Tube Morphogenesis Research Articles

Human neural rosettes secrete bioactive extracellular vesicles enriched in neuronal and glial cellular components

Extracellular vesicles (EVs) play a critical role in the development of neural cells in the central nervous system (CNS). Human neural rosettes (hNRs) are radial cell structures that assemble from induced pluripotent stem cells (hiPSCs) and recapitulate some stages of neural tube morphogenesis. Here we show that hiPSCs and hNRs secrete EVs (hiPSC-EVs and hNR-EVs) with distinctive protein cargoes. Remarkably, hNR-EVs carry neuronal and glial cellular components involved in human CNS development. Importantly, hNR-EVs stimulate stem cells to change their cellular morphology and promote neurite growth in human and murine neurons with a significant dysregulation of SOX2 levels. This transcription factor modulates both neural differentiation and pluripotency. Interestingly, these effects were inhibited by antibodies against an unexpected neuroglial cargo of hNR-EVs: the major proteolipid protein (PLP). These findings show that hNRs secrete bioactive EVs containing neural components and might contribute as trophic factors during human neurodevelopment.

The Critical Role of YAP/BMP/ID1 Axis on Simulated Microgravity-Induced Neural Tube Defects in Human Brain Organoids.

Integrated biochemical and biophysical signals regulate embryonic development. Correct neural tube formation is critical for the development of central nervous system. However, the role of microgravity in neurodevelopment and its underlying molecular mechanisms remain unclear. In this study, the effects of stimulated microgravity (SMG) on the development of human brain organoids are investigated. SMG impairs N-cadherin-based adherens junction formation, leading to neural tube defects associated with dysregulated self-renewal capacity and neuroepithelial disorganization in human brain organoids. Bulk gene expression analyses reveal that SMG alters Hippo and BMP signaling in brain organoids. The neuropathological deficits in SMG-treated organoids can be rescued by regulating YAP/BMP/ID1 axis. Furthermore, sing-cell RNA sequencing data show that SMG results in perturbations in the number and function of neural stem and progenitor cell subpopulations. One of these subpopulations senses SMG cues and transmits BMP signals to the subpopulation responsible for tube morphogenesis, ultimately affecting the proliferating cell population. Finally, SMG intervention leads to persistent neurologic damage even after returning to normal gravity conditions. Collectively, this study reveals molecular and cellular abnormalities associated with SMG during human brain development, providing opportunities for countermeasures to maintain normal neurodevelopment in space.

Identification of trait-associated microRNA modules in liver transcriptome of pig fed with PUFAs-enriched supplementary diet.

Dietary lipids provide energy, are cellular structural components, and are involved in physiological processes. Lipids are the dietary source in supplementary diet experiments in pigs. This study aims to investigate the dietary effects of PUFAs on the hepatic transcriptome and physiological pathways of two diets on two pig breeds. Polish Landrace (PL: n = 6) and six PLxDuroc (PLxD: n = 6) pigs were fed with a normal diet (n = 3) or PUFAs-enriched healthy diet (n = 3), and the hepatic miRNA profiles were studied for weighted gene co-expression network analysis biological interactions between gene networks and metabolic pathways of DE miRNA genes. The study identified trait-associated modules that were significantly associated with four phenotypic traits in the dietary groups of PL and PLxD: meat colour (a*), shoulder subcutaneous fat thickness, conductivity 24h post-mortem (PE24), and ashes. Trait-wise, a large set of co-expressed miRNAs of porcine liver were identified in these trait-associated significant modules (9, 7, 2, and 8) in PL and PLxD. Each module is represented by a module eigengene (ME). Forty-four miRNAs out of 94 miRNAs interacted with 6719 statistically significant target genes with a target score > 90. The GO/pathway analysis showed association with pathways including regulation of metallopeptidase activity, sebaceous gland development, collagen fibril organization, WNT signalling, epithelial tube morphogenesis, etc. The study showed the differences in miRNA expression between the dietary groups of PL and PLxD breeds. Hub genes of discovered miRNA clusters can be considered predicted miRNA genes associated with PE24, meat colour, shoulder subcutaneous fat thickness, and ashes. Discovered target genes for miRNA clusters play significant roles in biological functions such as (i) muscle and body growth development, (ii) different cellular processes and developments, (iii) system development, and (iv) metabolic processes.

