Mesodermal Precursors Research Articles

Temporal BMP4 effects on mouse embryonic and extraembryonic development.

The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamicsof the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and forallantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro.

Fgf17: A regulator of the mid/hind brain boundary in mammals

The Fibroblast growth factor (FGFs) family consists of at least 22 members that exert their function by binding and activating fibroblast growth factor receptors (FGFRs). The Fgf8/FgfD subfamily member, Fgf17, is located on human chromosome 8p21.3 and mouse chromosome 14 D2. In humans, FGF17 can be alternatively spliced to produce two isoforms (FGF17a and b) whereas three isoforms are present in mice (Fgf17a, b, and c), however, only Fgf17a and Fgf17b produce functional proteins. Fgf17 is a secreted protein with a cleavable N-terminal signal peptide and contains two binding domains, namely a conserved core region and a heparin binding site. Fgf17 mRNA is expressed in a wide range of different tissues during development, including the rostral patterning centre, midbrain-hindbrain boundary, tailbud mesoderm, olfactory placode, mammary glands, and smooth muscle precursors of major arteries. Given its broad expression pattern during development, it is surprising that adult Fgf17−/− mice displayed a rather mild phenotype; such that mutants only exhibited morphological changes in the frontal cortex and mid/hind brain boundary and changes in certain social behaviours. In humans, FGF17 mutations are implicated in several diseases, including Congenital Hypogonadotropic Hypogonadism and Kallmann Syndrome. FGF17 mutations contribute to CHH/KS in 1.1% of affected individuals, often presenting in conjunction with mutations in other FGF pathway genes like FGFR1 and FLRT3. FGF17 mutations were also identified in patients diagnosed with Dandy-Walker malformation and Pituitary Stalk Interruption Syndrome, however, it remains unclear how FGF17 is implicated in these diseases. Altered FGF17 expression has been observed in several cancers, including prostate cancer, hematopoietic cancers (acute myeloid leukemia and acute lymphoblastic leukemia), glioblastomas, perineural invasion in cervical cancer, and renal cell carcinomas. Furthermore, FGF17 has demonstrated neuroprotective effects, particularly during ischemic stroke, and has been shown to improve cognitive function in ageing mice.

Transcriptional profiling reveals fundamental differences in iPS-derived CD34+ Cells versus adult circulating CD34+

CD34+ cells hold significant promise in regenerative medicine and the treatment of various vascular degenerative diseases primarily because of their ability to regenerate and differentiate into various cell types. These cells can be derived from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or adult stem cells like blood and bone marrow, and can differentiate into endothelial, hematopoietic, myeloid, and other cell types. However, their characteristics vary based on the source, making detailed definitions essential for specific therapeutic applications. In this study, it was aimed to compare these two different CD34-positive cell populations by full genome transcriptional profiling (RNAseq). To this end, we first optimized a CD34+ cell differentiation protocol and found that Vascular Endothelial Growth Factor (VEGF) is critical for the transition of cells from mesodermal precursors to CD34+ cells. Additionally, principal component analysis (PCA) of RNAseq data showed that blood-derived CD34+ cells clustered far from iPS-derived CD34+ cells which illustrates these populations are fundamentally different. This data will be useful to better define these cell populations and facilitate the translation of regenerative approaches in this field as well as providing potentially novel diagnostic tools.

Transcriptome and open chromatin analysis reveals the process of myocardial cell development and key pathogenic target proteins in Long QT syndrome type 7

