Developmental Signaling Pathways Research Articles

Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage.

Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

Hedgehog signaling is required for larval muscle development and larval metamorphosis of the mussel Mytilus coruscus

Understanding the developmental processes and signaling pathways involved in larval myogenesis and metamorphosis is crucial for comprehending the life history and adaptive strategies of marine organisms. In this study, we investigated the temporal and spatial patterns of myogenesis in the mussel Mytilus coruscus (Mc), focusing on the emergence and transformation of major muscle groups during different larval stages. We also explored the role of the Hedgehog (Hh) signaling pathway in regulating myogenesis and larval metamorphosis. The results revealed distinct developmental stages characterized by the emergence of specific muscular components, such as velum retractor muscles and anterior adductor muscles, in D-veliger and umbo larvae, which are responsible for the planktonic stage. In the pediveliger stage, posterior ventral, posterior adductor, and foot muscles appeared. After larval metamorphosis, the velum structure and its corresponding retractor muscles degenerate, indicating the transition from planktonic to benthic life. We observed a conserved pattern of larval musculature development and revealed a high degree of conservation across bivalve species, with comparable emergence times during myogenesis. Furthermore, exposure to the Hh signaling inhibitor cyclopamine impaired larval muscle development, reduced larval swimming activity, and inhibited larval metamorphosis in M. coruscus. Cyclopamine-mediated inhibition of Hh signaling led to reduced expression of four key genes within the Hh signaling pathway (McHh, McPtc, McSmo, and McGli) and the striated myosin heavy chain gene (McMHC). It is hypothesised that the abnormal larval muscle development in cyclopamine-treated groups may be an indirect effect due to disrupted McMHC expression. We provide evidence for the first time that cyclopamine treatment inhibited larval metamorphosis in bivalves, highlighting the potential involvement of Hh signaling in mediating larval muscle development and metamorphosis in M. coruscus. The present study provides insights into the dynamic nature of myogenesis and the regulatory role of the Hh signaling pathway during larval development and metamorphosis in M. coruscus. The results obtained in this study contribute to a better understanding of the evolutionary significance of Hh signaling in bivalves and shed light on the mechanisms underlying larval muscle development and metamorphosis in marine invertebrates.

Risk and Resilience Variants in the Retinoic Acid Metabolic and Developmental Pathways Associated with Risk of FASD Outcomes.

Fetal Alcohol Spectrum Disorder (FASD) is a common neurodevelopmental disorder that affects an estimated 2-5% of North Americans. FASD is induced by prenatal alcohol exposure (PAE) during pregnancy and while there is a clear genetic contribution, few genetic factors are currently identified or understood. In this study, using a candidate gene approach, we performed a genetic variant analysis of retinoic acid (RA) metabolic and developmental signaling pathway genes on whole exome sequencing data of 23 FASD-diagnosed individuals. We found risk and resilience alleles in ADH and ALDH genes known to normally be involved in alcohol detoxification at the expense of RA production, causing RA deficiency, following PAE. Risk and resilience variants were also identified in RA-regulated developmental pathway genes, especially in SHH and WNT pathways. Notably, we also identified significant variants in the causative genes of rare neurodevelopmental disorders sharing comorbidities with FASD, including STRA6 (Matthew-Wood), SOX9 (Campomelic Dysplasia), FDG1 (Aarskog), and 22q11.2 deletion syndrome (TBX1). Although this is a small exploratory study, the findings support PAE-induced RA deficiency as a major etiology underlying FASD and suggest risk and resilience variants may be suitable biomarkers to determine the risk of FASD outcomes following PAE.

FLT4 causes developmental disorders of the cardiovascular and lymphovascular systems via pleiotropic molecular mechanisms.

