Construction and Identification of a Novel Mice Model of Microphthalmia.

Purpose: Microphthalmia is a rare developmental eye disease that affects 1 in 7000 births. Currently, there is no cure for this condition. This study aimed to construct a stable mouse model of microphthalmia, thus providing a new tool for the study of the etiology of microphthalmia. Methods: The Hedgehog signaling pathway plays a crucial role in eye development. One of the key mechanisms of the Sonic Hedgehog signaling is the strong transcriptional activation ability of GLI3, a major mediator of this pathway. This study used CRISPR/Cas9 system to construct a novel TgGli3Ki/Ki lens-specific over-expression mouse line. To identify the ocular characteristics of this line, quantitative PCR, Western blot, hematoxylin and eosin staining, immunofluorescent staining, and RNA-seq were performed on the ocular tissues of this line and normal mice. Results: The TgGli3Ki/Ki lens-specific over-expression mouse model exhibits the ocular phenotype of microphthalmia. In the TgGli3Ki/Ki mouse, Gli3 is over-expressed in the lens, and the size of the eyeball and lens is significantly smaller than the normal one. RNA-seq analysis using the lens and the retina samples from TgGli3Ki/Ki and normal mice indicates that the phototransduction pathway is ectopically activated in the lens. Immunofluorescent staining of the lens samples confirmed this activation. Conclusions: The TgGli3Ki/Ki mouse model consistently manifests the stereotypical microphthalmia phenotype across generations, making it an excellent tool for studying this severe eye disease. Translational Relevance: This study developed a novel animal model to facilitate clinical research on microphthalmia.

Positive regulation of Hedgehog signaling via phosphorylation of GLI2/GLI3 by DYRK2 kinase.

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.

A novel missense variant located within the zinc finger domain of the GLI3 gene was identified in a Vietnamese pedigree with index finger polydactyly.

BACKGROUND: Polydactyly, particularly of the index finger, remains an intriguing anomaly for which no specific gene or locus has been definitively linked to this phenotype. In this study, we conducted an investigation of a three-generation family displaying index finger polydactyly. METHODS: Exome sequencing was conducted on the patient, with a filtration to identify potential causal variation. Validation of the obtained variant was conducted by Sanger sequencing, encompassing all family members. RESULTS: Exome analysis uncovered a novel heterozygous missense variant (c.1482A>T; p.Gln494His) at the zinc finger DNA-binding domain of the GLI3 protein within the proband and all affected family members. Remarkably, the variant was absent in unaffected individuals within the pedigree, underscoring its association with the polydactyly phenotype. Computational analyses revealed that GLI3 p.Gln494His impacts a residue that is highly conserved across species. CONCLUSION: The GLI3 zinc finger DNA-binding region is an essential part of the Sonic hedgehog signaling pathway, orchestrating crucial aspects of embryonic development through the regulation of target gene expression. This novel finding not only contributes valuable insights into the molecular pathways governing polydactyly during embryonic development but also has the potential to enhance diagnostic and screening capabilities for this condition in clinical settings.

TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.

Distinct expression patterns of Hedgehog signaling components in mouse gustatory system during postnatal tongue development and adult homeostasis.

The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development.

A Pilot Immunohistochemical Study Identifies Hedgehog Pathway Expression in Sinonasal Adenocarcinoma.

Tumors of the head and neck, more specifically the squamous cell carcinoma, often show upregulation of the Hedgehog signaling pathway. However, almost nothing is known about its role in the sinonasal adenocarcinoma, either in intestinal or non-intestinal subtypes. In this work, we have analyzed immunohistochemical staining of six Hedgehog pathway proteins, sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched1 (PTCH1), Gli family zinc finger 1 (GLI1), Gli family zinc finger 2 (GLI2), and Gli family zinc finger 3 (GLI3), on 21 samples of sinonasal adenocarcinoma and compared them with six colon adenocarcinoma and three salivary gland tumors, as well as with matching healthy tissue, where available. We have detected GLI2 and PTCH1 in the majority of samples and also GLI1 in a subset of samples, while GLI3 and the ligands SHH and IHH were generally not detected. PTCH1 pattern of staining shows an interesting pattern, where healthy samples are mostly positive in the stromal compartment, while the signal shifts to the tumor compartment in tumors. This, taken together with a stronger signal of GLI2 in tumors compared to non-tumor tissues, suggests that the Hedgehog pathway is indeed activated in sinonasal adenocarcinoma. As Hedgehog pathway inhibitors are being tested in combination with other therapies for head and neck squamous cell carcinoma, this could provide a therapeutic option for patients with sinonasal adenocarcinoma as well.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

The BAF chromatin complex component SMARCC1 does not mediate GLI transcriptional repression of Hedgehog target genes in limb buds.