Systematic review and meta-analysis of bulk RNAseq studies in human Alzheimer's disease brain tissue.

To systematically review and meta-analyze bulk RNA sequencing studies comparing Alzheimer's disease (AD) patients with controls in human brain tissue, assessing study quality and identifying key genes and pathways. We searched PubMed, Web of Science, and Scopus on September 23, 2023, for studies using bulk RNAseq on primary human brain tissue from AD patients and controls. Excluded were non-primary tissue, re-analyses without new data, limited RNA types and gene panels. Quality was assessed with a 10-category tool. Meta-analysis used high-quality datasets. From 3,266 records, 24 studies met criteria. Meta-analysis found 571 differentially expressed genes (DEGs) in temporal lobe and 189 in frontal lobe; overlapping pathways included "Tube morphogenesis" and "Neuroactive ligand-receptor interaction." Study heterogeneity and limited data tables constrained the review. Rigorous methods are vital in AD transcriptomic studies. Findings enhance understanding of transcriptomic changes, aiding biomarker and therapeutic development. PROSPERO (CRD42023466522).

Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C.

The transcription factor MEF2C plays a critical role in the development of the linear heart tube, but the specific transcriptional networks controlled by MEF2C remain largely undefined. To address this, we performed combined single-nucleus RNA-and ATAC-sequencing on wild type and MEF2C-null embryos at distinct stages of development. We identified a broadly "posteriorized" cardiac gene signature and chromatin landscape throughout the heart tube in the absence of MEF2C. By integrating our gene expression and chromatin accessibility data in a deep-learning based model, we were able to construct developmental trajectories for each of the outflow tract, ventricular, and inflow tract lineages and determined how each of these segment-specific trajectories were distinctly altered in the MEF2C-null embryos. We computationally identified potential segment-specific MEF2C-dependent enhancers, and from these candidates, identified novel enhancers with activity in the developing heart tube using transgenesis in zebrafish. Finally, using inferred gene regulatory networks we discovered a genetic interaction between Mef2c and the atrial nuclear hormone receptor Nr2f2 , revealing that the MEF2C-null heart malformations are partly driven by a transcriptional network with increased NR2F2 activity. These studies not only provide a rich description of the genomic regulation of early heart tube development, but provide a generalizable framework for using genetic mutants to dissect the transcriptional networks that govern developmental processes.

M1 macrophages deliver CASC19 via exosomes to inhibit the proliferation and migration of colon cancer cells.

Colorectal cancer (CRC) continues to be one of the leading causes of cancer-related death worldwide. Exosomes have been established to play an important role in intercellular communication and that long non-coding RNA (lncRNA) CASC19 is enriched within M1 macrophage-derived exosomes (M1-exo). However, the biological functions and underlying molecular mechanisms of exosomal CASC19 from macrophages on CRC remain unknown. Cell proliferation and migration were evaluated by MTS and transwell assays. The exosomes were characterized by western blot, nanoparticle tracking analysis (NTA) and electron microscope imaging. The expression levels of CASC19 and its putative target miR-410-3p were quantified by reverse-transcription polymerase chain reaction (RT-qPCR). The interaction between CASC19 and miR-410-3p was detected by the pull-down assay. We found that the non-contact inhibition of M1 macrophages on the proliferation of colon cancer cells is largely dependent on the CASC19 released from M1 exosomes. M1 exosomes successfully delivered CASC19 to colon cancer cells, exerting an inhibitory effect on cell proliferation and migration. The exosomes secreted by M1 cells with CASC19 knockdown showed less inhibition effect on cell proliferation and migration. Mechanically, CASC19 exerted an inhibitory effect on colon cancer cells by sponging miR-410-3p via tube morphogenesis and TGF-β signaling pathway. We first proved that CASC19 in M1 macrophages is delivered into colon cancer cells via exosomes, exerting an inhibitory effect on their proliferation and migration by sponging miR-410-3p. The study may provide mechanistic insights into the roles of lncRNAs in CRC progression and a potential therapeutic target for the treatment of CRC.

Pathway to Independence - an interview with Eirini Maniou.

