundles and synapses could survive at least to P30. The EdU assay failed to detect any EdU+ SCs, while the SC number was significantly decreased in Foxg1 cKD mice, and lineage tracing data showed that much more tdTomato+ HCs originated from Sox2+ SCs in Foxg1 cKD mice compared to the control mice. Moreover, the sphere-forming assay showed that Foxg1 cKD in Lgr5+ progenitors did not significantly change their sphere-forming ability. All these results suggest that Foxg1 cKD promotes HC regeneration and leads to large numbers of extra HCs probably by inducing direct trans-differentiation of SCs and progenitors to HCs. Real-time qPCR showed that cell cycle and Notch signaling pathways were significantly down-regulated in Foxg1 cKD mice cochlear SCs. Together, this study provides new evidence for the role of Foxg1 in regulating HC regeneration from SCs and progenitors in the neonatal mouse cochlea.
... The dorsally adjoining dorsal pallium (DP) gives rise to the neocortex, which is responsible for higher mental functions, including memory, speech, value judgments, and sociality. The adjoining LP and DP have different cytoarchitectures; LP is a three-layered structure, while DP is a six-layered structure [5][6][7]. The regulatory mechanisms underlying the building of the six-layered cortex, which is a mammal-specific feature, are well established; sequential expression of transcription factors is involved in determining cell identities, and late-born neurons migrate to pass through and take their place outside earlier-born neurons, i.e., in an inside-out manner [8][9][10][11][12]. ...
The piriform cortex (paleocortex) is the olfactory cortex or the primary cortex for the sense of smell. It receives the olfactory input from the mitral and tufted cells of the olfactory bulb and is involved in the processing of information pertaining to odors. The piriform cortex and the adjoining neocortex have different cytoarchitectures; while the former has a three-layered structure, the latter has a six-layered structure. The regulatory mechanisms underlying the building of the six-layered neocortex are well established; in contrast, less is known about of the regulatory mechanisms responsible for structure formation of the piriform cortex. The differences as well as similarities in the regulatory mechanisms between the neocortex and the piriform cortex remain unclear. Here, the expression of neocortical layer-specific genes in the piriform cortex was examined. Two sublayers were found to be distinguished in layer II of the piriform cortex using Ctip2/Bcl11b and Brn1/Pou3f3. The sequential expression pattern of Ctip2 and Brn1 in the piriform cortex was similar to that detected in the neocortex, although the laminar arrangement in the piriform cortex exhibited an outside-in arrangement, unlike that observed in the neocortex.
... Lhx2 and Foxg1, which together repress dorsal midline fates. In the absence of Lhx2, a dramatic expansion of dorsal medial structures takes places (Bulchand et al., 2001, Monuki et al., 2001, Vyas et al., 2003. ...
Cerebral cortical development is a finely regulated process, depending on diverse progenitor cells. Abnormal behavior of the latter can give rise to cortical malformations. Mutations in Eml1/EML1 were identified in the HeCo mouse, as well as in three families presenting severe subcortical heterotopia (SH). SH is characterized by the presence of mislocalized neurons in the white matter. At early stages of corticogenesis, abnormally positioned apical radial glia progenitors (aRG) were found cycling outside the proliferative ventricular zone (VZ) in the HeCo cortical wall. I focused my research on characterizing aRG in the VZ to assess why some cells leave this region and thus to further understand SH mechanisms. Combining confocal and electron microscopy (EM), I uncovered abnormalities of centrosomes and primary cilia in Eml1-mutant aRGs: primary cilia are shorter, and often remain basally oriented within vesicles. Searching for Eml1-interacting partners using mass spectrometry (MS), combined with exome sequencing of SH patient DNAs, allowed us to identify a ciliary Eml1-interacting partner, RPGRIP1L, showing mutations in a SH patient. Gene ontology analyses of MS data pointed to Golgi apparatus and protein transport as enriched categories. Indeed, Golgi abnormalities were identified in HeCo aRGs. Altogether, these data indicate that the Golgi-to-primary cilium axis is perturbed in Eml1mutant conditions, pointing to new intracellular pathways involved in severe neurodevelopmental disorders.
... Based on studies of individual null mutant phenotypes, the transcription factors FOXG1 and LHX2 have been shown to regulate the formation of the hem. When either of these factors is constitutively lost, much of the dorsal telencephalic neuroepithelium transforms into hem instead of cortical primordium (Bulchand et al., 2001;Monuki et al., 2001;Vyas et al., 2003;Muzio and Mallamaci, 2005). There are, however, important differences in the two null mutant phenotypes. ...
