Morphogenic Pathways Research Articles

Discovery of Pyrazolopyrazines as Selective, Potent, and Mutant-Active MET Inhibitors with Intracranial Efficacy.

Mesenchymal-epithelial transition factor (MET) is a receptor tyrosine kinase that serves a critical function in numerous developmental, morphogenic, and proliferative signaling pathways. If dysregulated, MET has been shown to be involved in the development and survival of several cancers, including non-small cell lung cancer (NSCLC), renal cancer, and other epithelial tumors. Currently, the clinical efficacy of FDA approved MET inhibitors is limited by on-target acquired resistance, dose-limiting toxicities, and less than optimal efficacy against brain metastasis. Therefore, there is still an unmet medical need for the development of MET inhibitors to address these issues. Herein we report the application of structure-based design for the discovery and development of a novel class of brain-penetrant MET inhibitors with enhanced activity against clinically relevant mutations and improved selectivity. Compound 13 with a MET D1228N cell line IC50 value of 23 nM showed good efficacy in an intracranial tumor model and increased the median overall survival of the animals to 100% when dosed orally at 100 mg/kg daily for 21 days.

Increasing Sufu gene dosage reveals its unorthodox role in promoting polydactyly and medulloblastoma tumorigenesis.

Suppressor of fused (SUFU) is widely regarded as a key negative regulator of the sonic hedgehog (SHH) morphogenic pathway and a known tumor suppressor of medulloblastoma (MB). However, we report here that SUFU expression was markedly increased in 75% of specimens compiled in a tissue array comprising 49 unstratified MBs. The SUFU and GLI1 expression levels in this MB array showed strong positive correlation, which was also identified in a large public data set containing 736 MBs. We further report that increasing Sufu gene dosage in mice caused preaxial polydactyly, which was associated with the expansion of the Gli3 domain in the anterior limb bud and heightened Shh signaling responses during embryonic development. Increasing Sufu gene dosage also led to accelerated cerebellar development and, when combined with ablation of the Shh receptor encoded by Patched1 (Ptch1), promoted MB tumorigenesis. These data reveal multifaceted roles of SUFU in promoting MB tumorigenesis by enhancing SHH signaling. This revelation clarifies potentially counterintuitive clinical observation of high SUFU expression in MBs and may pave way for novel strategies to reduce or reverse MB progression.

A100 HEREDITARY HEMORRHAGIC TELANGIECTASIA: MORE THAN JUST ANOTHER RECTAL BLEED

Abstract Background Hereditary hemorrhagic telangiectasia (HHT), previously known as Osler-Weber-Rendu syndrome, is an autosomal dominant disorder characterized by widespread telangiectasia and visceral arteriovenous (AV) malformations. Diagnosis of HHT is commonly guided by The Curaçao Diagnostic Criteria. Molecular genetics can help confirm the clinical diagnosis. Genes involved include ENG, ACVRL1, SMAD4, and GDF2, which affect the bone morphogenic protein signaling pathway important in maintaining vascular endothelial integrity. Aims This report aims to review the case of a young male from a rural community in Northern Alberta with a possible new diagnosis of HHT, presenting with rectal bleeding in the setting of family history of telangiectasias. Methods Retrospective review of one patient. Results We report the case of a 33-year-old male who presented with a 1-day history of rectal bleeding (bright red turning to black) with hemoglobin at 116g/L. Past medical history was significant for occasional epistaxis since age 8, previous gastrointestinal bleed and laparotomy with pathology showing small intestinal vascular malformation at the age of 15, and small bowel obstruction secondary to adhesions requiring resection at the age of 26. There was no history of iron deficiency anemia. Family history was positive for a mother who had Von Willebrand disease and partial colectomy at the age of 28 due to bowel telangiectasias. His mother had no formal HHT diagnosis, but it was unclear if molecular genetics had been performed. Inpatient colonoscopy showed no culprit lesions but free-floating blood clots throughout the colon (Image). EGD and push enteroscopy were normal. CT enterography was then performed showing AV fistula in the distal ileum and venous malformations in the jejunum. Video capsule endoscopy followed revealed a few non-bleeding telangiectasia and petechiae. The patient was assessed to have possible HHT by The Curaçao Diagnostic Criteria. He was supplemented with IV iron and started on short-acting octreotide. On discharge, he received funding approval for long-acting octreotide and was referred to the Edmonton HHT clinic for follow up and further screening. Conclusions HHT remains underdiagnosed in both children and adults. Maintaining a strong suspicion and prompt diagnosis via the Curaçao Criteria is crucial for appropriate screening of the multisystem complications which stem from the AV malformations. This multidisciplinary approach can aid in reducing morbidity and mortality. Funding Agencies None

