Distinct requirements for Tcf3 and Tcf12 during oligodendrocyte development in the mouse telencephalon.

BACKGROUND: E-proteins encoded by Tcf3, Tcf4, and Tcf12 are class I basic helix-loop-helix (bHLH) transcription factors (TFs) that are thought to be widely expressed during development. However, their function in the developing brain, specifically in the telencephalon remains an active area of research. Our study examines for the first time if combined loss of two E-proteins (Tcf3 and Tcf12) influence distinct cell fates and oligodendrocyte development in the mouse telencephalon. METHODS: We generated Tcf3/12 double conditional knockouts (dcKOs) using Olig2Cre/+ or Olig1Cre/+ to overcome compensatory mechanisms between E-proteins and to understand the specific requirement for Tcf3 and Tcf12 in the ventral telencephalon and during oligodendrogenesis. We utilized a combination of in situ hybridization, immunohistochemistry, and immunofluorescence to address development of the telencephalon and oligodendrogenesis at embryonic and postnatal stages in Tcf3/12 dcKOs. RESULTS: We show that the E-proteins Tcf3 and Tcf12 are expressed in progenitors of the embryonic telencephalon and throughout the oligodendrocyte lineage in the postnatal brain. Tcf3/12 dcKOs showed transient defects in progenitor cells with an enlarged medial ganglionic eminence (MGE) region which correlated with reduced generation of embryonic oligodendrocyte progenitor cells (OPCs) and increased expression of MGE interneuron genes. Postnatal Tcf3/12 dcKOs showed a recovery of OPCs but displayed a sustained reduction in mature oligodendrocytes (OLs). Interestingly, Tcf4 remained expressed in the dcKOs suggesting that it cannot compensate for the loss of Tcf3 and Tcf12. Generation of Tcf3/12 dcKOs with Olig1Cre/+ avoided the MGE morphology defect caused by Olig2Cre/+ but dcKOs still exhibited reduced embryonic OPCs and subsequent reduction in postnatal OLs. CONCLUSION: Our data reveal that Tcf3 and Tcf12 play a role in controlling OPC versus cortical interneuron cell fate decisions in MGE progenitors in addition to playing roles in the generation of embryonic OPCs and differentiation of postnatal OLs in the oligodendrocyte lineage.

Conserved and Distinct Functions of the Autism-Related Chromatin Remodeler CHD8 in Embryonic and Adult Forebrain Neurogenesis.

The chromatin remodeler CHD8 represents a high-confidence risk factor in autism, a multistage progressive neurologic disorder, however the underlying stage-specific functions remain elusive. In this study, by analyzing Chd8 conditional knock-out mice (male and female), we find that CHD8 controls cortical neural stem/progenitor cell (NSC) proliferation and survival in a stage-dependent manner. Strikingly, inducible genetic deletion reveals that CHD8 is required for the production and fitness of transit-amplifying intermediate progenitors (IPCs) essential for upper-layer neuron expansion in the embryonic cortex. p53 loss of function partially rescues apoptosis and neurogenesis defects in the Chd8-deficient brain. Further, transcriptomic and epigenomic profiling indicates that CHD8 regulates the chromatin accessibility landscape to activate neurogenesis-promoting factors including TBR2, a key regulator of IPC neurogenesis, while repressing DNA damage- and p53-induced apoptotic programs. In the adult brain, CHD8 depletion impairs forebrain neurogenesis by impeding IPC differentiation from NSCs in both subventricular and subgranular zones; however, unlike in embryos, it does not affect NSC proliferation and survival. Treatment with an antidepressant approved by the Federal Drug Administration (FDA), fluoxetine, partially restores adult hippocampal neurogenesis in Chd8-ablated mice. Together, our multistage functional studies identify temporally specific roles for CHD8 in developmental and adult neurogenesis, pointing to a potential strategy to enhance neurogenesis in the CHD8-deficient brain.SIGNIFICANCE STATEMENT The role of the high-confidence autism gene CHD8 in neurogenesis remains incompletely understood. Here, we identify a stage-specific function of CHD8 in development of NSCs in developing and adult brains by conserved, yet spatiotemporally distinct, mechanisms. In embryonic cortex, CHD8 is critical for the proliferation, survival, and differentiation of both NSC and IPCs during cortical neurogenesis. In adult brain, CHD8 is required for IPC generation but not the proliferation and survival of adult NSCs. Treatment with FDA-approved antidepressant fluoxetine partially rescues the adult neurogenesis defects in CHD8 mutants. Thus, our findings help resolve CHD8 functions throughout life during embryonic and adult neurogenesis and point to a potential avenue to promote neurogenesis in CHD8 deficiency.