Transcriptional responses to the Hedgehog (HH) signaling pathway are primarily modulated by GLI repression in the mouse limb. Previous studies suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. However, in contrast to GLI repression which reduces chromatin accessibility, SMARCC1 maintains chromatin accessibility at most enhancers, including those bound by GLI. Moreover, SMARCC1 binding at GLI-regulated enhancers occurs independently of GLI3. Consistent with previous studies, some individual GLI target genes are mis-regulated in Smarcc1 conditional knockouts, though most GLI target genes are unaffected. Moreover, SMARCC1 is not necessary for mediating constitutive GLI repression in HH mutant limb buds. We conclude that SMARCC1 does not mediate GLI3 repression, which we propose utilizes alternative chromatin remodeling complexes.

A novel mechanism of regulation of the oncogenic transcription factor GLI3 by toll-like receptor signaling.

The transcription factor GLI3 is a member of the GLI family and has been shown to be regulated by canonical hedgehog (HH) signaling through smoothened (SMO). Little is known about SMO-independent regulation of GLI3. Here, we identify TLR signaling as a novel pathway regulating GLI3 expression. We show that GLI3 expression is induced by LPS/TLR4 in human monocyte cell lines and peripheral blood CD14+ cells. Further analysis identified TRIF, but not MyD88, signaling as the adapter used by TLR4 to regulate GLI3. Using pharmacological and genetic tools, we identified IRF3 as the transcription factor regulating GLI3 downstream of TRIF. Furthermore, using additional TLR ligands that signal through TRIF such as the TLR4 ligand, MPLA and the TLR3 ligand, Poly(I:C), we confirm the role of TRIF-IRF3 in the regulation of GLI3. We found that IRF3 directly binds to the GLI3 promoter region and this binding was increased upon stimulation of TRIF-IRF3 with Poly(I:C). Furthermore, using Irf3 -/- MEFs, we found that Poly(I:C) stimulation no longer induced GLI3 expression. Finally, using macrophages from mice lacking Gli3 expression in myeloid cells (M-Gli3-/- ), we found that in the absence of Gli3, LPS stimulated macrophages secrete less CCL2 and TNF-α compared with macrophages from wild-type (WT) mice. Taken together, these results identify a novel TLR-TRIF-IRF3 pathway that regulates the expression of GLI3 that regulates inflammatory cytokines and expands our understanding of the non-canonical signaling pathways involved in the regulation of GLI transcription factors.

The epigenetic state of EED-Gli3-Gli1 regulatory axis controls embryonic cortical neurogenesis.

Mutations in the embryonic ectoderm development (EED) cause Weaver syndrome, but whether and how EED affects embryonic brain development remains elusive. Here, we generated a mouse model in which Eed was deleted in the forebrain to investigate the role of EED. We found that deletion of Eed decreased the number of upper-layer neurons but not deeper-layer neurons starting at E16.5. Transcriptomic and genomic occupancy analyses revealed that the epigenetic states of a group of cortical neurogenesis-related genes were altered in Eed knockout forebrains, followed by a decrease of H3K27me3 and an increase of H3K27ac marks within the promoter regions. The switching of H3K27me3 to H3K27ac modification promoted the recruitment of RNA-Pol2, thereby enhancing its expression level. The small molecule activator SAG or Ptch1 knockout for activating Hedgehog signaling can partially rescue aberrant cortical neurogenesis. Taken together, we proposed a novel EED-Gli3-Gli1 regulatory axis that is critical for embryonic brain development.

GLI3 and androgen receptor are mutually dependent for their malignancy-promoting activity in ovarian and breast cancer cells.

Hedgehog signaling pathway has been previously elucidated to be inappropriately activated in many human cancers, including ovarian and breast cancer. However, mechanistic contribution of GLI3, one of the terminal effectors of the pathway, to ovarian and mammary cancer development is underexplored. In this study, we investigated whether GLI3 is necessary for the growth and migration of ovarian and breast cancer cells and further explored the underlying mechanism of GLI3-mediated oncogenesis. We report that GLI3 knockdown inhibited growth and migration of androgen receptor (AR)-positive ovarian and breast cancer cells, but not AR-negative ovarian and breast cancer cells. Furthermore, knockdown of AR expression was effective in inhibiting the growth and migration of AR-positive ovarian and breast cancer cells in the presence of GLI3, but not in GLI3 knockdown cells. Similarly, ectopic expression of AR promoted the growth and migration of AR-negative ovarian and breast cancer cells in the presence of GLI3, but not in GLI3 knockdown cells. GLI3 and AR co-immunoprecipitated each other. GLI3 expression was negatively associated with overall survival of ovarian or breast patients whose tumors expressed a high level of AR. Our findings suggest that GLI3 and AR not only physically interact, but also are mutually dependent for their malignancy-promoting activity in ovarian and breast cancer cells. GLI3-specific inhibitors may be novel therapeutics for AR-expressing ovarian and breast cancers.