ObjectiveLong QT syndrome type 7 (Andersen–Tawil syndrome, ATS), which is caused by KCNJ2 gene mutation, often leads to ventricular arrhythmia, periodic paralysis and skeletal malformations. The development, differentiation and electrophysiological maturation of cardiomyocytes (CMs) changes promote the pathophysiology of Long QT syndrome type 7(LQT7). We aimed to specifically reproduce the ATS disease phenotype and study the pathogenic mechanism.Methods and resultsWe established a cardiac cell model derived from human induced pluripotent stem cells (hiPSCs) to the phenotypes and electrophysiological function, and the establishment of a human myocardial cell model that specifically reproduces the symptoms of ATS provides a reliable platform for exploring the mechanism of this disease or potential drugs. The spontaneous pulsation rate of myocardial cells in the mutation group was significantly lower than that in the repair CRISPR group, the action potential duration was prolonged, and the Kir2.1 current of the inward rectifier potassium ion channel was decreased, which is consistent with the clinical symptoms of ATS patients. Only ZNF528, a chromatin-accessible TF related to pathogenicity, was continuously regulated beginning from the cardiac mesodermal precursor cell stage (day 4), and continued to be expressed at low levels, which was identified by WGCNA method and verified with ATAC-seq data in the mutation group. Subsequently, it indicated that seven pathways were downregulated (all p < 0.05) by used single sample Gene Set Enrichment Analysis to evaluate the overall regulation of potassium-related pathways enriched in the transcriptome and proteome of late mature CMs. Among them, the three pathways (GO: 0008076, GO: 1990573 and GO: 0030007) containing the mutated gene KCNJ2 is involved that are related to the whole process by which a potassium ion enters the cell via the inward rectifier potassium channel to exert its effect were inhibited. The other four pathways are related to regulation of the potassium transmembrane pathway and sodium:potassium exchange ATPase (p < 0.05). ZNF528 small interfering (si)-RNA was applied to hiPSC-derived cardiomyocytes for CRISPR group to explore changes in potassium ion currents and growth and development related target protein levels that affect disease phenotype. Three consistently downregulated proteins (KCNJ2, CTTN and ATP1B1) associated with pathogenicity were verificated through correlation and intersection analysis.ConclusionThis study uncovers TFs and target proteins related to electrophysiology and developmental pathogenicity in ATS myocardial cells, obtaining novel targets for potential therapeutic candidate development that does not rely on gene editing.Graphical

Gastrulation: Its Principles and Variations.

As epiblast cells initiate development into various somatic cells, they undergo a large-scale reorganization, called gastrulation. The gastrulation of the epiblast cells produces three groups of cells: the endoderm layer, the collection of miscellaneous mesodermal tissues, and the ectodermal layer, which includes the neural, epidermal, and associated tissues. Most studies of gastrulation have focused on the formation of the tissues that provide theprimary route for cell reorganization, that is, the primitive streak, in the chicken and mouse. In contrast, how gastrulation alters epiblast-derived cells has remained underinvestigated. This chapter highlights the regulation of cell and tissue fate via the gastrulation process. The roles and regulatory functions of neuromesodermal progenitors (NMPs) in the gastrulation process, elucidated in the last decade, are discussed in depth to resolve points of confusion. Chicken and mouse embryos, which form a primitive streak as the site of mesoderm precursor ingression, have been investigated extensively. However, primitive streak formation is an exception, even among amniotes. The roles of gastrulation processes in generating various somatic tissues will be discussed broadly.

Lung metastasis at initial presentation in a 3 year old girl with stage IV Wilms tumor

Background: Wilms tumor (nephroblastoma) is a malignant pediatric tumor that arises from mesodermal embryological precursors of renal parenchyma. Lung metastasis as the first presentation of nephroblastoma occurs in 20% of cases. However, haematogenous spread to the lungs or to regional lymphnodes from a previously diagnosed nephroblastoma occurs in of 85% of cases.&#x0D; Case report: A 3-year old girl presented at children emergency unit with cough and weight loss. Examination revealed an emaciated child, with a huge, right sided abdominal mass. Abdominal ultrasound scan showed a heterogenous, spherical mass with irregular anechoic areas and echogenic solid areas in kidney. Chest radiograph revealed bilateral cannon ball opacities. A diagnosis of nephroblastoma with lung metastasis was made. She underwent combined therapy but relapsed one year later.