Rare, deleterious genetic variants in FLT4 are associated with Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD). Distinct genetic variants in FLT4 are also an established cause of Milroy disease, the most prevalent form of primary hereditary lymphoedema. Phenotypic features of these two conditions are non-overlapping, implying pleiotropic cellular mechanisms during development. Here, we show that FLT4 variants identified in TOF patients, when expressed in primary human endothelial cells, cause aggregation of FLT4 protein in the perinuclear endoplasmic reticulum, activating proteostatic and metabolic signalling, whereas lymphoedema-associated FLT4 variants and wildtype FLT4 do not. FLT4 TOF variants display characteristic gene expression profiles in key developmental signalling pathways, revealing a role for FLT4 in cardiogenesis distinct from its role in lymphatic development. Inhibition of proteostatic signalling abrogates these effects, identifying potential avenues for therapeutic intervention. Depletion of flt4 in zebrafish caused cardiac phenotypes of reduced heart size and altered heart looping. These phenotypes were rescued with coinjection of wildtype human FLT4 mRNA, but incompletely or not at all by mRNA harbouring FLT4 TOF variants. Taken together, we identify a pathogenic mechanism for FLT4 variants predisposing to TOF that is distinct from the known dominant negative mechanism of Milroy-causative variants. FLT4 variants give rise to conditions of the two circulatory subdivisions of the vascular system via distinct developmental pleiotropic molecular mechanisms. Proteostatic dysfunction, if confirmed as a mechanism of CHD pathogenesis for other predisposing genes, may identify pathways to therapeutic interventions. Distinguishing mechanistically how variants in FLT4 give rise to CHD may have potential to individualise genetic counselling in affected families.

Transcription of microRNAs is regulated by developmental signaling pathways and transcription factors.

In early embryonic development, the cross-regulation of transcription factors and signaling pathways are critical in mediating developmental and physiological processes. Additionally, many studies have shown the importance of post-transcriptional regulation of signaling and network components mediated by microRNAs (miRNAs); however, how miRNAs are transcriptionally regulated is poorly understood. miRNAs are critical fine-tuners of many biological processes and their dysregulation leads to a variety of diseases and developmental defects. Previously, we have shown that miRNAs are dynamically expressed throughout sea urchin development, suggesting that miRNAs are likely to be under transcriptional regulation. Here, we used pharmacological inhibitors, genetic constructs, and loss-of-function reagents to assess the impact of key signaling pathways (Wnt, Nodal, MAPK, Sonic Hedgehog, Delta/Notch, VEGF, and BMP) and transcription factors (Alx1, Ets1/2, and Tbr) on the transcript levels of the evolutionarily conserved miR-1, miR-31, miR-92 and miR-124; the invertebrate-specific miR-71; and the echinoderm-specific miR-2002, miR-2007, and miR-2012. We also used computational methods to identify potential transcription factor binding sites of these miRNAs. Lists of binding motifs for transcription factors (TFs) were acquired from the MEME-Suite Motif Database and used as inputs for the algorithm FIMO (Find Individual Motif Occurrences), which detects short nucleotide motifs within larger sequences. Based on experimental data on miRNA expression in conjunction with bioinformatic predictions, we propose that the transcription factors Tbr, Alx1, and Ets1 regulate SpmiR-1, SpmiR-31, and SpmiR-71, respectively. We additionally observed significant effects on miRNA levels as a result of perturbations to Wnt, Nodal, MAPK, and Sonic Hedgehog signaling pathways, while no significant change on miRNA levels were observed with perturbations to Delta/Notch, VEGF, or BMP signaling pathways. Overall, this study provides insights into the transcriptional regulation of miRNAs by signaling pathways and transcription factors and contribute to our overall understanding of the genetic regulation of developmental processes.

Small Molecules Promote the Rapid Generation of Dental Epithelial Cells from Human-Induced Pluripotent Stem Cells.