P097 Spatially resolved insights into fistulating Crohn's disease pathogenesis: Unveiling molecular heterogeneity

Abstract Background Crohn's disease (CD) frequently results in fistula development in approximately 40% of patients due to sustained, transmural inflammation within the bowel wall. Despite advanced treatments, recurrence of fistulae affects one third of patients. Understanding its pathogenesis is crucial for targeted treatments, yet remains poorly defined. This study employs spatial transcriptomic and single-cell RNA-sequencing (scRNA-seq) technologies to characterise human fistulating CD tissue pathology. Methods Unbiased FFPE spatial transcriptomics (10x Visium) were applied to surgically resected, full-thickness (FT) tissue from 20 CD-associated fistulae cases and 15 controls. Subsequently, selected cases underwent further analysis using custom 500-plex MERFISH subcellular resolution spatial transcriptomics (Vizgen MERSCOPE). Additionally, a reference single-cell cohort encompassing patients with fistulating, stricturing, and inflammatory CD phenotypes, along with healthy controls, was generated. Optimisation of scRNA-seq for resected FT ileal tissue enabled the isolation and profiling of epithelial, immune, and stromal populations (10x Chromium). Computational analysis facilitated the spatial localisation of single-cell clusters enriched in fistula CD tissue, identifying key parameters linked to fistula development. Results The scRNA-seq data revealed diverse CD-specific cellular states adopted by intestinal fibroblasts, with IL11+ fibroblasts prominently expressing CD82, COL7A1, MMP1, CHI3L1, suggestive of pro-fibrotic signalling and regenerative morphogen pathways. This coincided with the expansion of a CD-specific subpopulation of pericytes (CCL19/CCL21) involved in leucocyte migration. Spatial profiling of CD fistula tracts by Visium and MERSCOPE unveiled an enrichment of key epithelial developmental transcription factors (e.g., GRHL3) and localised the pro-fibrotic signature displaying increased IL11 and MMP expression, along with perturbed WNT signalling within the fistula stroma. Active proliferation (HOPX, MKI67) was observed at the base of fistula tracts and within the stroma. Transcriptomic characterisation depicted a gradual loss in stem cell signature (LGR5, ASCL2, SMOC2) and supporting telocytes (POSTN) toward the leading edge of the fistula, signifying abnormal epithelium loss. Conclusion This pioneering study integrates multi-modal spatial transcriptomics and single-cell profiling to unveil the intricate cellular and molecular landscape in FT, fistulating CD pathology. Spatial analyses elucidate specific mechanisms involved in epithelial loss, stromal remodelling, and perturbed WNT signalling pathways within fistulating tissue, outlining a course of disrupted regenerative processes in fistulating CD.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.

Identification of Maleimide-Fused Carbazoles as Novel Noncanonical Bone Morphogenetic Protein Synergizers.