Population-level antagonism between FGF and BMP signaling steers mesoderm differentiation in embryonic stem cells.

The mesodermal precursor populations for different internal organ systems are specified during gastrulation by the combined activity of extracellular signaling systems such as BMP, Wnt, Nodal, and FGF. The BMP, Wnt and Nodal signaling requirements for the differentiation of specific mesoderm subtypes in mammals have been mapped in detail, but how FGF shapes mesodermal cell type diversity is not precisely known. It is also not clear how FGF signaling integrates with the activity of other signaling systems involved in mesoderm differentiation. Here, we address these questions by analyzing the effects of targeted signaling manipulations in differentiating stem cell populations with single cell resolution. We identify opposing functions of BMP and FGF, and map FGF-dependent and -independent mesodermal lineages. Stimulation with exogenous FGF boosts the expression of endogenous Fgfs while repressing Bmp ligand genes. This positive autoregulation of FGF signaling, coupled to the repression of BMP signaling, may contribute to the specification of reproducible and coherent cohorts of cells with the same identity via a community effect, both in the embryo and in synthetic embryo-like systems.

RAS-independent ERK activation by constitutively active KSR3 in non-chordate metazoa

During early development of the sea urchin embryo, activation of ERK signalling in mesodermal precursors is not triggered by extracellular RTK ligands but by a cell-autonomous, RAS-independent mechanism that was not understood. We discovered that in these cells, ERK signalling is activated through the transcriptional activation of a gene encoding a protein related to Kinase Suppressor of Ras, that we named KSR3. KSR3 belongs to a family of catalytically inactive allosteric activators of RAF. Phylogenetic analysis revealed that genes encoding kinase defective KSR3 proteins are present in most non-chordate metazoa but have been lost in flies and nematodes. We show that the structure of KSR3 factors resembles that of several oncogenic human RAF mutants and that KSR3 from echinoderms, cnidarians and hemichordates activate ERK signalling independently of RAS when overexpressed in cultured cells. Finally, we used the sequence of KSR3 factors to identify activating mutations of human B-RAF. These findings reveal key functions for this family of factors as activators of RAF in RAS-independent ERK signalling in invertebrates. They have implications on the evolution of the ERK signalling pathway and suggest a mechanism for its co-option in the course of evolution.

FGF signaling promotes spreading of fat body precursors necessary for adult adipogenesis in Drosophila.

Knowledge of adipogenetic mechanisms is essential to understand and treat conditions affecting organismal metabolism and adipose tissue health. In Drosophila, mature adipose tissue (fat body) exists in larvae and adults. In contrast to the well-known development of the larval fat body from the embryonic mesoderm, adult adipogenesis has remained mysterious. Furthermore, conclusive proof of its physiological significance is lacking. Here, we show that the adult fat body originates from a pool of undifferentiated mesodermal precursors that migrate from the thorax into the abdomen during metamorphosis. Through in vivo imaging, we found that these precursors spread from the ventral midline and cover the inner surface of the abdomen in a process strikingly reminiscent of embryonic mesoderm migration, requiring fibroblast growth factor (FGF) signaling as well. FGF signaling guides migration dorsally and regulates adhesion to the substrate. After spreading is complete, precursor differentiation involves fat accumulation and cell fusion that produces mature binucleate and tetranucleate adipocytes. Finally, we show that flies where adult adipogenesis is impaired by knock down of FGF receptor Heartless or transcription factor Serpent display ectopic fat accumulation in oenocytes and decreased resistance to starvation. Our results reveal that adult adipogenesis occurs de novo during metamorphosis and demonstrate its crucial physiological role.