Formation of the Mouse Internal Capsule and Cerebral Peduncle: A Pioneering Role for Striatonigral Axons as Revealed in Isl1 Conditional Mutants.

The projection neurons of the striatum, the principal nucleus of the basal ganglia, belong to one of the following two major pathways: the striatopallidal (indirect) pathway or the striatonigral (direct) pathway. Striatonigral axons project long distances and encounter ascending tracts (thalamocortical) while coursing alongside descending tracts (corticofugal) as they extend through the internal capsule and cerebral peduncle. These observations suggest that striatal circuitry may help to guide their trajectories. To investigate the developmental contributions of striatonigral axons to internal capsule formation, we have made use of Sox8-EGFP (striatal direct pathway) and Fezf2-TdTomato (corticofugal pathway) BAC transgenic reporter mice in combination with immunohistochemical markers to trace these axonal pathways throughout development. We show that striatonigral axons pioneer the internal capsule and cerebral peduncle and are temporally and spatially well positioned to provide guidance for corticofugal and thalamocortical axons. Using Isl1 conditional knock-out (cKO) mice, which exhibit disrupted striatonigral axon outgrowth, we observe both corticofugal and thalamocortical axon defects with either ventral forebrain- or telencephalon-specific Isl1 inactivation, despite Isl1 not being expressed in either cortical or thalamic projection neurons. Striatonigral axon defects can thus disrupt internal capsule formation. Our genome-wide transcriptomic analysis in Isl1 cKOs reveals changes in gene expression relevant to cell adhesion, growth cone dynamics, and extracellular matrix composition, suggesting potential mechanisms by which the striatonigral pathway exerts this guidance role. Together, our data support a novel pioneering role for the striatal direct pathway in the correct assembly of the ascending and descending axon tracts within the internal capsule and cerebral peduncle.SIGNIFICANCE STATEMENT The basal ganglia are a group of subcortical nuclei with established roles in the coordination of voluntary motor programs, aspects of cognition, and the selection of appropriate social behaviors. Hence, disruptions in basal ganglia connectivity have been implicated in the motor, cognitive, and social dysfunction characterizing common neurodevelopmental disorders such as attention-deficit/hyperactivity disorder, autism spectrum disorder, obsessive-compulsive disorder, and tic disorder. Here, we identified a novel role for the striatonigral (direct) pathway in pioneering the internal capsule and cerebral peduncle, and in guiding axons extending to and from the cortex. Our findings suggest that the abnormal development of basal ganglia circuits can drive secondary internal capsule defects and thereby may contribute to the pathology of these disorders.

Generation of a Mouse Model to Study the Noonan Syndrome Gene Lztr1 in the Telencephalon.

The leucine zipper-like transcriptional regulator 1 (Lztr1) is a BTB-Kelch domain protein involved in RAS/MAPK pathway regulation. Mutations in LZTR1 are associated with cancers and Noonan syndrome, the most common RASopathy. The expression and function of Lztr1 in the developing brain remains poorly understood. Here we show that Lztr1 is expressed in distinct regions of the telencephalon, the most anterior region of the forebrain. Lztr1 expression was robust in the cortex, amygdala, hippocampus, and oligodendrocytes in the white matter. To gain insight into the impact of Lztr1 deficiency, we generated a conditional knockout (cKO) restricted to the telencephalon using Foxg1 IREScre/+. Lztr1 cKOs are viable to postnatal stages and show reduced Lztr1 expression in the telencephalon. Interestingly, Lztr1 cKOs exhibit an increase in MAPK pathway activation in white matter regions and subsequently show an altered expression of stage-specific markers in the oligodendrocyte lineage with increased oligodendrocyte progenitor cells (OPCs) and decreased markers of oligodendrocyte differentiation. Moreover, Lztr1 cKOs also exhibit an increased expression of the astrocyte marker GFAP. These results highlight the generation of a new mouse model to study Lztr1 deficiency in the brain and reveal a novel role for Lztr1 in normal oligodendrocyte and astrocyte development in the telencephalon.