Human-induced pluripotent stem cells (hiPSCs) offer a promising source for generating dental epithelial (DE) cells. Whereas the existing differentiation protocols were time-consuming and relied heavily on growth factors, herein, we developed a three-step protocol to convert hiPSCs into DE cells in 8 days. In the first phase, hiPSCs were differentiated into non-neural ectoderm using SU5402 (an FGF signaling inhibitor). The second phase involved differentiating non-neural ectoderm into pan-placodal ectoderm and simultaneously inducing the formation of oral ectoderm (OE) using LDN193189 (a BMP signaling inhibitor) and purmorphamine (a SHH signaling activator). In the final phase, OE cells were differentiated into DE through the application of Purmorphamine, XAV939 (a WNT signaling inhibitor), and BMP4. qRT-PCR and immunostaining were performed to examine the expression of lineage-specific markers. ARS staining was performed to evaluate the formation of the mineralization nodule. The expression of PITX2, SP6, and AMBN, the emergence of mineralization nodules, and the enhanced expression of AMBN and AMELX in spheroid culture implied the generation of DE cells. This study delineates the developmental signaling pathways and uses small molecules to streamline the induction of hiPSCs into DE cells. Our findings present a simplified and quicker method for generating DE cells, contributing valuable insights for dental regeneration and dental disease research.

Rewiring of the epigenome and chromatin architecture by exogenously induced retinoic acid signaling during zebrafish embryonic development.

Retinoic acid (RA) is the ligand of RA receptors (RARs), transcription factors that bind to RA response elements. RA signaling is required for multiple processes during embryonic development, including body axis extension, hindbrain antero-posterior patterning and forelimb bud initiation. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during in vivo embryonic development. Here, we stimulate the RA pathway by treating zebrafish embryos with all-trans-RA (atRA) and use a combination of RNA-seq, ATAC-seq, ChIP-seq and HiChIP to gain insight into the molecular mechanisms by which exogenously induced RA signaling controls gene expression. We find that RA signaling is involved in anterior/posterior patterning, central nervous system development, and the transition from pluripotency to differentiation. AtRA treatment also alters chromatin accessibility during early development and promotes chromatin binding of RARαa and the RA targets Hoxb1b, Meis2b and Sox3, which cooperate in central nervous system development. Finally, we show that exogenous RA induces a rewiring of chromatin architecture, with alterations in chromatin 3D interactions involving target genes. Altogether, our findings identify genome-wide targets of RA signaling and provide a molecular mechanism by which developmental signaling pathways regulate target gene expression by altering chromatin topology.

APR-246 enhances the anticancer effect of doxorubicin against p53-mutant AsPC-1 pancreatic cancer cells

Pancreatic cancer (PC) is a disease with a high mortality rate, and the early diagnosis of PC is still challenging. The 5-year relative survival rate remains below 8%, and therapeutic strategies are ineffective at increasing patient survival rates. In PC cells, resistance to treatment has been associated with genetic changes that initiate aberrant developmental signaling pathways; therefore, new strategies for treating PC are warranted. Here, we sought to explore the small molecule p53 reactivator, APR-246, either as a mono-drug or combined with doxorubicin (Doxo) in the p53 mutant (p53-mut) AsPC-1 PC cell line. Cytotoxicity of APR-246 and Doxo was studied alone or in combination with the AsPC-1 cell line with MTT assay. CalcuSyn software was used to calculate the combination index (CI), which was based on the Chou and Talalay technique. Apoptosis was assessed after staining with Acridine orange/ethidium bromide (AO/EB). Morphological alterations were examined under a 200x magnification by an inverted microscope. Gene expression was quantified using quantitative reverse transcription PCR assay (RT-qPCR). APR-246 alone exhibited modest anti-proliferative effects, while Doxo combined with APR-246 showed synergistic effects associated with morphological change; no more enhanced apoptosis was observed, as supported by NOXA gene levels. In conclusion, APR-246, whether monotherapy or combined with Doxo, was hindering the proliferation of p53-mut AsPC-1 PC cells, and further in vitro and in vivo research is warranted.