In vivo clonal tracking reveals evidence of haemangioblast and haematomesoblast contribution to yolk sac haematopoiesis

During embryogenesis, haematopoietic and endothelial lineages emerge closely in time and space. It is thought that the first blood and endothelium derive from a common clonal ancestor, the haemangioblast. However, investigation of candidate haemangioblasts in vitro revealed the capacity for mesenchymal differentiation, a feature more compatible with an earlier mesodermal precursor. To date, no evidence for an in vivo haemangioblast has been discovered. Using single cell RNA-Sequencing and in vivo cellular barcoding, we have unravelled the ancestral relationships that give rise to the haematopoietic lineages of the yolk sac, the endothelium, and the mesenchyme. We show that the mesodermal derivatives of the yolk sac are produced by three distinct precursors with dual-lineage outcomes: the haemangioblast, the mesenchymoangioblast, and a previously undescribed cell type: the haematomesoblast. Between E5.5 and E7.5, this trio of precursors seeds haematopoietic, endothelial, and mesenchymal trajectories.

Abstract 198: Unveiling chromatin accessibility landscape and convergent oncogenic pathway in angiosarcoma models using induced pluripotent stem cells

Abstract Angiosarcoma is a rare soft tissue sarcoma that forms malignant vessels. Angiosarcomas are aggressive and highly metastatic, resulting in a poor prognosis. Recurrent somatic mutations in TP53 and genes involved in PI3K/AKT/mTOR pathway such as PIK3CA and PIK3R1 are identified in angiosarcomas. However, angiosarcomas are genomically complex, and the oncogenic mechanisms are virtually unknown. Due to its rarity, establishment of experimental tumor models is an unmet need for angiosarcoma research. In this study, we used human induced pluripotent stem cells (iPSCs) to develop a novel, reliable model for angiosarcoma recapitulating the genomic complexity and the tumor immune landscape. Specifically, we induced TP53 mutations in human iPSCs using CRISPR/Cas9 with validation of p53 deficiency by gene sequencing and Western blotting. We then established protocols to differentiate genetically engineered iPSCs to mesoderm and subsequently bi-potential hemangioblasts, which are considered the putative cell-of-origin of angiosarcoma. We found that isogenic wild-type (WT) and TP53 mutant iPSCs were capable of generating hemangioblasts, with no significant differences in morphology or growth patterns observed between WT and mutant cells. Putative iPSC-derived hemangioblasts were CD34+ by flow cytometry and contained primitive endothelial cells with the capacity to form tube-like structures in Matrigel. RNA-seq data libraries were generated to profile global gene expression in non-differentiated cells, mesodermal precursors, hemangioblasts, and endothelial cells derived from WT and p53 mutant iPSCs during differentiation over time (day 0, 2, 5, 8). Principal component analysis revealed that gene expression patterns were altered between WT and p53 mutant cells during differentiation, representing distinct gene signatures unique to each cell type. Intriguingly, transcriptomic alteration of p53 mutant iPSC-derived cells was more variable than that of WT. Our data also showed that p53 mutation induced dysregulation of genes associated with chromosome maintenance, extracellular matrix organization, hemostasis, and receptor tyrosine kinase signaling in iPSC-derived hemangioblasts when compared isogenic WT controls. Our data highlights the role that mutant p53 plays in the induction of genomic instability and transcriptional programs that regulate hemogenic and endothelial function during differentiation. Additionally, ATAC-seq data were generated from WT and p53 mutant iPSC-derived cells to determine chromatin accessibility dynamics and identify key transcription factors that activate convergent vascular tumorigenic pathways. We are currently generating iPSC harboring co-mutations in TP53 and PIK3CA in order to determine the phenotype and tumorigenic capacity of engineered iPSC-derived hemangioblasts in xenograft models. Citation Format: Sophia Wenthe, Kelsie Becklin, Brett Napiwocki, Emma Kozurek, Branden Moriarity, Jong Hyuk Kim. Unveiling chromatin accessibility landscape and convergent oncogenic pathway in angiosarcoma models using induced pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 198.

PDGFRα-lineage origin directs monocytes to trafficking proficiency to support peripheral immunity.