Eirini Maniou completed her undergraduate degree at the University of Patras, Greece, before moving to the UK to complete a Master's at the University of Bath and a PhD at the University of Dundee. For her postdoctoral work at University College London, Eirini turned her attention to neural tube morphogenesis, and she is now employing engineering principles to explore this developmental process at the University of Padua, Italy, where she is a Marie Skłodowska-Curie Actions postdoctoral fellow. Eirini was selected as one of our 2024 PI fellows, a group of researchers who will be supported by Development's Pathway to Independence Programme as they aim to secure independent positions. We spoke to Eirini to hear more about her career so far, why she decided to apply to the programme and what research questions she would like to address with her own group.

β-H-Spectrin is a key component of an apical-medial hub of proteins during cell wedging in tube morphogenesis.

Coordinated cell shape changes are a major driver of tissue morphogenesis, with apical constriction of epithelial cells leading to tissue bending. We previously identified that interplay between the apical-medial actomyosin, which drives apical constriction, and the underlying longitudinal microtubule array has a key role during tube budding of salivary glands in the Drosophila embryo. At this microtubule-actomyosin interface, a hub of proteins accumulates, and we have shown before that this hub includes the microtubule-actin crosslinker Shot and the microtubule minus-end-binding protein Patronin. Here, we identify two actin-crosslinkers, β-heavy (H)-Spectrin (also known as Karst) and Filamin (also known as Cheerio), and the multi-PDZ-domain protein Big bang as components of the protein hub. We show that tissue-specific degradation of β-H-Spectrin leads to reduction of apical-medial F-actin, Shot, Patronin and Big bang, as well as concomitant defects in apical constriction, but that residual Patronin is still sufficient to assist microtubule reorganisation. We find that, unlike Patronin and Shot, neither β-H-Spectrin nor Big bang require microtubules for their localisation. β-H-Spectrin is instead recruited via binding to apical-medial phosphoinositides, and overexpression of the C-terminal pleckstrin homology domain-containing region of β-H-Spectrin (β-H-33) displaces endogenous β-H-Spectrin and leads to strong morphogenetic defects. This protein hub therefore requires the synergy and coincidence of membrane- and microtubule-associated components for its assembly and function in sustaining apical constriction during tubulogenesis.

The ion channel Anoctamin 10/TMEM16K coordinates organ morphogenesis across scales in the urochordate notochord.

During embryonic development, tissues and organs are gradually shaped into their functional morphologies through a series of spatiotemporally tightly orchestrated cell behaviors. A highly conserved organ shape across metazoans is the epithelial tube. Tube morphogenesis is a complex multistep process of carefully choreographed cell behaviors such as convergent extension, cell elongation, and lumen formation. The identity of the signaling molecules that coordinate these intricate morphogenetic steps remains elusive. The notochord is an essential tubular organ present in the embryonic midline region of all members of the chordate phylum. Here, using genome editing, pharmacology and quantitative imaging in the early chordate Ciona intestinalis we show that Ano10/Tmem16k, a member of the evolutionarily ancient family of transmembrane proteins called Anoctamin/TMEM16 is essential for convergent extension, lumen expansion, and connection during notochord morphogenesis. We find that Ano10/Tmem16k works in concert with the plasma membrane (PM) localized Na+/Ca2+ exchanger (NCX) and the endoplasmic reticulum (ER) residing SERCA, RyR, and IP3R proteins to establish developmental stage specific Ca2+ signaling molecular modules that regulate notochord morphogenesis and Ca2+ dynamics. In addition, we find that the highly conserved Ca2+ sensors calmodulin (CaM) and Ca2+/calmodulin-dependent protein kinase (CaMK) show an Ano10/Tmem16k-dependent subcellular localization. Their pharmacological inhibition leads to convergent extension, tubulogenesis defects, and deranged Ca2+ dynamics, suggesting that Ano10/Tmem16k is involved in both the "encoding" and "decoding" of developmental Ca2+ signals. Furthermore, Ano10/Tmem16k mediates cytoskeletal reorganization during notochord morphogenesis, likely by altering the localization of 2 important cytoskeletal regulators, the small GTPase Ras homolog family member A (RhoA) and the actin binding protein Cofilin. Finally, we use electrophysiological recordings and a scramblase assay in tissue culture to demonstrate that Ano10/Tmem16k likely acts as an ion channel but not as a phospholipid scramblase. Our results establish Ano10/Tmem16k as a novel player in the prevertebrate molecular toolkit that controls organ morphogenesis across scales.

