Olfactory bulb anomalies in KBG syndrome mouse model and patients.

ANKRD11 (ankyrin repeat domain 11) is a chromatin regulator and the only gene associated with KBG syndrome, a rare neurodevelopmental disorder. We have previously shown that Ankrd11 regulates murine embryonic cortical neurogenesis. Here, we show a novel olfactory bulb phenotype in a KBG syndrome mouse model and two diagnosed patients. Conditional knockout of Ankrd11 in murine embryonic neural stem cells leads to aberrant postnatal olfactory bulb development and reduced size due to reduction of the olfactory bulb granule cell layer. We further show that the rostral migratory stream has incomplete migration of neuroblasts, reduced cell proliferation as well as aberrant differentiation of neurons. This leads to reduced neuroblasts and neurons in the olfactory bulb granule cell layer. In vitro, Ankrd11-deficient neural stem cells from the postnatal subventricular zone display reduced migration, proliferation, and neurogenesis. Finally, we describe two clinically and molecularly confirmed KBG syndrome patients with anosmia and olfactory bulb and groove hypo-dysgenesis/agenesis. Our report provides evidence that Ankrd11 is a novel regulator of olfactory bulb development and neuroblast migration. Moreover, our study highlights a novel clinical sign of KBG syndrome linked to ANKRD11 perturbations in mice and humans.

Documentation and prevalence of prenatal and neonatal outcomes in a cohort of individuals with KBG syndrome.

Ankyrin Repeat Domain 11 (ANKRD11) gene mutations are associated with KBG syndrome, a developmental disability that affects multiple organ systems. The function of ANKRD11 in human growth and development is not clear, but gene knockout or mutation are lethal in mice embryos and/or pups. In addition, it plays a vital role in chromatin regulation and transcription. Individuals with KBG syndrome are often misdiagnosed or remain undiagnosed until later in life. This is largely due to KBG syndrome's varying and nonspecific phenotypes as well as a lack of accessible genetic testing and prenatal screening. This study documents perinatal outcomes for individuals with KBG syndrome. We obtained data from 42 individuals through videoconferences, medical records, and emails. 45.2% of our cohort was born by C-section, 33.3% had a congenital heart defect, 23.8% were born prematurely, 23.8% were admitted to the NICU, 14.3% were small for gestational age, and 14.3% of the families had a history of miscarriage. These rates were higher in our cohort compared to the overall population, including non-Hispanic and Hispanic populations. Other reports included feeding difficulties (21.4%), neonatal jaundice (14.3%), decreased fetal movement (7.1%), and pleural effusions in utero (4.7%). Comprehensive perinatal studies about KBG syndrome and updated documentation of its phenotypes are important in ensuring prompt diagnosis and can facilitate correct management.

BRG1 interacts with GLI2 and binds Mef2c gene in a hedgehog signalling dependent manner during in vitro cardiomyogenesis.

BACKGROUND: The Hedgehog (HH) signalling pathway regulates cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (mES) cell model. To further assess the transcriptional role of HH signalling during cardiomyogenesis in stem cells, we studied the effects of overexpressing GLI2, a primary transducer of the HH signalling pathway, in mES cells. RESULTS: Stable GLI2 overexpression resulted in an enhancement of cardiac progenitor-enriched genes, Mef2c, Nkx2-5, and Tbx5 during mES cell differentiation. In contrast, pharmacological blockade of the HH pathway in mES cells resulted in lower expression of these genes. Mass spectrometric analysis identified the chromatin remodelling factor BRG1 as a protein which co-immunoprecipitates with GLI2 in differentiating mES cells. We then determined that BRG1 is recruited to a GLI2-specific Mef2c gene element in a HH signalling-dependent manner during cardiomyogenesis in P19 EC cells, a mES cell model. CONCLUSIONS: Thus, we propose a mechanism where HH/GLI2 regulates the expression of Mef2c by recruiting BRG1 to the Mef2c gene, most probably via chromatin remodelling, to ultimately regulate in vitro cardiomyogenesis.

CBP regulates the differentiation of interneurons from ventral forebrain neural precursors during murine development.