A Novel Mutation in Cse1l Disrupts Brain and Eye Development with Specific Effects on Pax6 Expression.

Forward genetics in the mouse continues to be a useful and unbiased approach to identifying new genes and alleles with previously unappreciated roles in mammalian development and disease. Here, we report a new mouse allele of Cse1l that was recovered from an ENU mutagenesis screen. Embryos homozygous for the anteater allele of Cse1l display a number of variable phenotypes, with craniofacial and ocular malformations being the most obvious. We provide evidence that Cse1l is the causal gene through complementation with a novel null allele of Cse1l generated by CRISPR-Cas9 editing. While the variability in the anteater phenotype was high enough to preclude a detailed molecular analysis, we demonstrate a very penetrant reduction in Pax6 levels in the developing eye along with significant ocular developmental phenotypes. The eye gene discovery tool iSyTE shows Cse1l to be significantly expressed in the lens from early eye development stages in embryos through adulthood. Cse1l has not previously been shown to be required for organogenesis as homozygosity for a null allele results in very early lethality. Future detailed studies of Cse1l function in craniofacial and neural development will be best served with a conditional allele to circumvent the variable phenotypes we report here. We suggest that human next-generation (whole genome or exome) sequencing studies yielding variants of unknown significance in CSE1L could consider these findings as part of variant analysis.

A role for sustained MAPK activity in the mouse ventral telencephalon.

The MAPK pathway is a major growth signal that has been implicated during the development of progenitors, neurons, and glia in the embryonic brain. Here, we show that the MAPK pathway plays an important role in the generation of distinct cell types from progenitors in the ventral telencephalon. Our data reveal that phospho-p44/42 (called p-ERK1/2) and the ETS transcription factor Etv5, both downstream effectors in the MAPK pathway, show a regional bias in expression during ventral telencephalic development, with enriched expression in the dorsal region of the LGE and ventral region of the MGE at E13.5 and E15.5. Interestingly, expression of both factors becomes more uniform in ventricular zone (VZ) progenitors by E18.5. To gain insight into the role of MAPK activity during progenitor cell development, we used a cre inducible constitutively active MEK1 allele (RosaMEK1DD/+) in combination with a ventral telencephalon enriched cre (Gsx2e-cre) or a dorsal telencephalon enriched cre (Emx1cre/+). Sustained MEK/MAPK activity in the ventral telencephalon (Gsx2e-cre; RosaMEK1DD/+) expanded dorsal lateral ganglionic eminence (dLGE) enriched genes (Gsx2 and Sp8) and oligodendrocyte progenitor cell (OPC) markers (Olig2, Pdgfrα, and Sox10), and also reduced markers in the ventral (v) LGE domain (Isl1 and Foxp1). Activation of MEK/MAPK activity in the dorsal telencephalon (Emx1cre/+; RosaMEK1DD/+) did not initially activate the expression of dLGE or OPC genes at E15.5 but ectopic expression of Gsx2 and OPC markers were observed at E18.5. These results support the idea that MAPK activity as readout by p-ERK1/2 and Etv5 expression is enriched in distinct subdomains of ventral telencephalic progenitors during development. In addition, sustained activation of the MEK/MAPK pathway in the ventral or dorsal telencephalon influences dLGE and OPC identity from progenitors.

Enhanced MAPK1 Function Causes a Neurodevelopmental Disorder within the RASopathy Clinical Spectrum.