BCL6B-dependent suppression of ETV2 hampers endothelial cell differentiation

BackgroundB-cell CLL/lymphoma 6 member B (BCL6B) operates as a sequence-specific transcriptional repressor within the nucleus, playing crucial roles in various biological functions, including tumor suppression, immune response, stem cell self-renew, and vascular angiogenesis. However, whether BCL6B is involved in endothelial cell (EC) development has remained largely unknown. ETS variant transcription factor 2 (ETV2) is well known to facilitate EC differentiation. This study aims to determine the important role of BCL6B in EC differentiation and its potential mechanisms.MethodsDoxycycline-inducible human induced pluripotent stem cell (hiPSC) lines with BCL6B overexpression or BCL6B knockdown were established and subjected to differentiate into ECs and vessel organoids (VOs). RNA sequencing analysis was performed to identify potential signal pathways regulated by BCL6B during EC differentiation from hiPSCs. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of pluripotency and vascular-specific marker genes expression. EC differentiation efficiency was determined by Flow cytometry analysis. The performance of EC was evaluated by in vitro Tube formation assay. The protein expression and the vessel-like structures were assessed using immunofluorescence analysis or western blot. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP)-PCR analysis were used to determine the regulatory relationship between BCL6B and ETV2.ResultsFunctional ECs and VOs were successfully generated from hiPSCs. Notably, overexpression of BCL6B suppressed while knockdown of BCL6B improved EC differentiation from hiPSCs. Additionally, the overexpression of BCL6B attenuated the capacity of derived hiPSC-ECs to form a tubular structure. Furthermore, compared to the control VOs, BCL6B overexpression repressed the growth of VOs, whereas BCL6B knockdown had little effect on the size of VOs. RNA sequencing analysis confirmed that our differentiation protocol induced landscape changes for cell/tissue/system developmental process, particularly vascular development and tube morphogenesis, which were significantly modulated by BCL6B. Subsequent experiments confirmed the inhibitory effect of BCL6B is facilitated by the binding of BCL6B to the promoter region of ETV2, led to the suppression of ETV2's transcriptional activity. Importantly, the inhibitory effect of BCL6B overexpression on EC differentiation from hiPSCs could be rescued by ETV2 overexpression.ConclusionsBCL6B inhibits EC differentiation and hinders VO development by repressing the transcriptional activity of ETV2.

The Differential Developmental Neurotoxicity of Valproic Acid on Anterior and Posterior Neural Induction of Human Pluripotent Stem Cells.

Valproic acid (VPA), widely used as an antiepileptic drug, exhibits developmental neurotoxicity when exposure occurs during early or late pregnancy, resulting in various conditions ranging from neural tube defects to autism spectrum disorders. However, toxicity during the very early stages of neural development has not been addressed. Therefore, we investigated the effects of VPA in a model where human pluripotent stem cells differentiate into anterior or posterior neural tissues. Exposure to VPA during the induction of neural stem cells induced different developmental toxic effects in a dose-dependent manner. For instance, VPA induced cell death more profoundly during anteriorly guided neural progenitor induction, while inhibition of cell proliferation and enhanced differentiation were observed during posteriorly guided neural induction. Furthermore, acute exposure to VPA during the posterior induction step also retarded the subsequent neurulation-like tube morphogenesis process in neural organoid culture. These results suggest that VPA exposure during very early embryonic development might exhibit cytotoxicity and subsequently disrupt neural differentiation and morphogenesis processes.

Identification and Characterization of the miRNA Transcriptome Controlling Green Pigmentation of Chicken Eggshells