Morphogenic signaling pathways govern embryonic development and tissue homeostasis on the cellular level. Precise control of such signaling events paves the way for innovative therapeutic approaches in the field of regenerative medicine. In line with these notions, bone morphogenic protein (BMP) is a major osteogenic driver and pharmacological stimulation of BMP signaling holds supreme potential for diseases and defects of the skeleton. Efforts to identify small-molecule modalities that activate or potentiate the BMP pathway have primarily been focused on the canonical signaling cascade. Here, we describe the phenotypic identification and development of specific carbazolomaleimides 2 as novel noncanonical BMP synergizers with submicromolar osteogenic cellular potency. The devised chemical tools are characterized to specifically regulate Id gene expression in a SMAD-independent, yet highly BMP-dependent fashion. Mechanistic studies revealed that GSK3 inhibition and increased β-catenin levels are partly responsible for this activity. The utility of the new BMP synergizer profile was further exemplified by showing how the synergistic action of canonical and noncanonical BMP enhancers additively amplifies BMP-dependent osteogenic outputs. Carbazolomaleimide 2b serves as a new and unique pharmacological tool for the modulation and study of the BMP pathway.

Reconstitution of morphogen shuttling circuits.

Developing tissues form spatial patterns by establishing concentration gradients of diffusible signaling proteins called morphogens. The bone morphogenetic protein (BMP) morphogen pathway uses a family of extracellular modulators to reshape signaling gradients by actively "shuttling" ligands to different locations. It has remained unclear what circuits are sufficient to enable shuttling, what other patterns they can generate, and whether shuttling is evolutionarily conserved. Here, using a synthetic, bottom-up approach, we compared the spatiotemporal dynamics of different extracellular circuits. Three proteins-Chordin, Twsg, and the BMP-1 protease-successfully displaced gradients by shuttling ligands away from the site of production. A mathematical model explained the different spatial dynamics of this and other circuits. Last, combining mammalian and Drosophila components in the same system suggests that shuttling is a conserved capability. Together, these results reveal principles through which extracellular circuits control the spatiotemporal dynamics of morphogen signaling.

Transcriptional metabolic reprogramming implements meiotic fate decision in mouse testicular germ cells

Nutrient starvation drives yeast meiosis, whereas retinoic acid (RA) is required for mammalian meiosis through its germline target Stra8. Here, by using single-cell transcriptomic analysis of wild-type and Stra8-deficient juvenile mouse germ cells, our data show that the expression of nutrient transporter genes, including Slc7a5, Slc38a2, and Slc2a1, is downregulated in germ cells during meiotic initiation, and this process requires Stra8, which binds to these genes and induces their H3K27 deacetylation. Consequently, Stra8-deficient germ cells sustain glutamine and glucose uptake in response to RA and exhibit hyperactive mTORC1/protein kinase A (PKA) activities. Importantly, expression of Slc38a2, a glutamine importer, is negatively correlated with meiotic genes in the GTEx dataset, and Slc38a2 knockdown downregulates mTORC1/PKA activities and induces meiotic gene expression. Thus, our study indicates that RA via Stra8, a chordate morphogen pathway, induces meiosis partially by generating a conserved nutrient restriction signal in mammalian germ cells by downregulating their nutrient transporter expression.

Multidimensional definition of the interferonopathy of Down syndrome and its response to JAK inhibition.

Individuals with Down syndrome (DS) display chronic hyperactivation of interferon signaling. However, the clinical impacts of interferon hyperactivity in DS are ill-defined. Here, we describe a multiomics investigation of interferon signaling in hundreds of individuals with DS. Using interferon scores derived from the whole blood transcriptome, we defined the proteomic, immune, metabolic, and clinical features associated with interferon hyperactivity in DS. Interferon hyperactivity associates with a distinct proinflammatory phenotype and dysregulation of major growth signaling and morphogenic pathways. Individuals with the highest interferon activity display the strongest remodeling of the peripheral immune system, including increased cytotoxic T cells, B cell depletion, and monocyte activation. Interferon hyperactivity accompanies key metabolic changes, most prominently dysregulated tryptophan catabolism. High interferon signaling stratifies a subpopulation with elevated rates of congenital heart disease and autoimmunity. Last, a longitudinal case study demonstrated that JAK inhibition normalizes interferon signatures with therapeutic benefit in DS. Together, these results justify the testing of immune-modulatory therapies in DS.