Zic5 stabilizes Gli3 via a non-transcriptional mechanism during retinal development.

The Zic family of zinc finger transcription factors plays a critical role in multiple developmental processes. Using loss-of-function studies, we find that Zic5 is important for the differentiation of retinal pigmented epithelium (RPE) and the rod photoreceptor layer through suppressing Hedgehog (Hh) signaling. Further, Zic5 interacts with the critical Hh signaling molecule, Gli3, through the zinc finger domains of both proteins. This Zic5-Gli3 interaction disrupts Gli3/Gli3 homodimerization, resulting in Gli3 protein stabilization via a reduction in Gli3 ubiquitination. During embryonic Hh signaling, the activator form of Gli is normally converted to a repressor form through proteosome-mediated processing of Gli3, and the ratio of Gli3 repressor to full-length (activator) form of Gli3 determines the Gli3 repressor output required for normal eye development. Our results suggest Zic5 is a critical player in regulating Gli3 stability for the proper differentiation of RPE and rod photoreceptor layer during Xenopus eye development.

GLI3 Is Required for OLIG2+ Progeny Production in Adult Dorsal Neural Stem Cells.

The ventricular-subventricular zone (V-SVZ) is a postnatal germinal niche. It holds a large population of neural stem cells (NSCs) that generate neurons and oligodendrocytes for the olfactory bulb and (primarily) the corpus callosum, respectively. These NSCs are heterogeneous and generate different types of neurons depending on their location. Positional identity among NSCs is thought to be controlled in part by intrinsic pathways. However, extrinsic cell signaling through the secreted ligand Sonic hedgehog (Shh) is essential for neurogenesis in both the dorsal and ventral V-SVZ. Here we used a genetic approach to investigate the role of the transcription factors GLI2 and GLI3 in the proliferation and cell fate of dorsal and ventral V-SVZ NSCs. We find that while GLI3 is expressed in stem cell cultures from both dorsal and ventral V-SVZ, the repressor form of GLI3 is more abundant in dorsal V-SVZ. Despite this high dorsal expression and the requirement for other Shh pathway members, GLI3 loss affects the generation of ventrally-, but not dorsally-derived olfactory interneurons in vivo and does not affect trilineage differentiation in vitro. However, loss of GLI3 in the adult dorsal V-SVZ in vivo results in decreased numbers of OLIG2-expressing progeny, indicating a role in gliogenesis.

GLI3 Is Stabilized by SPOP Mutations and Promotes Castration Resistance via Functional Cooperation with Androgen Receptor in Prostate Cancer.

Although the Sonic hedgehog (SHH) signaling pathway has been implicated in promoting malignant phenotypes of prostate cancer, details on how it is activated and exerts its oncogenic role during prostate cancer development and progression is less clear. Here, we show that GLI3, a key SHH pathway effector, is transcriptionally upregulated during androgen deprivation and posttranslationally stabilized in prostate cancer cells by mutation of speckle-type POZ protein (SPOP). GLI3 is a substrate of SPOP-mediated proteasomal degradation in prostate cancer cells and prostate cancer driver mutations in SPOP abrogate GLI3 degradation. Functionally, GLI3 is necessary and sufficient for the growth and migration of androgen receptor (AR)-positive prostate cancer cells, particularly under androgen-depleted conditions. Importantly, we demonstrate that GLI3 physically interacts and functionally cooperates with AR to enrich an AR-dependent gene expression program leading to castration-resistant growth of xenografted prostate tumors. Finally, we identify an AR/GLI3 coregulated gene signature that is highly correlated with castration-resistant metastatic prostate cancer and predictive of disease recurrence. Together, these findings reveal that hyperactivated GLI3 promotes castration-resistant growth of prostate cancer and provide a rationale for therapeutic targeting of GLI3 in patients with castration-resistant prostate cancer (CRPC). IMPLICATIONS: We describe two clinically relevant mechanisms leading to hyperactivated GLI3 signaling and enhanced AR/GLI3 cross-talk, suggesting that GLI3-specific inhibitors might prove effective to block prostate cancer development or delay CRPC.