The ascorbate-glutathione cycle coming of age.

Concepts regarding the operation of the ascorbate-glutathione cycle and the associated water/water cycle in the processing of metabolically generated hydrogen peroxide and other forms of reactive oxygen species (ROS) are well established in the literature. However, our knowledge of the functions of these cycles and their component enzymes continues to grow and evolve. Recent insights include participation in the intrinsic environmental and developmental signalling pathways that regulate plant growth, development, and defence. In addition to ROS processing, the enzymes of the two cycles not only support the functions of ascorbate and glutathione, they also have 'moonlighting' functions. They are subject to post-translational modifications and have an extensive interactome, particularly with other signalling proteins. In this assessment of current knowledge, we highlight the central position of the ascorbate-glutathione cycle in the network of cellular redox systems that underpin the energy-sensitive communication within the different cellular compartments and integrate plant signalling pathways.

Unbalancing cAMP and Ras/MAPK pathways as a therapeutic strategy for cutaneous neurofibromas.

Cutaneous neurofibromas (cNFs) are benign Schwann cell (SC) tumors arising from subepidermal glia. Individuals with neurofibromatosis type 1 (NF1) may develop thousands of cNFs, which greatly affect their quality of life. cNF growth is driven by the proliferation of NF1-/- SCs and their interaction with the NF1+/- microenvironment. We analyzed the crosstalk between human cNF-derived SCs and fibroblasts (FBs), identifying an expression signature specific to the SC-FB interaction. We validated the secretion of proteins involved in immune cell migration, suggesting a role of SC-FB crosstalk in immune cell recruitment. The signature also captured components of developmental signaling pathways, including the cAMP elevator G protein-coupled receptor 68 (GPR68). Activation of Gpr68 by ogerin in combination with the MEK inhibitor (MEKi) selumetinib reduced viability and induced differentiation and death of human cNF-derived primary SCs, a result corroborated using an induced pluripotent stem cell-derived 3D neurofibromasphere model. Similar results were obtained using other Gpr68 activators or cAMP analogs/adenylyl cyclase activators in combination with selumetinib. Interestingly, whereas primary SC cultures restarted their proliferation after treatment with selumetinib alone was stopped, the combination of ogerin-selumetinib elicited a permanent halt on SC expansion that persisted after drug removal. These results indicate that unbalancing the Ras and cAMP pathways by combining MEKi and cAMP elevators could be used as a potential treatment for cNFs.

Emerging trends in gastrointestinal cancers: Targeting developmental pathways in carcinogenesis and tumor progression.

Gastrointestinal carcinomas are a group of cancers associated with the digestive system and its accessory organs. The most prevalent cancers related to the gastrointestinal tract are colorectal, gall bladder, gastric, hepatocellular, and esophageal cancers, respectively. Molecular aberrations in different signaling pathways, such as signal transduction systems or developmental pathways are the chief triggering mechanisms in different cancers Though a massive advancement in diagnostic and therapeutic interventions results in improved survival of patients with gastrointestinal cancer; the lower malignancy stages of these carcinomas are comparatively asymptomatic. Various gastrointestinal-related cancers are detected at advanced stages, leading to deplorable prognoses and increased rates of recurrence. Recent molecular studies have elucidated the imperative roles of several signaling pathways, namely Wnt, Hedgehog, and Notch signaling pathways, play in the progression, therapeutic responsiveness, and metastasis of gastrointestinal-related cancers. This book chapter gives an interesting update on recent findings on the involvement of developmental signaling pathways their mechanistic insight in gastrointestinalcancer. Subsequently, evidences supporting the exploration of gastrointestinal cancer related molecular mechanisms have also been discussed for developing novel therapeutic strategies against these debilitating carcinomas.

Phase Separation as a Driver of Stem Cell Organization and Function during Development.

