Faculty Bibliography

The embryonic tissue origins of the skull vault result in difference in the properties of the bone component. However, the specific molecular differences behind the bone elements are not clear. In this study, we analyzed the differences in osteoblast activities from frontal and parietal bone at embryonic stage. The results showed that the osteoblasts of neural crest derived frontal bone are more capable of less bone matrix, higher proliferative ability and robust ALP activities, while the osteoblasts of mesoderm derived parietal tissue are with higher bone matrix, poor ALP activities and less ability of ossification. Our data provides a specific molecular difference in osteoblasts of frontal and parietal bone during the development.
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Medulloblastoma (MB), the most common malignant pediatric brain tumor, is a classic example of dysregulation of developmental pathways leading to tumorogenesis. Despite advancements in multi-modal therapies, most patients suffer from long-term neurocognitive and neuroendocrine disabilities. The Sonic Hedgehog subgroup of MB (SHH-MB) accounts for ~30% of all cases and originates from ATOH1+ cerebellar granule cell precursors (GCPs). Experimental data in mice has shown that activating mutations in the SHH pathway induce tumors only in rare GCPs, suggesting that additional mutations and epigenetic changes are required to influence tumor progression. The KMT2D gene, encoding the histone-lysine N-methyltransferase 2D, is amongst the ten most frequently mutated genes in MB, with somatic mutations seen in ~15% of all SHH-MB patients. We developed sporadic mouse models of SHH-MB with a low penetrance to enable studies of secondary mutations (Tan, PNAS, 2018). Immunofluorescence staining for KMT2D on early-stage SHH-MB lesions, mid-stage and late-stage tumors revealed that a subset of lesions/tumors (16/98) do not express KMT2D and are negative for H3K4me3. Interestingly, P53 and KMT2D expression showed a positive correlation in ~94% of tumors/lesions and NeuN and KMT2D showed a positive correlation in ~92% of tumors/lesions. In order to determine the roles for KMT2D in GCP proliferation and differentiation, and uncover whether and how KMT2D promotes SHH-MB tumorigenesis, we are using genetic mouse-models whereby Kmt2d is heterozygously or homozygously deleted alone, or in conjunction with activation of the SHH pathway. Mice with SHH-MB tumors expressing SmoM2 and a loss of Kmt2d develop aggressive tumors at high penetrance, with metastatic leptomeningeal spread in the brain stem and spinal cord. Thus, loss of Kmt2d increases SHH-MB tumor progression and leads to malignancy. Ongoing studies are determining how the chromatin landscape and gene expression are changed when Kmt2d is deleted in GCPs

We previously found GPR133 (ADGRD1), an orphan adhesion GPCR, is De novo expressed in glioblastoma (GBM) and enriched in patient-derived glioblastoma stem cells (GSCs). Knockdown of GPR133 reduces GBM cell proliferation and tumorsphere formation, and abolishes orthotopic tumor initiation in vivo in mice. Analysis of TCGA data indicates that increased GPR133 transcription inversely correlates with patient survival in GBM. While these findings underscore the importance of GPR133 in GBM and suggest an essential role in tumor growth, its ligand and mechanism of activation remain unknown. Toward identifying GPR133 ligands, we used GPR133's N-terminal ectodomain as bait and performed affinity co-immunoprecipitation (CoIP) followed by mass spectrometry as an unbiased screening approach. We identified 490 extracellular proteins with enriched binding to GPR133 compared to control. Reverse CoIP using the 15 most abundant candidate ligands as bait to purify the receptor confirmed this interaction reproducibly in 4 candidates. Despite this binding, overexpression of these candidate ligands, or addition of purified recombinant protein, is not sufficient to increase receptor signaling as assessed by cAMP levels in HEK293 cells. This suggests that ligand binding to the GPR133 ectodomain may not be sufficient by itself to induce receptor activation. We hypothesize receptor activation requires mechanical forces in addition to ligand binding. Consistent with this hypothesis, the GPR133 binding proteins we have identified may be anchored to the extracellular matrix, mediating such mechanical force. To test whether mechanical shearing of the extracellular domain is sufficient for receptor activation, we used Dynabeads coupled to antibody against GPR133's N-terminal ectodomain, and indeed observed receptor activation leading to elevated cAMP levels. No activation was observed when Dynabeads devoid of antibody were used. This mode of GPR133 activation might indicate a role in sensing mechanical/viscoelastic properties of GBM extracellular matrix, which may be relevant to tumor cell migration and invasion