The Role of Plant Transcription Factors in the Fight against Plant Viruses.

Plants are vulnerable to the challenges of unstable environments and pathogen infections due to their immobility. Among various stress conditions, viral infection is a major threat that causes significant crop loss. In response to viral infection, plants undergo complex molecular and physiological changes, which trigger defense and morphogenic pathways. Transcription factors (TFs), and their interactions with cofactors and cis-regulatory genomic elements, are essential for plant defense mechanisms. The transcriptional regulation by TFs is crucial in establishing plant defense and associated activities during viral infections. Therefore, identifying and characterizing the critical genes involved in the responses of plants against virus stress is essential for the development of transgenic plants that exhibit enhanced tolerance or resistance. This article reviews the current understanding of the transcriptional control of plant defenses, with a special focus on NAC, MYB, WRKY, bZIP, and AP2/ERF TFs. The review provides an update on the latest advances in understanding how plant TFs regulate defense genes expression during viral infection.

Unravelling morphoea aetiopathogenesis by next-generation sequencing of paired skin biopsies

BackgroundMorphoea can have a significant disease burden. Aetiopathogenesis remains poorly understood, with very limited existing genetic studies. Linear morphoea (LM) may follow Blascho’s lines of epidermal development, providing potential pathogenic clues.ObjectiveThe first objective of this study was to identify the presence of primary somatic epidermal mosaicism in LM. The second objective was tTo explore differential gene expression in morphoea epidermis and dermis to identify potential pathogenic molecular pathways and tissue layer cross-talk.MethodologySkin biopsies from paired affected and contralateral unaffected skin were taken from 16 patients with LM. Epidermis and dermis were isolated using a 2-step chemical-physical separation protocol. Whole Genome Sequencing (WGS; n = 4 epidermal) and RNA-seq (n = 5-epidermal, n = 5-dermal) with gene expression analysis via GSEA-MSigDBv6.3 and PANTHER-v14.1 pathway analyses, were performed. RTqPCR and immunohistochemistry were used to replicate key results.ResultsSixteen participants (93.8% female, mean age 27.7 yrs disease-onset) were included. Epidermal WGS identified no single affected gene or SNV. However, many potential disease-relevant pathogenic variants were present, including ADAMTSL1 and ADAMTS16. A highly proliferative, inflammatory and profibrotic epidermis was seen, with significantly-overexpressed TNFα-via-NFkB, TGFβ, IL6/JAKSTAT and IFN-signaling, apoptosis, p53 and KRAS-responses. Upregulated IFI27 and downregulated LAMA4 potentially represent initiating epidermal ‘damage’ signals and enhanced epidermal-dermal communication. Morphoea dermis exhibited significant profibrotic, B-cell and IFN-signatures, and upregulated morphogenic patterning pathways such as Wnt.ConclusionThis study supports the absence of somatic epidermal mosaicism in LM, and identifies potential disease-driving epidermal mechanisms, epidermal-dermal interactions and disease-specific dermal differential-gene-expression in morphoea. We propose a potential molecular narrative for morphoea aetiopathogenesis which could help guide future targeted studies and therapies.

Abstract A007: Pancreatic cancer comprises co-existing transcriptional states regulated by distinct master regulator programs