A properly organized subcellular composition is essential to cell function. The canonical organizing principle within eukaryotic cells involves membrane-bound organelles; yet, such structures do not fully explain cellular complexity. Furthermore, discrete non-membrane-bound structures have been known for over a century. Liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mode of cellular organization without the need for formal lipid membranes, with an ever-expanding and diverse list of cellular functions that appear to be regulated by this process. In comparison to traditional organelles, LLPS can occur across wider spatial and temporal scales and involves more distinct protein and RNA complexes. In this review, we discuss the impacts of LLPS on the organization of stem cells and their function during development. Specifically, the roles of LLPS in developmental signaling pathways, chromatin organization, and gene expression will be detailed, as well as its impacts on essential processes of asymmetric cell division. We will also discuss how the dynamic and regulated nature of LLPS may afford stem cells an adaptable mode of organization throughout the developmental time to control cell fate. Finally, we will discuss how aberrant LLPS in these processes may contribute to developmental defects and disease.

Oncofetal reprogramming in tumor development and progression: novel insights into cancer therapy.

Emerging evidence indicates that cancer cells can mimic characteristics of embryonic development, promoting their development and progression. Cancer cells share features with embryonic development, characterized by robust proliferation and differentiation regulated by signaling pathways such as Wnt, Notch, hedgehog, and Hippo signaling. In certain phase, these cells also mimic embryonic diapause and fertilized egg implantation to evade treatments or immune elimination and promote metastasis. Additionally, the upregulation of ATP-binding cassette (ABC) transporters, including multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and breast cancer-resistant protein (BCRP), in drug-resistant cancer cells, analogous to their role in placental development, may facilitate chemotherapy efflux, further resulting in treatment resistance. In this review, we concentrate on the underlying mechanisms that contribute to tumor development and progression from the perspective of embryonic development, encompassing the dysregulation of developmental signaling pathways, the emergence of dormant cancer cells, immune microenvironment remodeling, and the hyperactivation of ABC transporters. Furthermore, we synthesize and emphasize the connections between cancer hallmarks and embryonic development, offering novel insights for the development of innovative cancer treatment strategies.

Novel Insights into the circRNA-Modulated Developmental Mechanism of Western Honey Bee Larval Guts.

Circular RNAs (circRNAs) are a class of novel non-coding RNAs (ncRNAs) that play essential roles in the development and growth of vertebrates through multiple manners. However, the mechanism by which circRNAs modulate the honey bee gut development is currently poorly understood. Utilizing the transcriptome data we obtained earlier, the highly expressed circRNAs in the Apis mellifera worker 4-, 5-, and 6-day-old larval guts were analyzed, which was followed by an in-depth investigation of the expression pattern of circRNAs during the process of larval guts development and the potential regulatory roles of differentially expressed circRNAs (DEcircRNAs). In total, 1728 expressed circRNAs were detected in the A. mellifera larval guts. Among the most highly expressed 10 circRNAs, seven (novel_circ_000069, novel_circ_000027, novel_circ_000438, etc.) were shared by the 4-, 5-, and 6-day-old larval guts. In addition, 21 (46) up-regulated and 22 (27) down-regulated circRNAs were, respectively, screened in the Am4 vs. Am5 (Am5 vs. Am6) comparison groups. Additionally, nine DEcircRNAs, such as novel_circ_000340, novel_circ_000758 and novel_circ_001116, were shared by these two comparison groups. These DEcircRNAs were predicted to be transcribed from 14 and 29 parental genes; these were respectively annotated to 15 and 22 GO terms such as biological regulation and catalytic activity as well as 16 and 21 KEGG pathways such as dorsoventral axis formation and apoptosis. Moreover, a complicated competing endogenous RNA (ceRNA) network was observed; novel_circ_000838 in the Am4 vs. Am5 comparison group potentially targeted ame-miR-6000a-3p, further targeting 518 mRNAs engaged in several developmental signaling pathways (e.g., TGF-beta, hedgehog, and wnt signaling pathway) and immune pathways (e.g., phagosome, lysosome, and MAPK signaling pathway). The results demonstrated that the novel_circ_000838-ame-miR-6000a-3p axis may plays a critical regulatory part in the larval gut development and immunity. Furthermore, back-splicing sites of six randomly selected DEcircRNAs were amplified and verified by PCR; an RT-qPCR assay of these six DEcircRNAs confirmed the reliability of the used high-throughput sequencing data. Our findings provide a novel insight into the honey bee gut development and pave a way for illustration of the circRNA-modulated developmental mechanisms underlying the A. mellifera worker larval guts.