Multiple embryonic precursors give rise to leukocytes in adults while the lineage‐based functional impacts are underappreciated. Mesodermal precursors expressing PDGFRα appear transiently during E7.5‐8.5 descend to a subset of Lin–Sca1+Kit+ hematopoietic progenitors found in adult BM. By analyzing a PDGFRα‐lineage tracing mouse line, we here report that PDGFRα‐lineage BM F4/80+SSClo monocytes/macrophages are solely Ly6C+LFA‐1hiMac‐1hi monocytes enriched on the abluminal sinusoidal endothelium while Ly6C–LFA‐1loMac‐1lo macrophages are mostly from non‐PDGFRα‐lineage in vivo. Monocytes with stronger integrin profiles outcompete macrophages for adhesion on an endothelial monolayer or surfaces coated with ICAM‐1‐Fc or VCAM‐1‐Fc. Egress of PDGFRα‐lineage‐rich monocytes and subsequent differentiation to peripheral macrophages spatially segregates them from non‐PDGFRα‐lineage BM‐resident macrophages and allows functional specialization since macrophages derived from these egressing monocytes differ in morphology, phenotype, and functionality from BM‐resident macrophages in culture. Extravasation preference for blood PDGFRα‐lineage monocytes varies by tissues and governs the local lineage composition of macrophages. More PDGFRα‐lineage classical monocytes infiltrated into skin and colon but not into peritoneum. Accordingly, transcriptomic analytics indicated augmented inflammatory cascades in dermatitis skin of BM‐chimeric mice harbouring only PDGFRα‐lineage leukocytes. Thus, the PDGFRα‐lineage origin biasedly generates monocytes predestined for BM exit to support peripheral immunity following extravasation and macrophage differentiation.

Wnt Signaling Induces Asymmetric Dynamics in the Actomyosin Cortex of the C. elegans Endomesodermal Precursor Cell.

During asymmetrical division of the endomesodermal precursor cell EMS, a cortical flow arises, and the daughter cells, endodermal precursor E and mesodermal precursor MS, have an enduring difference in the levels of F-actin and non-muscular myosin. Ablation of the cell cortex suggests that these observed differences lead to differences in cortical tension. The higher F-actin and myosin levels in the MS daughter coincide with cell shape changes and relatively lower tension, indicating a soft, actively moving cell, whereas the lower signal in the E daughter cell is associated with higher tension and a more rigid, spherical shape. The cortical flow is under control of the Wnt signaling pathway. Perturbing the pathway removes the asymmetry arising during EMS division and induces subtle defects in the cellular movements at the eight-cell stage. The perturbed cellular movement appears to be associated with an asymmetric distribution of E-cadherin across the EMS cytokinesis groove. ABpl forms a lamellipodium which preferentially adheres to MS by the E-cadherin HMR-1. The HMR-1 asymmetry across the groove is complete just at the moment cytokinesis completes. Perturbing Wnt signaling equalizes the HMR-1 distribution across the lamellipodium. We conclude that Wnt signaling induces a cortical flow during EMS division, which results in a transition in the cortical contractile network for the daughter cells, as well as an asymmetric distribution of E-cadherin.

The nephric mesenchyme lineage of intermediate mesoderm is derived from Tbx6-expressing derivatives of neuro-mesodermal progenitors via BMP-dependent Osr1 function

In vertebrate embryos, the kidney primordium metanephros is formed from two distinct cell lineages, Wolffian duct and metanephric mesenchyme, which were classically grouped as intermediate mesoderm. Whereas the reciprocal interactions between these two cell populations in kidney development have been studied extensively, the mechanisms generating them remain elusive. Here, we show that the mouse cell lineage that forms nephric mesenchyme develops as a subpopulation of Tbx6-expressing mesodermal precursor derivatives of neuro-mesodermal progenitors (NMPs) under the condition of bone morphogenetic protein (BMP)-signal-dependent Osr1 expression. The Osr1-expressing nephric mesenchyme precursors were confirmed as descendants of NMPs because they were labeled by Sox2 N1 enhancer–EGFP. In Tbx6 mutant embryos, nephric mesenchyme changed its fate into neural tissues, which reflected its NMP origin. In Osr1 mutant embryos, the specific region of the Tbx6-expressing mesoderm precursor, which normally expresses Osr1 and develops into the nephric mesenchyme, instead expressed the somite marker FoxC2. BMP signaling activated Osr1 expression in a region of TBX6-expressing mesoderm and elicited nephric mesenchyme development. This study suggested a new model of cell lineage segregation during gastrulation.