Green eggs are mainly caused by inserting an avian endogenous retrovirus (EVA-HP) fragment into the SLCO1B3 gene. Although the genotypes for this insertion allele are consistent, eggshell color (ESC) may vary after a peak laying period; light-colored eggs are undesired by consumers and farmers and result in financial loss, so it is necessary to resolve this problem. miRNAs are small non-coding RNAs that exert essential functions in animal development and diseases. However, the regulatory miRNAs and detailed molecular mechanisms regulating eggshell greenness remain unclear. In the present study, we determined the genotype of green-eggshell hens through the detection of a homozygous allele insertion in the SLCO1B3 gene. The shell gland epithelium was obtained from green-eggshell hens that produced white and green shell eggs to perform transcriptome sequencing and investigate the important regulatory mechanisms that influence the ESC. Approximately 921 miRNAs were expressed in these two groups, which included 587 known miRNAs and 334 novel miRNAs, among which 44 were differentially expressed. There were 22 miRNAs that were significantly upregulated in the green and white groups, respectively, which targeted hundreds of genes, including KIT, HMOX2, and several solute carrier family genes. A Gene Ontology enrichment analysis of the target genes showed that the differentially expressed miRNA-targeted genes mainly belonged to the functional categories of homophilic cell adhesion, gland development, the Wnt signaling pathway, and epithelial tube morphogenesis. A KEGG enrichment analysis showed that the Hedgehog signaling pathway was significantly transformed in this study. The current study provides an overview of the miRNA expression profiles and the interaction between the miRNAs and their target genes. It provides valuable insights into the molecular mechanisms underlying green eggshell pigmentation, screening more effective hens to produce stable green eggs and obtaining higher economic benefits.

The dishevelled associated activator of morphogenesis protein 2 (Daam2) regulates neural tube closure.

The Wnt signaling pathway is highly conserved in metazoans and regulates a large array of cellular processes including motility, polarity and fate determination, and stem cell homeostasis. Modulation of the actin cytoskeleton via the non-canonical Wnt pathway regulate cell polarity and cell migration that are required for proper vertebrate gastrulation and subsequent neurulation. However, the mechanism(s) of how the non-canonical pathway mediates actin cytoskeleton modulation is not fully understood. Herein, we characterize the role of the Formin-homology protein; dishevelled associated activator of morphogenesis 2 (Daam2) protein in the Wnt signaling pathway. Co-immunoprecipitation assays confirm the binding of Daam2 to dishevelled2 (Dvl2) as well as the domains within these proteins required for interaction; additionally, the interaction between Daam2 and Dvl2 was Wnt-regulated. Sub-cellular localization studies reveal Daam2 is cytoplasmic and regulates the cellular actin cytoskeleton by modulating actin filament formation. During Xenopus development, a knockdown or loss of Daam2 specifically produces neural tube closure defects indicative of a role in non-canonical signaling. Additionally, our studies did not identify any role for Daam2 in canonical Wnt signaling in mammalian culture cells or the Xenopus embryo. Our studies together identify Daam2 as a component of the non-canonical Wnt pathway and Daam2 is a regulator of neural tube morphogenesis during vertebrate development.

Cannabidiol Exposure During Rat Pregnancy Leads to Labyrinth-Specific Vascular Defects in the Placenta and Reduced Fetal Growth.

Introduction: Cannabis use is increasing among pregnant people, and cannabidiol (CBD), a constituent of cannabis, is often perceived as "natural" and "safe" as it is non-intoxicating. In utero, cannabis exposure is associated with negative health outcomes, including fetal growth restriction (FGR). The placenta supplies oxygen and nutrients to the fetus, and alterations in placental development can lead to FGR. While there has been some investigation into the effects of Δ9-THC, there has been limited investigation into the impacts of in utero gestational CBD exposure on the placenta. Methods: This study used histological and transcriptomic analysis of embryonic day (E)19.5 rat placentas from vehicle and CBD (3 mg/kg intraperitoneal injection) exposed pregnancies (E6.5-18.5). Results: The study revealed that pups from CBD-exposed pregnancies were 10% smaller, with the placentae displaying a decreased fetal blood space perimeter-to-area ratio. The transcriptomic analysis supported compromised angiogenesis and blood vessel formation with downregulated biological processes, including tube morphogenesis, angiogenesis, blood vessel morphogenesis, blood vessel development and vasculature development. Further, the CBD-exposed placentas displayed changed expression of glucose transporters (decreased GLUT1 and GR expression and increased GLUT3 expression). Transcriptomic analysis further revealed upregulated biological processes associated with metabolism. Finally, histological and transcriptomic analysis revealed altered cell populations within the placenta, specifically to syncytiotrophoblast layer II and endothelial cells. Conclusion: Together these results suggest that the structural changes in CDB-exposed placentae, including the altered expression of nutrient transporters and the changes to the placental fetal vasculature, may underlie the reduced fetal growth.

Lysyl oxidase-mediated elastin upregulation promotes the proliferation and migration of human retinal endothelial cells.