Abstract Despite extensive efforts, reproducible assessment of pancreatic ductal adenocarcinoma (PDA) heterogeneity and plasticity at the single cell level remains elusive. Systematic, network-based analysis of single cell RNA-seq profiles showed that most PDA tumors comprise three coexisting lineages whose aberrant transcriptional state is mechanistically controlled by distinct regulatory programs. These lineages were characterized by the aberrant activation of either gastrointestinal lineage markers (GLS), transcriptional effectors of morphogen pathways (MOS) and acinar to ductal metaplasia markers (ALS). Each lineage was characterized by cells in two different cell states determined by the differential activation of MEK signaling (M+/M-) and high cellular plasticity. These states were confirmed in multiple cohorts, cell lines, PDX models and harmonized with bulk profile analyses. Master regulators (MRs) of GLS and MOS state were predictive of patient’s survival in bulk profiles. Cross-state plasticity was confirmed by lineage tracing assays, while pooled CRISPR/Cas9 assays confirmed the essentiality of identified MR proteins. Finally, mechanistic MR-mediated cell state control was confirmed by MR expression-mediated reprogramming of MOS cells to a GLS state. Our work provided a mechanistic model of pancreatic cancer heterogeneity and testable hypothesis to target cell state-specific pancreatic cancer dependencies. Citation Format: Pasquale Laise, Mikko Turunen, Hans Carlo Maurer, Alvaro Curiel Garcia, Ela Elyada, Bernhard Schmierer, Lorenzo Tomassoni, Jeremy Worley, Mariano J. Alvarez, Jordan Kesner, Xiangtian Tan, Somnath Tagore, Ester Calvo Fernandez, Kelly Wong, Alexander L. E. Wang, Sabrina Ge, Alina C. Iuga, Aaron T. Griffin, Winston Wong, Gulam A. Manji, Faiyaz Notta, David A. Tuveson, Kenneth P. P. Olive, Andrea Califano. Pancreatic cancer comprises co-existing transcriptional states regulated by distinct master regulator programs [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A007.

PSXIII-B-20 Variants within Genes Tspan18 and NRG3 Associated with Angular Limb Deformity in Rambouillet Rams

Abstract Incidence of angular limb deformities (ALD) have been described in many species of livestock and companion animals. Young, rapidly growing animals are at an increased risk for developing ALD. Although work has been done to identify the casual genetic basis of ALD, bone deformities are likely influenced by the environment, making clear genetic answers challenging to discover. Rambouillet rams from Wyoming, North Dakota and South Dakota annual ram performance tests have been observed to develop limb abnormalities consistent with ALD. Historical incidence of ALD in test rams has ranged from 3 to 18%. A genome-wide association study (GWAS) was conducted with 342 ram-test animals, including 40 ALD-affected rams and 302 unaffected rams collected over three consecutive years. Significant markers were identified within the genes tetraspanin 18 (TSPAN18; p-value = 4.87e-07) and neuregulin 3 (NRG3; p-value = 8.06e-07). In mice, Tspan18 has been proposed as a regulator of endothelial cell Orai1/Ca2+ signaling. Orai1 has also been shown to mediate osteogenic differentiation via the bone morphogenic protein signaling pathway, which suggests the possibility for TSPAN18 involvement in differentiation of chondrocytes and osteocytes. The gene NRG3 is a member of the neuregulin family, which encodes ligands for epidermal growth factor receptors ERBB3 and ERBB4. The epidermal growth factor receptor system has described roles in the regulation of proliferation and differentiation of osteoblast, chondrocyte and osteoclast cells. Identifying genetic risk factors for ALD has the potential to improve animal welfare and animal production in at-risk breeds. This study proposes TSPAN18 and NRG3 as targets for further research towards identifying genetic risk factors for ALD in Rambouillet sheep of the NW United States.

Hepatic Hedgehog Signaling Participates in the Crosstalk between Liver and Adipose Tissue in Mice by Regulating FGF21.