The mechanisms that regulate appropriate genesis and differentiation of interneurons in the developing mammalian brain are of significant interest not only because interneurons play key roles in the establishment of neural circuitry, but also because when they are deficient, this can cause epilepsy. In this regard, one genetic syndrome that is associated with deficits in neural development and epilepsy is Rubinstein-Taybi Syndrome (RTS), where the transcriptional activator and histone acetyltransferase CBP is mutated and haploinsufficient. Here, we have asked whether CBP is necessary for the appropriate genesis and differentiation of interneurons in the murine forebrain, since this could provide an explanation for the epilepsy that is associated with RTS. We show that CBP is expressed in neural precursors within the embryonic medial ganglionic eminence (MGE), an area that generates the vast majority of interneurons for the cortex. Using primary cultures of MGE precursors, we show that knockdown of CBP causes deficits in differentiation of these precursors into interneurons and oligodendrocytes, and that overexpression of CBP is by itself sufficient to enhance interneuron genesis. Moreover, we show that levels of the neurotransmitter synthesis enzyme GAD67, which is expressed in inhibitory interneurons, are decreased in the dorsal and ventral forebrain of neonatal CBP(+/-) mice, indicating that CBP plays a role in regulating interneuron development in vivo. Thus, CBP normally acts to ensure the differentiation of appropriate numbers of forebrain interneurons, and when its levels are decreased, this causes deficits in interneuron development, providing a potential explanation for the epilepsy seen in individuals with RTS.

Hedgehog signaling regulates MyoD expression and activity.

The inhibition of MyoD expression is important for obtaining muscle progenitors that can replenish the satellite cell niche during muscle repair. Progenitors could be derived from either embryonic stem cells or satellite cells. Hedgehog (Hh) signaling is important for MyoD expression during embryogenesis and adult muscle regeneration. To date, the mechanistic understanding of MyoD regulation by Hh signaling is unclear. Here, we demonstrate that the Hh effector, Gli2, regulates MyoD expression and associates with MyoD gene elements. Gain- and loss-of-function experiments in pluripotent P19 cells show that Gli2 activity is sufficient and required for efficient MyoD expression during skeletal myogenesis. Inhibition of Hh signaling reduces MyoD expression during satellite cell activation in vitro. In addition to regulating MyoD expression, Hh signaling regulates MyoD transcriptional activity, and MyoD activates Hh signaling in myogenic conversion assays. Finally, Gli2, MyoD, and MEF2C form a protein complex, which enhances MyoD activity on skeletal muscle-related promoters. We therefore link Hh signaling to the function and expression of MyoD protein during myogenesis in stem cells.

Gli2 and MEF2C activate each other's expression and function synergistically during cardiomyogenesis in vitro.

The transcription factors Gli2 (glioma-associated factor 2), which is a transactivator of Sonic Hedgehog (Shh) signalling, and myocyte enhancer factor 2C (MEF2C) play important roles in the development of embryonic heart muscle and enhance cardiomyogenesis in stem cells. Although the physiological importance of Shh signalling and MEF2 factors in heart development is well known, the mechanistic understanding of their roles is unclear. Here, we demonstrate that Gli2 and MEF2C activated each other's expression while enhancing cardiomyogenesis in differentiating P19 EC cells. Furthermore, dominant-negative mutant proteins of either Gli2 or MEF2C repressed each other's expression, while impairing cardiomyogenesis in P19 EC cells. In addition, chromatin immunoprecipitation (ChIP) revealed association of Gli2 to the Mef2c gene, and of MEF2C to the Gli2 gene in differentiating P19 cells. Finally, co-immunoprecipitation studies showed that Gli2 and MEF2C proteins formed a complex, capable of synergizing on cardiomyogenesis-related promoters containing both Gli- and MEF2-binding elements. We propose a model whereby Gli2 and MEF2C bind each other's regulatory elements, activate each other's expression and form a protein complex that synergistically activates transcription, enhancing cardiac muscle development. This model links Shh signalling to MEF2C function during cardiomyogenesis and offers mechanistic insight into their in vivo functions.

The chromatin regulator Ankrd11 controls cardiac neural crest cell-mediated outflow tract remodeling and heart function.