Abstract 15510: Generation of the Cardiac Valve Lineage From Human Pluripotent Stem Cells

Background: Heart valves are living structures whose sophisticated functions are mediated by a specialized population of valvular interstitial cells (VICs). Given their central role in valve homeostasis, VICs represent a promising cell population for studying valve diseases and developing novel therapies to treat them. Herein, we describe the generation of VIC-like cells from human pluripotent stem cells (hPSCs). Methods and Results: Using a previously established protocol, we first generated endocardial cells from cardiovascular mesoderm. Within this endocardial population, we identified a subset of cells — marked by the expression of PDGFRβ — that express valvular endocardial cell (VEC) markers and demonstrate VEC-like functional properties, namely the ability to generate calcium transients in response to ATP and the ability to undergo endothelial-to-mesenchymal transition (EndoMT). Through stage-specific manipulation of developmental signaling pathways, we established a protocol that promotes the development of VIC-like cells from these hPSC-derived VEC progenitors (Figure 1A). The cells thus generated transcriptionally matched primary human fetal VICs by scRNAseq. When embedded in a tissue-engineered scaffold, hPSC-VICs secreted collagen and glycosaminoglycans, and demonstrated compact tissue organization analogous to that of native valve leaflets (Figure 1B, C). Finally, we show that VICs generated from an hPSC line with a Noonan syndrome mutation displayed excessive endocardial cell proliferation, diminished EndoMT, and dysregulated pERK activity, recapitulating key aspects of the disease. Conclusions: Together, the findings presented in this report provide a reproducible method for the scaled generation of bona fide VICs from hPSCs. The generation of hPSC-VICs addresses an important gap in the field and provides a platform to study valvulogenesis and heart valve disease, as well as a novel avenue for heart valve tissue engineering.

OR08-02 In Situ Reconstruction Of Distinct Normal And Pathological Cell Populations Within The Human Adrenal Gland Using Spatial Transcriptomics

Abstract Disclosure: F. Rui: None. K. Walters: None. K. Koc: None. A. Baldwin: Stock Owner; Self; Tempus Labs. K. Kiseljak-Vassiliades: Advisory Board Member; Self; HRA Pharma. L.M. Fishbein: Consulting Fee; Self; Lantheus/Progenics Azedra. N. Mukherjee: None. The human adrenal gland is a vital endocrine organ controlling a multitude of functions through the secretion of cortical steroid and medullary catecholamine hormones. Several outstanding questions remain about the adrenal spatial organization and related differential developmental signaling pathways which have functional consequences within and between the cortex and medulla. The current model of the functional zonation of the cortex posits a centripetal differentiation process in which the cortical cells arise in the capsule and migrate inwards changing their gene expression patterns as they progress to the three different functional layers of the cortex. The evidence for this model is based on mouse lineage tracing experiments, and it is unclear to what extent it applies to human adrenal glands. To address this, we performed spatially resolved transcriptomics on four adult adrenal sections from two different human specimens to identify cell populations and their spatial relationship in this highly organized and functionally diverse organ. We identified 14 distinct cell populations; 79% of these spots represented the cortex or medulla and exhibited the concentric expression pattern reflecting the known spatial organization of the human adrenal gland. Trajectory inference analysis of the cortical cells showed continuous expression transitions from cells of the capsule to zona glomerulosa to zona fasciculata to zona reticularis. These data demonstrate for the first time that the centripetal differentiation model for functional zonation in mice is consistent for adult human adrenal tissue. We also validated the spatial expression pattern of a subset of WNT-related genes specifically expressed with distinct patterns in the capsule and zona glomerulosa cells. These genes represent novel regulatory factors that may be crucial in the differentiation of adult human adrenal cortex. Finally, we observed two CYP11B2 positive aldosterone producing cell clusters (APCC) in the adrenal tissue section from a 72-year-old woman. APCCs are thought to play a role in the pathological progression of primary aldosteronism; however, it is unclear if there are unifying expression signatures other than CYP11B2 protein expression. Our analysis found both similarities and differences to previously published data sets of APCC signatures. It is unclear to what extent these expression differences are technical (i.e. different platforms) or due to biological heterogeneity (between individuals or APCCs). The subgroup of similar gene expression in APCCs across all studies thus far may represent a core APCC signature which should be validated on a larger cohort. Altogether, our study uses spatial transcriptomics of the normal human adrenal and the results have important implications for the differentiation and functional zonation,as well as, for the early dysregulation leading to primary aldosteronism. Presentation: Friday, June 16, 2023