Proliferative diabetic retinopathy (PDR) is a major cause of irreversible blindness in the working age population. The dysfunction of retinal vascular endothelial cells (RVECs) is the primary cause of PDR. Extracellular matrix (ECM) accumulation promotes intracellular signaling required for RVEC proliferation, migration, survival, and tube morphogenesis. This study aimed to investigate the role of lysyl oxidase (LOX) in the cellular function of RVECs and PDR pathogenesis and to identify the underlying mechanisms. Protein expression was determined with western blot. The interaction between LOX and elastin (ELN) was detected using a co-immunoprecipitation (Co-IP) assay, and the Cell Counting Kit-8 (CCK-8) assay evaluated cell viability. A colony formation assay was employed to assess the proliferation of human RVECs (hRVECs), and a transwell assay to determine their migration ability. Streptozotocin was used to establish PDR in mice in vivo. A histological analysis was conducted using hematoxylin and eosin (H&E) staining. The results showed that LOX was overexpressed in PDR patients. The LOX knockdown suppressed ECM formation and hRVEC proliferation and migration. Additionally, LOX upregulated ELN expression. However, overexpressed ELN promoted hRVEC proliferation and migration. In vivo experiments showed that curcumin-mediated LOX deficiency restored retinal tissue structure. The LOX-knockdown suppressed ECM formation and hRVEC proliferation and migration by inactivating ELN. Therefore, LOX/ELN signaling may be a potential PDR biomarker.

Commonly disrupted pathways in brain and kidney in a pig model of systemic endotoxemia

Sepsis is a life-threatening state that arises due to a hyperactive inflammatory response stimulated by infection and rarely other insults (e.g., non-infections tissue injury). Although changes in several proinflammatory cytokines and signals are documented in humans and small animal models, far less is known about responses within affected tissues of large animal models. We sought to understand the changes that occur during the initial stages of inflammation by administering intravenous lipopolysaccharide (LPS) to Yorkshire pigs and assessing transcriptomic alterations in the brain, kidney, and whole blood. Robust transcriptional alterations were found in the brain, with upregulated responses enriched in inflammatory pathways and downregulated responses enriched in tight junction and blood vessel functions. Comparison of the inflammatory response in the pig brain to a similar mouse model demonstrated some overlapping changes but also numerous differences, including oppositely dysregulated genes between species. Substantial changes also occurred in the kidneys following LPS with several enriched upregulated pathways (cytokines, lipids, unfolded protein response, etc.) and downregulated gene sets (tube morphogenesis, glomerulus development, GTPase signal transduction, etc.). We also found significant dysregulation of genes in whole blood that fell into several gene ontology categories (cytokines, cell cycle, neutrophil degranulation, etc.). We observed a strong correlation between the brain and kidney responses, with significantly shared upregulated pathways (cytokine signaling, cell death, VEGFA pathways) and downregulated pathways (vasculature and RAC1 GTPases). In summary, we have identified a core set of shared genes and pathways in a pig model of systemic inflammation.

Plate-curving cell culture as a model for assessing curvature-induced cellular responses during neural tube morphogenesis

ABSTRACT Neurulation is an important shape-transforming event during embryonic development where a flat neural plate is converted into a neural tube. Failure in this morphogenetic process accounts for one of the most common birth defects. Mechanical biology has provided key insights into neural tube formation and curvature among many physical properties that are eliciting attention. However, the lack of a proper model to study the effect of curvature has limited the potential to reveal its role in neurulation. In this study, we introduce a novel cell culture method called plate-curving cell culture where a polydimethylsiloxane (PDMS) plate of desired physical properties is curved in either a concave or convex form while the human pluripotent stem cell culture induced to have early neural plate identity is placed on top of its surface. With this method, we observed the elongation of cell colony morphology, as well as the perpendicular alignment of the cell division axis in the concave surface; the oriented cell division does not seem to explain the colony elongation. Transcriptome comparison in search of alternate possibilities suggested selectively altered pathways in the concave surface culture. Our new method is widely available, easy-to-use and culture-friendly, facilitating future mechanobiological studies of neurulation.