The Hedgehog signaling pathway regulates many processes during embryogenesis and the homeostasis of adult organs. Recent data suggest that central metabolic processes and signaling cascades in the liver are controlled by the Hedgehog pathway and that changes in hepatic Hedgehog activity also affect peripheral tissues, such as the reproductive organs in females. Here, we show that hepatocyte-specific deletion of the Hedgehog pathway is associated with the dramatic expansion of adipose tissue in mice, the overall phenotype of which does not correspond to the classical outcome of insulin resistance-associated diabetes type 2 obesity. Rather, we show that alterations in the Hedgehog signaling pathway in the liver lead to a metabolic phenotype that is resembling metabolically healthy obesity. Mechanistically, we identified an indirect influence on the hepatic secretion of the fibroblast growth factor 21, which is regulated by a series of signaling cascades that are directly transcriptionally linked to the activity of the Hedgehog transcription factor GLI1. The results of this study impressively show that the metabolic balance of the entire organism is maintained via the activity of morphogenic signaling pathways, such as the Hedgehog cascade. Obviously, several pathways are orchestrated to facilitate liver metabolic status to peripheral organs, such as adipose tissue.

Syndecan-1 and -4 influence Wnt signaling and cell migration in human breast cancers

Heparan sulfate proteoglycans (HSPGs) participate in numerous normal and pathophysiological cellular functions. HSPGs are crucial components of the extracellular matrix (ECM) binding signalling molecules such as fibroblast growth factors (FGF) and Wnts to mediate various cellular processes including cell proliferation, migration, and cancer invasion. The syndecans (SDCs1-4) are a major family of four HSPGs, implicated in the development of breast carcinomas. This study examined syndecan-1 (SDC1) and syndecan-4 (SDC4; SDC1/4) in breast cancer (BC) in vitro cell models and their role in tumorigenesis. Gene expression of HSPG core proteins, biosynthesis and modification enzymes along with Wnt/FGF morphogen pathway components were examined following inhibition of SDC1 and SDC4 via small interfering RNA (siRNA), and enhancement of HSPGs via addition of heparin and FGF. siRNAs knockdowns (KDs) were performed in the MCF-7 (lowly invasive and poorly metastatic) and the MDA-MB-231 (highly invasive and metastatic) human BC cell lines. Significantly decreased gene expression of SDC1 and SDC4 was observed in both cell lines following KD. Additionally, via gene expression analysis, downregulation of SDC1/4 decreased the biosynthesis of heparan sulfate modification enzymes and reduced expression of Wnt signalling molecules. Following the enhancement/inhibition of HSPGs via heparin/siRNA treatment, heparin increased the migratory characteristics of MCF-7 cells while KD of SDC1 increased cell migration in both MCF-7 and MDA-MB-231 cells when compared to scramble negative control conditions. Our findings suggest that a niche-specific function exists for SDC1/4 in the BC microenvironment, mediating Wnt signalling cascades and potentially regulating migration of BC cells.

Overview of Somatic Embryogenesis.

Somatic embryogenesis is a natural phenomenon through which somatic embryos are produced from somatic cells although. It is considered the most efficient morphogenic pathways for plant multiplication. One of the key features of somatic embryogenesis is the use of cellular totipotency, where dedifferentiation is induced to foster cell proliferation, followed by the induction of differentiation using plant growth regulators to produce new plants. There is a cell group with the potential to undergo the somatic embryogenesis pathway through adequate stimulation (plant growth regulators, incubation conditions, and supplementation of the culture medium). There are two somatic embryogenesis pathways in plants: direct and indirect embryogenesis. Direct somatic embryogenesis consists of the formation of embryos directly from isolated cells, without the formation of "callous" tissue. Indirect somatic embryogenesis is characterized by the formation of a callus as a stage that precedes the formation of somatic embryos. It should be stressed that not all plant cells have this morphogenic capacity; consequently, determining the type of factors that drive this type of response has been challenging. This book provides the reader with updated available information on the techniques, relevant protocols, and tools to perform somatic embryogenesis in different plant species for economic purposes.