Molecular Etiology in Thumb Polydactyly in Mutated GLI3 Mouse Gene.

Sporadic thumb polydactyly with nonfamily inheritance is the most common in clinical work. This study focused on characterization of GLI3 gene function. We constructed the plasmid with p.m948i point mutation of GLI3 and transfected it into mouse embryonic fibroblasts (MEFs) to study the effects and potential mechanism of the mutant gene. The RNA of GLI3 mutant cells was extracted and analyzed by transcriptome sequencing and bioinformatics. Finally, we constructed cbx3 overexpression plasmid, designed siRNA for gene silencing, and transfected it into the MEFs. Cell proliferation and invasion ability of the MEFs were examined. The results showed that there were 2,452 differential expression genes in the MEFs transfected with GLI3 mutant plasmid compared with wild-type MEFs. The results of differential expression analysis showed that the cbx3 gene was significantly up-regulated. Overexpression of cbx3 in MEFs promoted cell proliferation and invasion, while siRNA knockdown of cbx3 expression reduced proliferation and invasion. GLI3 gene mutation in MEFs resulted in cbx3 up-regulation and promoted MEF proliferation and invasion. This study further clarified the potential function of GLI3 in limb development, established a new relationship between gene mutation and polydactyly, and preliminarily clarified the possible signal pathway, all of which have laid a foundation for further study on the etiology of polydactyl.

Conserved and species-specific chromatin remodeling and regulatory dynamics during mouse and chicken limb bud development.

Chromatin remodeling and genomic alterations impact spatio-temporal regulation of gene expression, which is central to embryonic development. The analysis of mouse and chicken limb development provides important insights into the morphoregulatory mechanisms, however little is known about the regulatory differences underlying their morphological divergence. Here, we identify the underlying shared and species-specific epigenomic and genomic variations. In mouse forelimb buds, we observe striking synchrony between the temporal dynamics of chromatin accessibility and gene expression, while their divergence in chicken wing buds uncovers species-specific regulatory heterochrony. In silico mapping of transcription factor binding sites and computational footprinting establishes the developmental time-restricted transcription factor-DNA interactions. Finally, the construction of target gene networks for HAND2 and GLI3 transcriptional regulators reveals both conserved and species-specific interactions. Our analysis reveals the impact of genome evolution on the regulatory interactions orchestrating vertebrate limb bud morphogenesis and provides a molecular framework for comparative Evo-Devo studies.

Long non-coding RNA SLC2A1-AS1 induced by GLI3 promotes aerobic glycolysis and progression in esophageal squamous cell carcinoma by sponging miR-378a-3p to enhance Glut1 expression.

BACKGROUND: Emerging evidence demonstrates that lncRNAs play pivotal roles in tumor energy metabolism; however, the detailed mechanisms of lncRNAs in the regulation of tumor glycolysis remain largely unknown. METHODS: The expression of SLC2A1-AS1 was investigated by TCGA, GEO dataset and qRT-PCR. The binding of GLI3 to SLC2A1-AS1 promoter was detected by Luciferase Reporter Assay System and Ago2-RIP assay. FISH was performed to determine the localization of SLC2A1-AS1 in ESCC cells. Double Luciferase Report assay was used to investigate the interaction of miR-378a-3p with SLC2A1-AS1 and Glut1. Gain-of-function and Loss-of-function assay were performed to dissect the function of SLC2A1-AS1/miR-378a-3p/Glut1 axis in ESCC progression in vitro and in vivo. RESULTS: We identified a novel lncRNA SLC2A1-AS1 in ESCC. SLC2A1-AS1 was frequently overexpressed in ESCC tissues and cells, and its overexpression was associated with TNM stage, lymph node metastasis and poor prognosis of ESCC patients. Importantly, GLI3 and SLC2A1-AS1 formed a regulatory feedback loop in ESCC cells. SLC2A1-AS1 promoted cell growth in vitro and in vivo, migration and invasion, and suppressed apoptosis, leading to EMT progression and increased glycolysis in ESCC cells. SLC2A1-AS1 functioned as ceRNA for sponging miR-378a-3p, resulting in Glut1 overexpression in ESCC cells. MiR-378a-3p inhibited cell proliferation and invasion as well as induced apoptosis, resulting in reduced glycolysis, which was partly reversed by SLC2A1-AS1 or Glut1 overexpression in ESCC cells. CONCLUSION: SLC2A1-AS1 plays important roles in ESCC development and progression by regulating glycolysis, and SLC2A1-AS1/miR-378a-3p/Glut1 regulatory axis may be a novel therapeutic target in terms of metabolic remodeling of ESCC patients.