SMAD1 Is Dispensable for CDX2 Induction but Required for the Repression of Ectopic Small-Intestinal Gene Expression in Human-Pluripotent-Stem-Cell-Derived Colonic Organoids

The generation of gastrointestinal tissues from human pluripotent stem cells has provided unprecedented insight into the molecular mechanisms that drive the patterning of the primitive gut tube. Previous work has identified bone-morphogenetic-protein (BMP) signaling as an important mediator of mid/hindgut versus foregut and hindgut versus midgut cell fate choice. Inhibition of BMP signaling during gut tube morphogenesis inhibits the expression of the pan-intestinal transcription factor CDX2. Treatment of CDX2+ mid/hindgut cultures with BMP patterns them into hindgut, which gives rise to colonic organoids (HCOs). While the role for BMP signaling is clear, the molecular mechanisms through which BMP signaling patterns the mid/hindgut and colon remain unclear. BMPs bind to BMP receptors, activating a signaling cascade that results in the activation of SMADs, which function as transcription factors. We hypothesized that one of these factors, SMAD1, would be necessary for establishing the CDX2 domain and the colon domain. Unexpectedly, endoderm derived from SMAD1-deficient induced pluripotent stem cells was capable of inducing CDX2 in response to WNT and FGF signaling. In addition, CDX2+ gut tube cultures could activate posterior HOX genes in response to BMP. However, examination of HCOs following cytodifferentiation revealed that SMAD1-deficient HCOs ectopically expressed small-intestinal markers despite expressing posterior HOX genes. These results indicate that there is redundancy of SMADs during early hindgut patterning but that SMAD1 is required for the inhibition of small-intestinal gene expression in HCOs.

The zebrafish paralog six2b is required for early proximal pronephros morphogenesis

The transcription factor Six2 plays a crucial role in maintaining self-renewing nephron progenitor cap mesenchyme (CM) during metanephric kidney development. In mouse and human, expression at single-cell resolution has detected Six2 in cells as they leave the CM pool and differentiate. The role Six2 may play in these cells as they differentiate remains unknown. Here, we took advantage of the zebrafish pronephric kidney which forms directly from intermediate mesoderm to test six2b function during pronephric tubule development and differentiation. Expression of six2b during early zebrafish development was consistent with a role in pronephros formation. Using morpholino knock-down and CRISPR/Cas9 mutagenesis, we show a functional role for six2b in the development of proximal elements of the pronephros. By 48 h post-fertilization, six2b morphants and mutants showed disrupted pronephric tubule morphogenesis. We observed a lower-than-expected frequency of phenotypes in six2b stable genetic mutants suggesting compensation. Supporting this, we detected increased expression of six2a in six2b stable mutant embryos. To further confirm six2b function, F0 crispant embryos were analyzed and displayed similar phenotypes as morphants and stable mutants. Together our data suggests a conserved role for Six2 during nephrogenesis and a role in the morphogenesis of the proximal tubule.

Elucidating the Morphogenic and Signaling Roles of Defined Growth Factors Controlling Human Endothelial Cell Lumen Formation Versus Sprouting Behavior

Five growth factors [ie, insulin, fibroblast growth factor-2 (FGF-2), stem cell factor, IL-3, and stromal-derived factor 1α] in combination are necessary for human endothelial cells (ECs) to undergo tube morphogenesis, a process requiring both lumen formation and sprouting behavior. This study investigated why these factors are required by subdividing the factors into 4 separate groups: insulin-only, insulinand FGF-2, no FGF-2 (all factors but without FGF-2), and all factors. The study found that the insulin-only condition failed to support EC morphogenesis or survival, the insulinand FGF-2 condition supported primarily EC lumen formation, and the no FGF-2 condition supported EC sprouting behavior. By comparison, the all-factors condition more strongly stimulated both EC lumen formation and sprouting behavior, and signaling analysis revealed prolonged stimulation of multiple promorphogenic signals coupled with inhibition of proregressive signals. Pharmacologic inhibition of Jak kinases more selectively blocked EC sprouting behavior, whereas inhibition of Raf, phosphatidylinositol 3-kinase, and Akt kinases showed selective blockade of lumen formation. Inhibition of Src family kinases and Notch led to increased sprouting coupled to decreased lumen formation, whereas inhibition of Pak, Mek, and mammalian target of rapamycin kinases blocked both sprouting and lumen formation. These findings reveal novel downstream biological and signaling activities of defined factors that are required for the assembly of human EC-lined capillary tube networks.