Signal transduction through the RAF-MEK-ERK pathway, the first described mitogen-associated protein kinase (MAPK) cascade, mediates multiple cellular processes and participates in early and late developmental programs. Aberrant signaling through this cascade contributes to oncogenesis and underlies the RASopathies, a family of cancer-prone disorders. Here, we report that de novo missense variants in MAPK1, encoding the mitogen-activated protein kinase 1 (i.e., extracellular signal-regulated protein kinase 2, ERK2), cause a neurodevelopmental disease within the RASopathy phenotypic spectrum, reminiscent of Noonan syndrome in some subjects. Pathogenic variants promote increased phosphorylation of the kinase, which enhances translocation to the nucleus and boosts MAPK signaling in vitro and in vivo. Two variant classes are identified, one of which directly disrupts binding to MKP3, a dual-specificity protein phosphatase negatively regulating ERK function. Importantly, signal dysregulation driven by pathogenic MAPK1 variants is stimulus reliant and retains dependence on MEK activity. Our data support a model in which the identified pathogenic variants operate with counteracting effects on MAPK1 function by differentially impacting the ability of the kinase to interact with regulators and substrates, which likely explains the minor role of these variants as driver events contributing to oncogenesis. After nearly 20 years from the discovery of the first gene implicated in Noonan syndrome, PTPN11, the last tier of the MAPK cascade joins the group of genes mutated in RASopathies.

Single-Cell Transcriptomics Uncovers Glial Progenitor Diversity and Cell Fate Determinants during Development and Gliomagenesis.

The identity and degree of heterogeneity of glial progenitors and their contributions to brain tumor malignancy remain elusive. By applying lineage-targeted single-cell transcriptomics, we uncover an unanticipated diversity of glial progenitor pools with unique molecular identities in developing brain. Our analysis identifies distinct transitional intermediate states and their divergent developmental trajectories in astroglial and oligodendroglial lineages. Moreover, intersectional analysis uncovers analogous intermediate progenitors during brain tumorigenesis, wherein oligodendrocyte-progenitor intermediates are abundant, hyper-proliferative, and progressively reprogrammed toward a stem-like state susceptible to further malignant transformation. Similar actively cycling intermediate progenitors are prominent components in human gliomas with distinct driver mutations. We further unveil lineage-driving networks underlying glial fate specification and identify Zfp36l1 as necessary for oligodendrocyte-astrocyte lineage transition and glioma growth. Together, our results resolve the dynamic repertoire of common and divergent glial progenitors during development and tumorigenesis and highlight Zfp36l1 as a molecular nexus for balancing glial cell-fate decision and controlling gliomagenesis.

Gsx transcription factors control neuronal versus glial specification in ventricular zone progenitors of the mouse lateral ganglionic eminence.

The homeobox gene Gsx2 has previously been shown to inhibit oligodendroglial specification in dorsal lateral ganglionic eminence (dLGE) progenitors of the ventral telencephalon. The precocious specification of oligodendrocyte progenitor cells (OPCs) observed in Gsx2 mutants, however, is transient and begins to normalize by late stages of embryogenesis. Interestingly, this normalization correlates with the expansion of Gsx1, a close homolog of Gsx2, in a subset of progenitors in the Gsx2 mutant LGE. Here, we interrogated the mechanisms underlying oligodendroglial specification in Gsx2 mutants in relation to Gsx1. We found that Gsx1/2 double mutant embryos exhibit a more robust expansion of Olig2+ cells (i.e. OPCs) in the subventricular zone (SVZ) of the dLGE than Gsx2 mutants. Moreover, misexpression of Gsx1 throughout telencephalic VZ progenitors from E15 and onward resulted in a significant reduction of cortical OPCs. These results demonstrate redundant roles of Gsx1 and Gsx2 in suppressing early OPC specification in LGE VZ progenitors. However, Gsx1/2 mutants did not show a significant increase in adjacent cortical OPCs at later stages compared to Gsx2 mutants. This is likely due to reduced proliferation of OPCs within the SVZ of the Gsx1/2 double mutant LGE, suggesting a novel role for Gsx1 in expansion of migrating OPCs in the ventral telencephalon. We further investigated the glial specification mechanisms downstream of Gsx2 by generating Olig2/Gsx2 double mutants. Consistent with the known essential role for Olig2 in OPC specification, ectopic production of cortical OPCs observed in Gsx2 mutants disappeared in Olig2/Gsx2 double mutants. These mutants, however, maintained the expanded expression of gliogenic markers Zbtb20 and Bcan in the VZ of the LGE similarly to Gsx2 single mutants, suggesting that Gsx2 suppresses gliogenesis via Olig2-dependent and -independent mechanisms.

Gain-of-function mutations in the gene encoding the tyrosine phosphatase SHP2 induce hydrocephalus in a catalytically dependent manner.