ANKRD11 (Ankyrin Repeat Domain 11) is a chromatin regulator and a causative gene for KBG syndrome, a rare developmental disorder characterized by multiple organ abnormalities, including cardiac defects. However, the role of ANKRD11 in heart development is unknown. The neural crest plays a leading role in embryonic heart development, and its dysfunction is implicated in congenital heart defects. We demonstrate that conditional knockout of Ankrd11 in the murine embryonic neural crest results in persistent truncus arteriosus, ventricular dilation, and impaired ventricular contractility. We further show these defects occur due to aberrant cardiac neural crest cell organization leading to outflow tract septation failure. Lastly, knockout of Ankrd11 in the neural crest leads to impaired expression of various transcription factors, chromatin remodelers and signaling pathways, including mTOR, BMP and TGF-ß in the cardiac neural crest cells. In this work, we identify Ankrd11 as a regulator of neural crest-mediated heart development and function.

Testosterone enhances cardiomyogenesis in stem cells and recruits the androgen receptor to the MEF2C and HCN4 genes.

Since a previous study (Goldman-Johnson et al., 2008 [4]) has shown that androgens can stimulate increased differentiation of mouse embryonic stem (mES) cells into cardiomyocytes using a genomic pathway, the aim of our study is to elucidate the molecular mechanisms regulating testosterone-enhanced cardiomyogenesis. Testosterone upregulated cardiomyogenic transcription factors, including GATA4, MEF2C, and Nkx2.5, muscle structural proteins, and the pacemaker ion channel HCN4 in a dose-dependent manner, in mES cells and P19 embryonal carcinoma cells. Knock-down of the androgen receptor (AR) or treatment with anti-androgenic compounds inhibited cardiomyogenesis, supporting the requirement of the genomic pathway. Chromatin immunoprecipitation (ChIP) studies showed that testosterone enhanced recruitment of AR to the regulatory regions of MEF2C and HCN4 genes, which was associated with increased histone acetylation. In summary, testosterone upregulated cardiomyogenic transcription factor and HCN4 expression in stem cells. Further, testosterone induced cardiomyogenesis, at least in part, by recruiting the AR receptor to the regulatory regions of the MEF2C and HCN4 genes. These results provide a detailed molecular analysis of the function of testosterone in stem cells and may offer molecular insight into the role of steroids in the heart.

Regulation of microglia function by neural stem cells.

Neural stem and precursor cells (NPCs) build and regenerate the central nervous system (CNS) by maintaining their pool (self-renewal) and differentiating into neurons, astrocytes, and oligodendrocytes (multipotency) throughout life. This has inspired research into pro-regenerative therapies that utilize transplantation of exogenous NPCs or recruitment of endogenous adult NPCs for CNS regeneration and repair. Recent advances in single-cell RNA sequencing and other "omics" have revealed that NPCs express not just traditional progenitor-related genes, but also genes involved in immune function. Here, we review how NPCs exert immunomodulatory function by regulating the biology of microglia, immune cells that are present in NPC niches and throughout the CNS. We discuss the role of transplanted and endogenous NPCs in regulating microglia fates, such as survival, proliferation, migration, phagocytosis and activation, in the developing, injured and degenerating CNS. We also provide a literature review on NPC-specific mediators that are responsible for modulating microglia biology. Our review highlights the immunomodulatory properties of NPCs and the significance of these findings in the context of designing pro-regenerative therapies for degenerating and diseased CNS.

Culture Protocol and Transcriptomic Analysis of Murine SVZ NPCs and OPCs.

The mammalian adult brain contains two neural stem and precursor (NPC) niches: the subventricular zone [SVZ] lining the lateral ventricles and the subgranular zone [SGZ] in the hippocampus. From these, SVZ NPCs represent the largest NPC pool. While SGZ NPCs typically only produce neurons and astrocytes, SVZ NPCs produce neurons, astrocytes and oligodendrocytes throughout life. Of particular importance is the generation and replacement of oligodendrocytes, the only myelinating cells of the central nervous system (CNS). SVZ NPCs contribute to myelination by regenerating the parenchymal oligodendrocyte precursor cell (OPC) pool and by differentiating into oligodendrocytes in the developing and demyelinated brain. The neurosphere assay has been widely adopted by the scientific community to facilitate the study of NPCs in vitro. Here, we present a streamlined protocol for culturing postnatal and adult SVZ NPCs and OPCs from primary neurosphere cells. We characterize the purity and differentiation potential as well as provide RNA-sequencing profiles of postnatal SVZ NPCs, postnatal SVZ OPCs and adult SVZ NPCs. We show that primary neurospheres cells generated from postnatal and adult SVZ differentiate into neurons, astrocytes and oligodendrocytes concurrently and at comparable levels. SVZ OPCs are generated by subjecting primary neurosphere cells to OPC growth factors fibroblast growth factor (FGF) and platelet-derived growth factor-AA (PDGF-AA). We further show SVZ OPCs can differentiate into oligodendrocytes in the absence and presence of thyroid hormone T3. Transcriptomic analysis confirmed the identities of each cell population and revealed novel immune and signalling pathways expressed in an age and cell type specific manner.