Structure, mechanism, and inhibition of Hedgehog acyltransferase

SummaryThe Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

Prenatal alcohol exposure disrupts Sonic hedgehog pathway and primary cilia genes in the mouse neural tube

Neurulation-stage alcohol exposure (NAE; embryonic day [E] 8–10) is associated with midline craniofacial and CNS defects that likely arise from disruption of morphogen pathways, such as Sonic hedgehog (Shh). Notably, midline anomalies are also a hallmark of genetic ciliopathies such as Joubert syndrome. We tested whether NAE alters Shh pathway signaling and the number and function of primary cilia, organelles critical for Shh pathway transduction. Female C57BL/6 J mice were administered two doses of alcohol (2.9 g/kg/dose) or vehicle on E9. Embryos were collected 6, 12, or 24 h later, and changes to Shh, cell cycle genes, and primary cilia were measured in the rostroventral neural tube (RVNT). Within the first 24 h post-NAE, reductions in Shh pathway and cell cycle gene expression and the ratio of Gli3 forms in the full-length activator state were observed. RVNT volume and cell layer width were reduced at 12 h. In addition, altered expression of multiple cilia-related genes was observed at 6 h post-NAE. As a further test of cilia gene-ethanol interaction, mice heterozygous for Kif3a exhibited perturbed behavior during adolescence following NAE compared to vehicle-treated mice, and Kif3a heterozygosity exacerbated the hyperactive effects of NAE on exploratory activity. These data demonstrate that NAE downregulates the Shh pathway in a region of the neural tube that gives rise to alcohol-sensitive brain structures and identifies disruption of primary cilia function, or a “transient ciliopathy”, as a possible cellular mechanism of prenatal alcohol pathogenesis.

Zonation in NASH - A key paradigm for understanding pathophysiology and clinical outcomes.

Non-alcoholic fatty liver disease (NAFLD) exists as a spectrum ranging from simple steatosis to histologically defined hepatocyte injury and inflammatory changes that define steatohepatitis (NASH), and increase risk for fibrosis. Although zonal differences in NASH have not been systematically studied, periportal involvement has been associated with worse metabolic outcomes and more hepatic fibrosis as compared to pericentral disease. These data suggest that hepatic zonation of disease may influence the diversity of clinical presentations. Similarly, several randomized clinical trials suggest a differential response based on zonation of disease, with preferential effects on periportal (cysteamine) or pericentral disease (obeticholic acid, pioglitazone). Intriguingly, morphogenic pathways known to affect zonal development and maintenance - WNT/β-Catenin, Hedgehog, HIPPO/Yap/TAZ and Notch - have been implicated in NASH pathogenesis, and nuclear hormone receptors downstream of potential NASH therapeutics show zonal preferences. In this review, we summarize these data and propose that patient-specific activation of these pathways may explain the variability in clinical presentation, and the zone-specific response observed in clinical trials.

Epidemiological overview of multidimensional chromosomal and genome toxicity of cannabis exposure in congenital anomalies and cancer development

Cannabis and cannabinoids are implicated in multiple genotoxic, epigenotoxic and chromosomal-toxic mechanisms and interact with several morphogenic pathways, likely underpinning previous reports of links between cannabis and congenital anomalies and heritable tumours. However the effects of cannabinoid genotoxicity have not been assessed on whole populations and formal consideration of effects as a broadly acting genotoxin remain unexplored. Our study addressed these knowledge gaps in USA datasets. Cancer data from CDC, drug exposure data from National Survey of Drug Use and Health 2003–2017 and congenital anomaly data from National Birth Defects Prevention Network were used. We show that cannabis, THC cannabigerol and cannabichromene exposure fulfill causal criteria towards first Principal Components of both: (A) Down syndrome, Trisomies 18 and 13, Turner syndrome, Deletion 22q11.2, and (B) thyroid, liver, breast and pancreatic cancers and acute myeloid leukaemia, have mostly medium to large effect sizes, are robust to adjustment for ethnicity, other drugs and income in inverse probability-weighted models, show prominent non-linear effects, have 55/56 e-Values > 1.25, and are exacerbated by cannabis liberalization (P = 9.67 × 10–43, 2.66 × 10–15). The results confirm experimental studies showing that cannabinoids are an important cause of community-wide genotoxicity impacting both birth defect and cancer epidemiology at the chromosomal hundred-megabase level.