Catalytically activating mutations in Ptpn11, which encodes the protein tyrosine phosphatase SHP2, cause 50% of Noonan syndrome (NS) cases, whereas inactivating mutations in Ptpn11 are responsible for nearly all cases of the similar, but distinct, developmental disorder Noonan syndrome with multiple lentigines (NSML; formerly called LEOPARD syndrome). However, both types of disease mutations are gain-of-function mutations because they cause SHP2 to constitutively adopt an open conformation. We found that the catalytic activity of SHP2 was required for the pathogenic effects of gain-of-function, disease-associated mutations on the development of hydrocephalus in the mouse. Targeted pan-neuronal knockin of a Ptpn11 allele encoding the active SHP2 E76K mutant resulted in hydrocephalus due to aberrant development of ependymal cells and their cilia. These pathogenic effects of the E76K mutation were suppressed by the additional mutation C459S, which abolished the catalytic activity of SHP2. Moreover, ependymal cells in NSML mice bearing the inactive SHP2 mutant Y279C were also unaffected. Mechanistically, the SHP2 E76K mutant induced developmental defects in ependymal cells by enhancing dephosphorylation and inhibition of the transcription activator STAT3. Whereas STAT3 activity was reduced in Ptpn11E76K/+ cells, the activities of the kinases ERK and AKT were enhanced, and neural cell-specific Stat3 knockout mice also manifested developmental defects in ependymal cells and cilia. These genetic and biochemical data demonstrate a catalytic-dependent role of SHP2 gain-of-function disease mutants in the pathogenesis of hydrocephalus.

Dek overexpression in murine epithelia increases overt esophageal squamous cell carcinoma incidence.

Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression in vivo, we generated and validated a tetracycline responsive Dek transgenic mouse model referred to as Bi-L-Dek. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing Bi-L-Dek mice to keratin 5 tetracycline transactivator (K5-tTA) mice. Conditional transgene expression was validated in the resulting Bi-L-Dek_K5-tTA mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these data demonstrate that Dek overexpression in the cell of origin for SCC is sufficient to promote esophageal SCC development in vivo.

A histone deacetylase 3-dependent pathway delimits peripheral myelin growth and functional regeneration.

Deficits in Schwann cell-mediated remyelination impair functional restoration after nerve damage, contributing to peripheral neuropathies. The mechanisms mediating block of remyelination remain elusive. Here, through small-molecule screening focusing on epigenetic modulators, we identified histone deacetylase 3 (HDAC3; a histone-modifying enzyme) as a potent inhibitor of peripheral myelinogenesis. Inhibition of HDAC3 enhanced myelin growth and regeneration and improved functional recovery after peripheral nerve injury in mice. HDAC3 antagonizes the myelinogenic neuregulin-PI3K-AKT signaling axis. Moreover, genome-wide profiling analyses revealed that HDAC3 represses promyelinating programs through epigenetic silencing while coordinating with p300 histone acetyltransferase to activate myelination-inhibitory programs that include the HIPPO signaling effector TEAD4 to inhibit myelin growth. Schwann cell-specific deletion of either Hdac3 or Tead4 in mice resulted in an elevation of myelin thickness in sciatic nerves. Thus, our findings identify the HDAC3-TEAD4 network as a dual-function switch of cell-intrinsic inhibitory machinery that counters myelinogenic signals and maintains peripheral myelin homeostasis, highlighting the therapeutic potential of transient HDAC3 inhibition for improving peripheral myelin repair.

Loss of Intercalated Cells (ITCs) in the Mouse Amygdala of Tshz1 Mutants Correlates with Fear, Depression, and Social Interaction Phenotypes.