Fractalkine enhances oligodendrocyte regeneration and remyelination in a demyelination mouse model.

Demyelinating disorders of the central nervous system (CNS) occur when myelin and oligodendrocytes are damaged or lost. Remyelination and regeneration of oligodendrocytes can be achieved from endogenous oligodendrocyte precursor cells (OPCs) that reside in the adult CNS tissue. Using a cuprizone mouse model of demyelination, we show that infusion of fractalkine (CX3CL1) into the demyelinated murine brain increases de novo oligodendrocyte formation and enhances remyelination in the corpus callosum and cortical gray matter. This is achieved by increased OPC proliferation in the cortical gray matter as well as OPC differentiation and attenuation of microglia/macrophage activation both in corpus callosum and cortical gray matter. Finally, we show that activated OPCs and microglia/macrophages express fractalkine receptor CX3CR1 in vivo, and that in OPC-microglia co-cultures fractalkine increases in vitro oligodendrocyte differentiation by modulating both OPC and microglia biology. Our results demonstrate a novel pro-regenerative role of fractalkine in a demyelinating mouse model.

MyoD directly up-regulates premyogenic mesoderm factors during induction of skeletal myogenesis in stem cells.

Gain- and loss-of-function experiments have illustrated that the family of myogenic regulatory factors is necessary and sufficient for the formation of skeletal muscle. Furthermore, MyoD required cellular aggregation to induce myogenesis in P19 embryonal carcinoma stem cells. To determine the mechanism by which stem cells can be directed into skeletal muscle, a time course of P19 cell differentiation was examined in the presence and absence of exogenous MyoD. By quantitative PCR, the first MyoD up-regulated transcripts were the premyogenic mesoderm factors Meox1, Pax7, Six1, and Eya2 on day 4 of differentiation. Subsequently, the myoblast markers myogenin, MEF2C, and Myf5 were up-regulated, leading to skeletal myogenesis. These results were corroborated by Western blot analysis, showing up-regulation of Pax3, Six1, and MEF2C proteins, prior to myogenin protein expression. To determine at what stage a dominant-negative MyoD/EnR mutant could inhibit myogenesis, stable cell lines were created and examined. Interestingly, the premyogenic mesoderm factors, Meox1, Pax3/7, Six1, Eya2, and Foxc1, were down-regulated, and as expected, skeletal myogenesis was abolished. Finally, to identify direct targets of MyoD in this system, chromatin immunoprecipitation experiments were performed. MyoD was observed associated with regulatory regions of Meox1, Pax3/7, Six1, Eya2, and myogenin genes. Taken together, MyoD directs stem cells into the skeletal muscle lineage by binding and activating the expression of premyogenic mesoderm genes, prior to activating myoblast genes.

Fine-tuning the stimulation of MLL1 methyltransferase activity by a histone H3-based peptide mimetic.

The SET1 family of methyltransferases carries out the bulk of histone H3 Lys-4 methylation in vivo. One of the common features of this family is the regulation of their methyltransferase activity by a tripartite complex composed of WDR5, RbBP5, and Ash2L. To selectively probe the role of the SET1 family of methyltransferases, we have developed a library of histone H3 peptide mimetics and report herein the characterization of an Nα acetylated form of histone H3 peptide (NαH3). Binding and inhibition studies reveal that the addition of an acetyl moiety to the N terminus of histone H3 significantly enhances its binding to WDR5 and prevents the stimulation of MLL1 methyltransferase activity by the WDR5-RbBP5-Ash2L complex. The crystal structure of NαH3 in complex with WDR5 reveals that a high-affinity hydrophobic pocket accommodates the binding of the acetyl moiety. These results provide the structural basis to control WDR5-RbBP5-Ash2L-MLL1 activity and a tool to manipulate stem cell differentiation programs.