Dissecting the Molecular Mechanisms Driving Electropathology in Atrial Fibrillation: Deployment of RNA Sequencing and Transcriptomic Analyses.

Despite many efforts to treat atrial fibrillation (AF), the most common progressive and age-related cardiac tachyarrhythmia in the Western world, the efficacy is still suboptimal. A plausible reason for this is that current treatments are not directed at underlying molecular root causes that drive electrical conduction disorders and AF (i.e., electropathology). Insights into AF-induced transcriptomic alterations may aid in a deeper understanding of electropathology. Specifically, RNA sequencing (RNA-seq) facilitates transcriptomic analyses and discovery of differences in gene expression profiles between patient groups. In the last decade, various RNA-seq studies have been conducted in atrial tissue samples of patients with AF versus controls in sinus rhythm. Identified differentially expressed molecular pathways so far include pathways related to mechanotransduction, ECM remodeling, ion channel signaling, and structural tissue organization through developmental and inflammatory signaling pathways. In this review, we provide an overview of the available human AF RNA-seq studies and highlight the molecular pathways identified. Additionally, a comparison is made between human RNA-seq findings with findings from experimental AF model systems and we discuss contrasting findings. Finally, we elaborate on new exciting RNA-seq approaches, including single-nucleotide variants, spatial transcriptomics and profiling of different populations of total RNA, small RNA and long non-coding RNA.

Root-knot nematodes produce functional mimics of tyrosine-sulfated plant peptides

Root-knot nematodes (Meloidogyne spp.) are highly evolved obligate parasites threatening global food security. These parasites have a remarkable ability to establish elaborate feeding sites in roots, which are their only source of nutrients throughout their life cycle. A wide range of nematode effectors have been implicated in modulation of host pathways for defense suppression and/or feeding site development. Plants produce a diverse array of peptide hormones including PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY)-family peptides, which promote root growth via cell expansion and proliferation. A sulfated PSY-like peptide RaxX (required for activation of XA21 mediated immunity X) produced by the biotrophic bacterial pathogen (Xanthomonas oryzae pv. oryzae) has been previously shown to contribute to bacterial virulence. Here, we report the identification of genes from root-knot nematodes predicted to encode PSY-like peptides (MigPSYs) with high sequence similarity to both bacterial RaxX and plant PSYs. Synthetic sulfated peptides corresponding to predicted MigPSYs stimulate root growth in Arabidopsis. MigPSY transcript levels are highest early in the infection cycle. Downregulation of MigPSY gene expression reduces root galling and egg production, suggesting that the MigPSYs serve as nematode virulence factors. Together, these results indicate that nematodes and bacteria exploit similar sulfated peptides to hijack plant developmental signaling pathways to facilitate parasitism.