... Although both display an expanded hem, the Lhx2 mutant displays an expanded hem juxtaposed to an expanded antihem, with no cortical primordium in between them (Mangale et al., 2008). In contrast, loss of Foxg1 spares only medial-dorsal fates, so the telencephalon contains an expanded hem and some hippocampal primordium, but no lateral cortical tissue or antihem (Vyas et al., 2003;Muzio and Mallamaci, 2005). Another important difference is that the specification of the ventral telencephalon appears normal in the Lhx2 mutant, whereas this structure is entirely lost in the Foxg1 mutant (Xuan et al., 1995;Huh et al., 1999;Martynoga et al., 2005). ...
During forebrain development, a telencephalic organizer called the cortical hem is critical for inducing hippocampal fate in adjacent cortical neuroepithelium. How the hem is restricted to its medial position is therefore a fundamental patterning question. Here, we demonstrate that Foxg1-Lhx2 interactions are critical for the formation of the hem. Loss of either gene causes a portion of the cortical neuroepithelium to transform into hem. We show that FOXG1 regulates Lhx2 expression in the cortical primordium. In the absence of Foxg1, the presence of Lhx2 is sufficient to suppress hem fate, and hippocampal markers appear selectively in Lhx2-expressing regions. FOXG1 also restricts the temporal window in which loss of Lhx2 results in a transformation of cortical primordium into hem. Therefore, Foxg1 and Lhx2 form a genetic hierarchy in the spatiotemporal regulation of cortical hem specification and positioning, and together ensure the normal development of this hippocampal organizer.
... However, in contrast to Dbx1, we find that sFrp2 does not reappear in the Lhx2 lox/lox ;Pax6 lox/lox double mutant (Additional file 4: Figure S4). Since loss of Lhx2 alone does not cause loss of sFrp2 expression [17], this suggests that sFrp2 critically requires PAX6 for its expression. This indicates that different molecular features of the antihem are regulated by distinct combinations of factors, underscoring the heterogeneity of this structure. ...
Patterning of the telencephalic neuroepithelium is a tightly regulated process controlled by transcription factors and signalling molecules. The cortical primordium is flanked by two signalling centres, the hem medially, and the antihem laterally. The hem induces the formation of the hippocampus in adjacent neuroepithelium. Therefore, the position of the hem defines the position of the hippocampus in the brain. The antihem is positioned at the boundary between the dorsal and ventral telencephalon and proposed to provide patterning cues during development.
LIM-homeodomain (LIM-HD) transcription factor LHX2 suppresses both hem and antihem fate in the cortical neuroepithelium. Upon loss of Lhx2, medial cortical neuroepithelium is transformed into hem, whereas lateral cortical neuroepithelium is transformed into antihem. Here, we show that transcription factor PAX6, known to regulate patterning of the lateral telencephalon, restricts this tissue from transforming into hem upon loss of Lhx2. When Lhx2 and Pax6 are both deleted, the cortical hem expands to occupy almost the complete extent of the cortical primordium, indicating that both factors act to suppress hem fate in the lateral telencephalon. Furthermore, the shift in the pallial-subpallial boundary and absence of the antihem, observed in the Pax6 mutant, are both restored in the Lhx2; Pax6 double mutant.
Together, these results not only reveal a novel function for LHX2 in regulating dorsoventral patterning in the telencephalon, but also identify PAX6 as a fundamental regulator of where the hem can form, and therefore implicate this molecule as a determinant of hippocampal positioning.
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... Having said this, the HF develops in the medial pallium, adjacent to the roof plate and a cortical organizer called the cortical hem. Molecular signals from both roof plate and cortical hem, including bone morphogenetic proteins and/or Wnt proteins [Grove et al., 1998], induce the expression of transcription factors (TFs) such as Emx1/2, Lef1, and Lhx2 in the adjacent pallium, in concentrationdependent gradients [Porter et al., 1997;Galceran et al., 2000;Bulchand et al., 2001;Vyas et al., 2003]. The ven- Comparison of chicken, lacertid lizard and mouse hippocampal and parahippocampal subdivisions. ...
The hippocampal formation is a highly conserved structure of the medial pallium that works in association with the entorhinal cortex, playing a key role in memory formation and spatial navigation. Although it has been described in several vertebrates, the presence of comparable subdivisions across species remained unclear. This panorama has started to change in recent years thanks to the identification of some of the genes that regulate the development of the hippocampal formation in the mouse and help to delineate its subdivisions based on molecular features. Some of these genes have been used to try to identify subdivisions in chicken and lizards comparable to those of the mammalian hippocampal formation and the entorhinal cortex. Here, we review some of these data, which suggest the existence of fields comparable to the dentate gyrus, CA3, CA1, subiculum, as well as medial and lateral parts of the entorhinal cortex in all amniotes. We also analyze available data suggesting the existence of serial connections between these fields, speculate on the possible existence of auto-associative loops in CA3, and discuss general principles governing the formation of the connections.