The intercalated cells (ITCs) of the amygdala have been shown to be critical regulatory components of amygdalar circuits, which control appropriate fear responses. Despite this, the molecular processes guiding ITC development remain poorly understood. Here we establish the zinc finger transcription factor Tshz1 as a marker of ITCs during their migration from the dorsal lateral ganglionic eminence through maturity. Using germline and conditional knock-out (cKO) mouse models, we show that Tshz1 is required for the proper migration and differentiation of ITCs. In the absence of Tshz1, migrating ITC precursors fail to settle in their stereotypical locations encapsulating the lateral amygdala and BLA. Furthermore, they display reductions in the ITC marker Foxp2 and ectopic persistence of the dorsal lateral ganglionic eminence marker Sp8. Tshz1 mutant ITCs show increased cell death at postnatal time points, leading to a dramatic reduction by 3 weeks of age. In line with this, Foxp2-null mutants also show a loss of ITCs at postnatal time points, suggesting that Foxp2 may function downstream of Tshz1 in the maintenance of ITCs. Behavioral analysis of male Tshz1 cKOs revealed defects in fear extinction as well as an increase in floating during the forced swim test, indicative of a depression-like phenotype. Moreover, Tshz1 cKOs display significantly impaired social interaction (i.e., increased passivity) regardless of partner genetics. Together, these results suggest that Tshz1 plays a critical role in the development of ITCs and that fear, depression-like and social behavioral deficits arise in their absence.SIGNIFICANCE STATEMENT We show here that the zinc finger transcription factor Tshz1 is expressed during development of the intercalated cells (ITCs) within the mouse amygdala. These neurons have previously been shown to play a crucial role in fear extinction. Tshz1 mouse mutants exhibit severely reduced numbers of ITCs as a result of abnormal migration, differentiation, and survival of these neurons. Furthermore, the loss of ITCs in mouse Tshz1 mutants correlates well with defects in fear extinction as well as the appearance of depression-like and abnormal social interaction behaviors reminiscent of depressive disorders observed in human patients with distal 18q deletions, including the Tshz1 locus.

Characterization of Glcci1 expression in a subpopulation of lateral ganglionic eminence progenitors in the mouse telencephalon.

BACKGROUND: The lateral ganglionic eminence (LGE) in the ventral telencephalon is a diverse progenitor domain subdivided by distinct gene expression into a dorsal region (dLGE) that gives rise to olfactory bulb and amygdalar interneurons and a ventral region (vLGE) that gives rise to striatal projection neurons. The homeobox gene, Gsx2, is an enriched marker of the LGE and is expressed in a high dorsal to low ventral gradient in the ventricular zone (VZ) as development proceeds. Aside from Gsx2, markers restricted to the VZ in the dLGE and/or vLGE remain largely unknown. RESULTS: Here, we show that the gene and protein expression of Glucocorticoid-induced transcript 1 (Glcci1) has a similar dorsal to ventral gradient of expression in the VZ as Gsx2. We found that Glcci1 gene and protein expression are reduced in Gsx2 mutants, and are increased in the cortex after early and late Gsx2 misexpression. Moreover, Glcci1 expressing cells are restricted to a subpopulation of Gsx2 positive cells on the basal side of the VZ and co-express Ascl1, but not the subventricular zone dLGE marker, Sp8. CONCLUSIONS: These findings suggest that Glcci1 is a new marker of a subpopulation of LGE VZ progenitor cells in the Gsx2 lineage. Developmental Dynamics 247:222-228, 2018. © 2017 Wiley Periodicals, Inc.

Septal contributions to olfactory bulb interneuron diversity in the embryonic mouse telencephalon: role of the homeobox gene Gsx2.

BACKGROUND: Olfactory bulb (OB) interneurons are known to represent diverse neuronal subtypes, which are thought to originate from a number of telencephalic regions including the embryonic dorsal lateral ganglionic eminence (dLGE) and septum. These cells migrate rostrally toward the OB, where they then radially migrate to populate different OB layers including the granule cell layer (GCL) and the outer glomerular layer (GL). Although previous studies have attempted to investigate regional contributions to OB interneuron diversity, few genetic tools have been used to address this question at embryonic time points when the earliest populations are specified. METHODS: In this study, we utilized Zic3-lacZ and Gsx2e-CIE transgenic mice as genetic fate-mapping tools to study OB interneuron contributions derived from septum and LGE, respectively. Moreover, to address the regional (i.e. septal) requirements of the homeobox gene Gsx2 for OB interneuron diversity, we conditionally inactivated Gsx2 in the septum, leaving it largely intact in the dLGE, by recombining the Gsx2 floxed allele using Olig2 Cre/+ mice. RESULTS: Our fate mapping studies demonstrated that the dLGE and septum gave rise to OB interneuron subtypes differently. Notably, the embryonic septum was found to give rise largely to the calretinin+ (CR+) GL subtype, while the dLGE was more diverse, generating all major GL subpopulations as well as many GCL interneurons. Moreover, Gsx2 conditional mutants (cKOs), with septum but not dLGE recombination, showed impaired generation of CR+ interneurons within the OB GL. These Gsx2 cKOs exhibited reduced proliferation within the septal subventricular zone (SVZ), which correlated well with the reduced number of CR+ interneurons observed. CONCLUSIONS: Our findings indicate that the septum and LGE contribute differently to OB interneuron diversity. While the dLGE provides a wide range of OB interneuron subtypes, the septum is more restricted in its contribution to the CR+ subtype. Gsx2 is required in septal progenitors for the correct expansion of SVZ progenitors specified toward the CR+ subtype. Finally, the septum has been suggested to be the exclusive source of CR+ interneurons in postnatal studies. Our results here demonstrate that dLGE progenitors in the embryo also contribute to this OB neuronal subtype.