#3078 MILD EXPERIMENTAL CKD IS ASSOCIATED WITH MYOCARDIAL FIBROSIS AND NOTCH/HEDGEHOG SIGNALING DYSREGULATION

Abstract Background and Aims Chronic kidney disease (CKD) is an independent cardiovascular risk factor. The initial mechanisms of myocardial remodeling (MR) in CKD are poorly understood. The major developmental signaling pathways Notch, Hedgehog, Wnt, and Bmp seem to be involved in many crucial steps of MR (cardiomyocytes survival and regeneration, fibrotic response, angiogenesis), while their role in MR due to CKD was not previously studied. Phosphate (Pi) is essential for cellular signaling and its redistribution to the cardiovascular compartment in CKD could be a one of the mechanisms of MR. Here we investigated the involvement of Pi/PPi transport, and reactivation of developmental signaling pathways in myocardial alterations associated with mild CKD. Method We induce mild CKD by 3/4 nephrectomy in adult male SHRs (SHR-Nx, n=8) with normal phosphate intake (0.6%) and 2-month follow-up. Controls were sham-operated SHR (SHR-Sham, n=8) and Wistar Kyoto rats (WKY-Sham, n=8). We analyzed chronic kidney injury and Pi exchange parameters, serum intact parathyroid hormone (PTH), intact fibroblast growth factor 23 (FGF23), myocardial phosphorus content (ICP-AES), histomorphometry, PiT1 and PiT2 expression (IHC), and mRNA expression of Slc20a1, Slc20a2, Ankh, Mapk1, Mapk3, Bmp2, Bmp4, Ctnnb1, Fzd2, Sfrp2, Dkk1, Wif1, Numb, Notch1, Jag1, Hes1, Lgr4, Tnfrsf11b, Tnfsf11, Ptch1. Results Systolic blood pressure and myocardial mass index (MMI) were higher in SHRs vs WKY-Sham (Table 1). Decrease in creatinine clearance by 30% and an increase in albuminuria corresponded to mild CKD in SHR-Nx model (Table 1). In SHR-Nx, myocardial interstitial fibrosis was significantly higher (Figure 1), while MMI and cardiomyocyte diameter did not differ compared to SHR-Sham (Table 1). Myocardial alterations were accompanied by the increase in myocardium phosphorus content (Figure 1), down-regulation in PPi-exporter Ankh, and up-regulation in Hes1 and decline in Ptch1 mRNA expression (Figure 2). We found no changes in Slc20a1 and Slc20a2 mRNA levels (Figure 2). An appearance of PiT2-positive fibroblasts-like cells in myocardial interstitium was apparent in SHR-Nx rats (data not shown). Conclusion Myocardial alterations in mild CKD are manifested in an increase in its phosphorus content, interstitial fibrosis, and dysregulation in genes likely related to the induction of the myofibroblasts and extracellular matrix remodeling.

Osteohematology: To be or Notch to be.

Osteohematology is an emerging research field that studies the crosstalk between hematopoietic and bone stromal cells, to elucidate the mechanisms of hematological and skeletal malignancies and diseases. The Notch is an evolutionary conserved developmental signaling pathway, with critical roles in embryonic development by controlling cell proliferation and differentiation. However, the Notch pathway is also critically involved in cancer initiation and progression, such as osteosarcoma, leukemia, and multiple myeloma. The Notch-mediated malignant cells dysregulate bone and bone marrow cells in the tumour microenvironment, resulting in disorders ranging from osteoporosis to bone marrow dysfunction. To date, the complex interplay of Notch signaling molecules in hematopoietic and bone stromal cells is still poorly understood. In this mini-review, we summarize the crosstalk between cells in bone and bone marrow and their influence under the Notch signaling pathway in physiological conditions and in tumour microenvironment.