Human placenta and trophoblasts simultaneously express three isoforms of atypical protein kinase-c.

Atypical protein kinase-c (aPKC) isoforms are important regulators of polarity and stem cell differentiation. There are three isoforms of aPKC: aPKC-ι, aPKC-ζ, and PKM-ζ. Recently, aPKC-ι was shown to regulate human trophoblast stem cell (TSC) differentiation. Compensation by remaining isoforms when a single aPKC isoform is lost can occur, but the expression pattern of aPKC-ζ in placenta is unknown. Here we show that aPKC-ι, aPKC-ζ and a new isoform, aPKC-ζ III, are expressed in trophoblasts. Therefore, studies examining the role of aPKC isoforms that control for potential compensation between aPKC isoforms are necessary to understand aPKC-mediated regulation of TSC differentiation.

Hepatoma Derived Growth Factor Enhances Oligodendrocyte Genesis from Subventricular Zone Precursor Cells.

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), perform vital functions in neural protection and communication, as well as cognition. Enhanced production of oligodendrocytes has been identified as a therapeutic approach for neurodegenerative and neurodevelopmental disorders. In the postnatal brain, oligodendrocytes are generated from the neural stem and precursor cells (NPCs) in the subventricular zone (SVZ) and parenchymal oligodendrocyte precursor cells (OPCs). Here, we demonstrate exogenous Hepatoma Derived Growth Factor (HDGF) enhances oligodendrocyte genesis from murine postnatal SVZ NPCs in vitro without affecting neurogenesis or astrogliogenesis. We further show that this is achieved by increasing proliferation of both NPCs and OPCs, as well as OPC differentiation into oligodendrocytes. In vivo results demonstrate that intracerebroventricular infusion of HDGF leads to increased oligodendrocyte genesis from SVZ NPCs, as well as OPC proliferation. Our results demonstrate a novel role for HDGF in regulating SVZ precursor cell proliferation and oligodendrocyte differentiation.

KBG syndrome: videoconferencing and use of artificial intelligence driven facial phenotyping in 25 new patients.

Genetic variants in Ankyrin Repeat Domain 11 (ANKRD11) and deletions in 16q24.3 are known to cause KBG syndrome, a rare syndrome associated with craniofacial, intellectual, and neurobehavioral anomalies. We report 25 unpublished individuals from 22 families with molecularly confirmed diagnoses. Twelve individuals have de novo variants, three have inherited variants, and one is inherited from a parent with low-level mosaicism. The mode of inheritance was unknown for nine individuals. Twenty are truncating variants, and the remaining five are missense (three of which are found in one family). We present a protocol emphasizing the use of videoconference and artificial intelligence (AI) in collecting and analyzing data for this rare syndrome. A single clinician interviewed 25 individuals throughout eight countries. Participants' medical records were reviewed, and data was uploaded to the Human Disease Gene website using Human Phenotype Ontology (HPO) terms. Photos of the participants were analyzed by the GestaltMatcher and DeepGestalt, Face2Gene platform (FDNA Inc, USA) algorithms. Within our cohort, common traits included short stature, macrodontia, anteverted nares, wide nasal bridge, wide nasal base, thick eyebrows, synophrys and hypertelorism. Behavioral issues and global developmental delays were widely present. Neurologic abnormalities including seizures and/or EEG abnormalities were common (44%), suggesting that early detection and seizure prophylaxis could be an important point of intervention. Almost a quarter (24%) were diagnosed with attention deficit hyperactivity disorder and 28% were diagnosed with autism spectrum disorder. Based on the data, we provide a set of recommendations regarding diagnostic and treatment approaches for KBG syndrome.

Agathisflavone Modifies Microglial Activation State and Myelination in Organotypic Cerebellar Slices Culture.