Mutations of claudin-19 cause Familial Hypomagnesaemia and Hypercalciuria, Nephrocalcinosis with Ocular Involvement. To study the ocular disease without the complications of the kidney disease, naturally occurring point mutations of human CLDN19 were recreated in human induced pluripotent cells or overexpressed in the retinae of newborn mice. In human induced pluripotent cells, we show that the mutation affects retinal neurogenesis and maturation of retinal pigment epithelium (RPE). In mice, the mutations diminish the P1 wave of the electroretinogram, activate apoptosis in the outer nuclear layer, and alter the morphology of bipolar cells. If mice are given 9-cis-retinal to counter the loss of retinal isomerase, the P1 wave is partially restored. The ARPE19 cell line fails to express claudin-19. Exogenous expression of wild type, but not mutant claudin-19, increases the expression of RPE signature genes. Mutated claudin-19 affects multiple stages of RPE and retinal differentiation through its effects on multiple functions of the RPE.

Identification of biomarkers is a major goal of personalized medicine. Large transcriptome screens have identified new targets and molecular signatures for disease sub-types. However, tissue spatial information, which fundamentally alters in vivo cell behavior and gene expression, is lost. To understand spatial context and validate bulk tissue screens, most researchers rely exclusively on antibodies and immunostaining assays. Unfortunately, this is either not the appropriate choice for some targets, such as long non-coding RNAs, or it is not feasible because no reliable antibodies exist. To address these issues, we have established an alternative work-flow incorporating RNA in situ hybridization (CISH and FISH), whole slide and/or multispectral scanning and image analysis. Methods: RNAscope technology; Leica SCN scanner or Vectra3 multispectral imaging system for image acquisition; ImageJ2/FIJI, R, InForm and Visiopharm software platforms for quantitative analysis. Results: Several laboratories have used this workflow to address their specific questions. For example, we established and validated RNAscope assays for signaling factor transcripts, which are now integrated into an ongoing clinical trial. In this case, all tested commercial antibodies failed the validation assay. We also assessed expression of LNC RNAs in prostate cancer and put in place protocols for normalizing probe quantification across samples. The analysis revealed that storage and/or sample preparation affected the detection of certain LNC RNAs more than others identifying important factors regarding banking specimens. Spatial heat map visualization of RNAscope probes revealed an unexpected distribution of inflammatory cytokine targets in the kidney which are now being further investigated. In conclusion, RNA ISH is a powerful alternative strategy for assessing the spatial distribution of specific cell populations and critical biomarkers within intact tissues. This approach coupled with sophisticated imaging modalities and downstream analysis support provides new collaborative opportunities for Core laboratories.

Several modalities of multiplex immunofluorescence histology currently available require significant time and resources to implement. Many research laboratories develop questions benefiting from multiplex staining and analysis but do not have the human resources and/or equipment to perform the assay. Furthermore, pilot studies use similar metrics to evaluate multiplex histology data making them ideal for a core laboratory setup. The objective of this study was to establish a semi-automated workflow for multiplex immunofluorescence staining and initial quantification of cell populations in whole slide microscopy scans. The requirements for the workflow included: A. minimal transfer of decision making from the researcher to core personnel (Semi-Automation), B. modifiable in terms of antigen targets and tissue types with minimal disruption to the process and C. reproducible across samples submitted at different time periods (eg patient samples).

Synapses are highly specialized for neurotransmitter signaling, yet activity-dependent growth factor release also plays critical roles at synapses. While efficient neurotransmitter signaling relies on precise apposition of release sites and neurotransmitter receptors, molecular mechanisms enabling high-fidelity growth factor signaling within the synaptic microenvironment remain obscure. Here we show that the auxiliary calcium channel subunit α₂δ-3 promotes the function of an activity-dependent autocrine Bone Morphogenetic Protein (BMP) signaling pathway at the Drosophila neuromuscular junction (NMJ). α₂δ proteins have conserved synaptogenic activity, although how they execute this function has remained elusive. We find that α₂δ-3 provides an extracellular scaffold for an autocrine BMP signal, suggesting a mechanistic framework for understanding α₂δ's conserved role in synapse organization. We further establish a transcriptional requirement for activity-dependent, autocrine BMP signaling in determining synapse density, structure, and function. We propose that activity-dependent, autocrine signals provide neurons with continuous feedback on their activity state for modulating both synapse structure and function.