Hedgehog gene expression patterns among intrinsic subtypes of breast cancer: Prognostic relevance.

PURPOSE: Hedgehog (Hh) signaling is a crucial developmental regulatory pathway recognized as a primary oncogenesis driver in various human cancers. However, its role in breast carcinoma (BC) has been underexplored. METHODS: We analyzed the expression of several Hh associated genes in a clinical series and breast cancer cell lines. We included 193 BC stratified according to intrinsic immunophenotypes. Gene expression profiling ofBOC, PTCH, SMO, GLI1, GLI2, and GLI3 was performed by qRT-PCR. Results were correlated with clinical-pathological variables and outcome. RESULTS: We observed expression ofGLI2 in triple-negative/basal-like (TN/BL) and GLI3 in luminal cells. In samples, BOC, GLI1, GLI2, and GLI3 expression correlated significantly with luminal tumors and good prognostic factors. In contrast, PTCH and SMO correlated with TN/BL phenotype and nodal involvement. Patients whose tumors expressed SMO had a poorer outcome, especially those with HER2 phenotype. Positive lymph-node status and high SMO remained independent poor prognostic factors. CONCLUSION: Our results support a differential Hh pathway activation in BC phenotypes.SMO levels stratified patients at risk of recurrence and death in HER2 phenotype, and it showed an independent prognostic value. Therefore, SMO could be a potential therapeutic target for a subset of BC patients.

Topoisomerase IIß immunoreactivity (IR) co-localizes with neuronal marker-IR but not glial fibrillary acidic protein-IR in GLI3-positive medulloblastomas: an immunohistochemical analysis of 124 medulloblastomas from the Japan Children's Cancer Group.

We previously reported observing GLI3 in medulloblastomas expressing neuronal markers (NM) and/or glial fibrillary acidic protein (GFAP). Furthermore, patients with medulloblastomas expressing NM or GFAP tended to show favorable or poor prognosis, respectively. In the present study, we focused on the role of topoisomerase IIß (TOP2ß) as a possible regulator for neuronal differentiation in medulloblastomas and examined the pathological roles of GLI3, NM, GFAP, and TOP2ß expressions in a larger population. We divided 124 medulloblastomas into three groups (NM-/GFAP-, NM +/GFAP-, and GFAP +) based on their immunoreactivity (IR) against NM and GFAP. The relationship among GLI3, NM, GFAP, and TOP2ß was evaluated using fluorescent immunostaining and a publicly available single-cell RNA sequencing dataset. In total, 87, 30, and 7 medulloblastomas were classified as NM-/GFAP-, NM + /GFAP-, and GFAP +, and showed intermediate, good, and poor prognoses, respectively. GLI3-IR was frequently observed in NM +/GFAP- and GFAP + , and TOP2ß-IR was frequently observed only in NM +/GFAP- medulloblastomas. In fluorescent immunostaining, TOP2ß-IR was mostly co-localized with NeuN-IR but not with GFAP-IR. In single-cell RNA sequencing, TOP2ß expression was elevated in CMAS/DCX-positive, but not in GFAP-positive, cells. NM-IR and GFAP-IR are important for estimating the prognosis of patients with medulloblastoma; hence they should be assessed in clinical practice.

An early Sox2-dependent gene expression programme required for hippocampal dentate gyrus development.

The hippocampus is a brain area central for cognition. Mutations in the human SOX2 transcription factor cause neurodevelopmental defects, leading to intellectual disability and seizures, together with hippocampal dysplasia. We generated an allelic series of Sox2 conditional mutations in mouse, deleting Sox2 at different developmental stages. Late Sox2 deletion (from E11.5, via Nestin-Cre) affects only postnatal hippocampal development; earlier deletion (from E10.5, Emx1-Cre) significantly reduces the dentate gyrus (DG), and the earliest deletion (from E9.5, FoxG1-Cre) causes drastic abnormalities, with almost complete absence of the DG. We identify a set of functionally interconnected genes (Gli3, Wnt3a, Cxcr4, p73 and Tbr2), known to play essential roles in hippocampal embryogenesis, which are downregulated in early Sox2 mutants, and (Gli3 and Cxcr4) directly controlled by SOX2; their downregulation provides plausible molecular mechanisms contributing to the defect. Electrophysiological studies of the Emx1-Cre mouse model reveal altered excitatory transmission in CA1 and CA3 regions.