Oligodendrocytes produce the myelin that is critical for rapid neuronal transmission in the central nervous system (CNS). Disruption of myelin has devastating effects on CNS function, as in the demyelinating disease multiple sclerosis (MS). Microglia are the endogenous immune cells of the CNS and play a central role in demyelination and repair. There is a need for new potential therapies that regulate myelination and microglia to promote repair. Agathisflavone (FAB) is a non-toxic flavonoid that is known for its anti-inflammatory and neuroprotective properties. Here, we examined the effects of FAB (5-50 µM) on myelination and microglia in organotypic cerebellar slices prepared from P10-P12 Sox10-EGFP and Plp1-DsRed transgenic mice. Immunofluorescence labeling for myelin basic protein (MBP) and neurofilament (NF) demonstrates that FAB significantly increased the proportion of MBP + /NF + axons but did not affect the overall number of oligodendroglia or axons, or the expression of oligodendroglial proteins CNPase and MBP. FAB is known to be a phytoestrogen, but blockade of α- or ß- estrogen receptors (ER) indicated the myelination promoting effects of FAB were not mediated by ER. Examination of microglial responses by Iba1 immunohistochemistry demonstrated that FAB markedly altered microglial morphology, characterized by smaller somata and reduced branching of their processes, consistent with a decreased state of activation, and increased Iba1 protein expression. The results provide evidence that FAB increases the extent of axonal coverage by MBP immunopositive oligodendroglial processes and has a modulatory effect upon microglial cells, which are important therapeutic strategies in multiple neuropathologies.

Canonical Wnt signaling regulates Foxc1/2 expression in P19 cells.

FOXC1 and FOXC2 are forkhead/winged-helix transcription factors expressed in paraxial mesoderm and somites. Emphasizing the importance of FOXC1/2 during embryonic development, double-knockout mice lacking the alleles for both Foxc1 and Foxc2 failed to form segmented somites and undergo myogenesis. The present study aims to determine upstream factors that regulate Foxc1/2 expression during the differentiation of P19 cells into skeletal muscle. Previous work had shown that dominant-negative forms of beta-catenin, Gli2, and Meox1 could inhibit distinct stages of skeletal myogenesis in P19 cells. In the presence of a dominant-negative beta-catenin fusion protein, Foxc1/2 transcripts were not upregulated and neither were markers of somitogenesis/myogenesis, including Meox1, Pax3 and MyoD. Conversely, inhibition of GSK3 by LiCl or overexpression of activated beta-catenin in aggregated P19 cells resulted in enhancement of Foxc1/2 expression, indicating that FOX transcription may be under the control of Wnt signaling. Supporting this hypothesis, beta-catenin bound to conserved regions upstream of Foxc1 during P19 cell differentiation and drove transcription from this region in a promoter assay. In addition, ectopic expression of a dominant-negative Meox1 or Gli2 resulted in decreased Foxc1/2 transcript levels, correlating with inhibition of skeletal myogenesis. Overexpression of Gli2 was also sufficient to upregulate Foxc1/2 transcript levels and induce skeletal myogenesis. In summary, Foxc1/2 expression is dependent on a complex interplay from various signaling inputs from the Wnt and Shh pathways during early stages of in vitro skeletal myogenesis.

Increasing phagocytosis of microglia by targeting CD33 with liposomes displaying glycan ligands.

CD33 is an immunomodulatory receptor expressed by microglia and genetically linked to Alzheimer's disease (AD) susceptibility. While antibodies targeting CD33 have entered clinical trials to treat neurodegeneration, it is unknown whether the glycan-binding properties of CD33 can be exploited to modulate microglia. Here, we use liposomes that multivalently display glycan ligands of CD33 (CD33L liposomes) to engage CD33. We find that CD33L liposomes increase phagocytosis of cultured monocytic cells and microglia in a CD33-dependent manner. Enhanced phagocytosis strongly correlates with loss of CD33 from the cell surface and internalization of liposomes. Increased phagocytosis by treatment with CD33L liposomes is dependent on a key intracellular signaling motif on CD33 as well as the glycan-binding ability of CD33. These effects are specific to trans engagement of CD33 by CD33L liposomes, as cis engagement through insertion of lipid-linked CD33L into cells produces the opposite effect on phagocytosis. Moreover, intracerebroventricular injection of CD33L liposomes into transgenic mice expressing human CD33 in the microglial cell lineage enhances phagocytosis of microglia in a CD33-dependent manner. These results demonstrate that multivalent engagement of CD33 with glycan ligands can modulate microglial cell function.