Selective neuronal expression of the SoxE factor, Sox8, in direct pathway striatal projection neurons of the developing mouse brain.

The striatum is the major component of the basal ganglia and is well known to play a key role in the control of motor function via balanced output from the indirect (iSPNs) and direct pathway striatal projection neurons (dSPNs). Little is known, however, about the molecular genetic mechanisms that control the formation of the iSPNs versus dSPNs. We show here that the SoxE family member, Sox8, is co-expressed with the dSPN markers, Isl1 and Ebf1, in the developing striatum. Moreover, dSPNs, as marked by Isl1-cre fate map, express Sox8 in the embryonic striatum and Sox8-EGFP BAC transgenic mice specifically reveal the direct pathway axons during development. These EGFP+ axons are first observed to reach their midbrain target, the substantia nigra pars reticulata (SNr), at E14 in the mouse with a robust connection observed already at birth. The selective expression of EGFP in dSPNs of Sox8-EGFP BAC mice is maintained at postnatal timepoints. Sox8 is known to be expressed in oligodendrocyte precursor cells (OPCs) together with other SoxE factors and we show here that the EGFP signal co-localizes with the OPC markers throughout the brain. Finally, we show that Sox8-EGFP BAC mice can be used to interrogate the altered dSPN development in Isl1 conditional mutants including aberrant axonal projections detected already at embryonic timepoints. Thus, Sox8 represents an early and specific marker of embryonic dSPNs and the Sox8-EGFP BAC transgenic mice are an excellent tool to study the development of basal ganglia circuitry.

The transcription factor Olig2 is important for the biology of diffuse intrinsic pontine gliomas.

Background: Diffuse intrinsic pontine glioma (DIPG) is a high-grade brainstem glioma of children with dismal prognosis. There is no single unifying model about the cell of origin of DIPGs. Proliferating cells in the developing human and mouse pons, the site of DIPGs, express neural stem/progenitor cell (NPC) markers, including Sox2, nestin, vimentin, Olig2, and glial fibrillary acidic protein, in an overlapping and non-overlapping manner, suggesting progenitor cell heterogeneity in the pons. It is thought that during a restricted window of postnatal pons development, a differentiation block caused by genetic/epigenetic changes leads to unrestrained progenitor proliferation and DIPG development. Nearly 80% of DIPGs harbor a mutation in the H3F3A or the related HIST1H3B gene. Supporting the impaired differentiation model, NPCs derived from human induced pluripotent stem cells expressing the H3F3A mutation showed complete differentiation block. However, the mechanisms regulating an altered differentiation program in DIPG are unknown. Methods: We established syngeneic serum-dependent and independent primary DIPG lines, performed molecular characterization of DIPG lines in vitro and in an orthotopic xenograft model, and used small hairpin RNA to examine Olig2 function in DIPG. Results: The transcription factor Olig2 is highly expressed in 70%-80% of DIPGs. Here we report that Olig2 expression and DIPG differentiation are mutually exclusive events in vitro, and only DIPG cells that retained Olig2 in vitro formed robust Olig2-positive brainstem glioma with 100% penetrance in a xenograft model. Conclusion: Our results indicate Olig2 as an onco-requisite factor in DIPG and propose investigation of Olig2 target genes as novel candidates in DIPG therapy.

Characterization of a new Gsx2-cre line in the developing mouse telencephalon.

In this study, we generated a transgenic mouse line driving Cre and EGFP expression with two putative cis-regulatory modules (CRMs) (i.e., hs687 and hs678) upstream of the homeobox gene Gsx2 (formerly Gsh2), a critical gene for establishing lateral ganglionic eminence (LGE) identity. The combination of these two CRMs drives transgene expression within the endogenous Gsx2 expression domains along the anterior-posterior neuraxis. By crossing this transgenic line with the RosatdTomato (Ai14) reporter mouse line, we observed a unique recombination pattern in the lateral ventral telencephalon, namely the LGE and the dorsal half of the medial GE (MGE), but not in the septum. We found robust recombination in many cell types derived from these embryonic regions, including olfactory bulb and amygdala interneurons and striatal projection neurons from the LGE, as well as cortical interneurons from the MGE and caudal GE (CGE). In summary, this transgenic mouse line represents a new tool for genetic manipulation in the LGE/CGE and the dorsal half of MGE.

The protein tyrosine phosphatase Shp2 is required for the generation of oligodendrocyte progenitor cells and myelination in the mouse telencephalon.

The protein tyrosine phosphatase Shp2 (PTPN11) is crucial for normal brain development and has been implicated in dorsal telencephalic neuronal and astroglia cell fate decisions. However, its roles in the ventral telencephalon and during oligodendrogenesis in the telencephalon remain largely unknown. Shp2 gain-of-function (GOF) mutations are observed in Noonan syndrome, a type of RASopathy associated with multiple phenotypes, including cardiovascular, craniofacial, and neurocognitive abnormalities. To gain insight into requirements for Shp2 (LOF) and the impact of abnormal Shp2 GOF mutations, we used a Shp2 conditional mutant allele (LOF) and a cre inducible Shp2-Q79R GOF transgenic mouse in combination with Olig2(cre/+) mice to target embryonic ventral telencephalic progenitors and the oligodendrocyte lineage. In the absence of Shp2 (LOF), neuronal cell types originating from progenitors in the ventral telencephalon were generated, but oligodendrocyte progenitor cell (OPC) generation was severely impaired. Late embryonic and postnatal Shp2 cKOs showed defects in the generation of OPCs throughout the telencephalon and subsequent reductions in white matter myelination. Conversely, transgenic expression of the Shp2 GOF Noonan syndrome mutation resulted in elevated OPC numbers in the embryo and postnatal brain. Interestingly, expression of this mutation negatively influenced myelination as mice displayed abnormal myelination and fewer myelinated axons in the white matter despite elevated OPC numbers. Increased proliferating OPCs and elevated MAPK activity were also observed during oligodendrogenesis after expression of Shp2 GOF mutation. These results support the notion that appropriate Shp2 activity levels control the number as well as the differentiation of oligodendrocytes during development.

Concomitant inactivation of foxo3a and fancc or fancd2 reveals a two-tier protection from oxidative stress-induced hydrocephalus.

AIMS: This study seeks at investigating the cause of hydrocephalus, and at identifying therapeutic targets for the prevention of hydrocephalus. RESULTS: In this study, we show that inactivation of the Foxo3a gene in two mouse models of Fanconi anemia (FA) leads to the development of hydrocephalus in late embryonic stage and after birth. More than 50% of Foxo3a(-/-) Fancc(-/-) or Foxo3a(-/-) Fancd2(-/-) mice die during embryonic development or within 6 months of life as a result of hydrocephalus characterized by cranial distortion, dilation of the ventricular system, reduced thickness of the cerebral cortex, and disorganization of the ependymal cilia and subcommissural organ. Combined deficiency of Foxo3a and Fancc or Fancd2 not only impairs the self-renewal capacity but also markedly increases the apoptosis of neural stem and progenitor cells (NSPCs), leading to defective neurogenesis. Increased accumulation of reactive oxygen species (ROS) and subsequently de-regulated mitosis and ultimately apoptosis in the neural stem or progenitor cells is identified as one of the potential mechanisms of congenital obstructive hydrocephalus. INNOVATION: The work unravels a two-tier protective mechanism for preventing oxidative stress-induced hydrocephalus. CONCLUSION: The deletion of Foxo3a in FA mice increased the accumulation of ROS and subsequently de-regulated mitosis and ultimately apoptosis in the NSPCs, leading to hydrocephalus development.