Dynamics of primitive streak regression controls the fate of neuromesodermal progenitors in the chicken embryo.

In classical descriptions of vertebrate development, the segregation of the three embryonic germ layers completes by the end of gastrulation. Body formation then proceeds in a head to tail fashion by progressive deposition of lineage-committed progenitors during regression of the primitive streak (PS) and tail bud (TB). The identification by retrospective clonal analysis of a population of neuromesodermal progenitors (NMPs) contributing to both musculoskeletal precursors (paraxial mesoderm) and spinal cord during axis formation challenged these notions. However, classical fate mapping studies of the PS region in amniotes have so far failed to provide direct evidence for such bipotential cells at the single-cell level. Here, using lineage tracing and single-cell RNA sequencing in the chicken embryo, we identify a resident cell population of the anterior PS epiblast, which contributes to neural and mesodermal lineages in trunk and tail. These cells initially behave as monopotent progenitors as classically described and only acquire a bipotential fate later, in more posterior regions. We show that NMPs exhibit a conserved transcriptomic signature during axis elongation but lose their epithelial characteristicsin the TB. Posterior to anterior gradients of convergence speed and ingression along the PS lead to asymmetric exhaustion of PS mesodermal precursor territories. Through limited ingression and increased proliferation, NMPs are maintained and amplified as a cell population which constitute the main progenitors in the TB. Together, our studies provide a novel understanding of the PS and TB contribution through the NMPs to the formation of the body of amniote embryos.

Differential Spatio-Temporal Regulation of T-Box Gene Expression by microRNAs during Cardiac Development.

Cardiovascular development is a complex process that starts with the formation of symmetrically located precardiac mesodermal precursors soon after gastrulation and is completed with the formation of a four-chambered heart with distinct inlet and outlet connections. Multiple transcriptional inputs are required to provide adequate regional identity to the forming atrial and ventricular chambers as well as their flanking regions; i.e., inflow tract, atrioventricular canal, and outflow tract. In this context, regional chamber identity is widely governed by regional activation of distinct T-box family members. Over the last decade, novel layers of gene regulatory mechanisms have been discovered with the identification of non-coding RNAs. microRNAs represent the most well-studied subcategory among short non-coding RNAs. In this study, we sought to investigate the functional role of distinct microRNAs that are predicted to target T-box family members. Our data demonstrated a highly dynamic expression of distinct microRNAs and T-box family members during cardiogenesis, revealing a relatively large subset of complementary and similar microRNA–mRNA expression profiles. Over-expression analyses demonstrated that a given microRNA can distinctly regulate the same T-box family member in distinct cardiac regions and within distinct temporal frameworks, supporting the notion of indirect regulatory mechanisms, and dual luciferase assays on Tbx2, Tbx3 and Tbx5 3′ UTR further supported this notion. Overall, our data demonstrated a highly dynamic microRNA and T-box family members expression during cardiogenesis and supported the notion that such microRNAs indirectly regulate the T-box family members in a tissue- and time-dependent manner.

Early embryogenesis and organogenesis in the annelid Owenia fusiformis

BackgroundAnnelids are a diverse group of segmented worms within Spiralia, whose embryos exhibit spiral cleavage and a variety of larval forms. While most modern embryological studies focus on species with unequal spiral cleavage nested in Pleistoannelida (Sedentaria + Errantia), a few recent studies looked into Owenia fusiformis, a member of the sister group to all remaining annelids and thus a key lineage to understand annelid and spiralian evolution and development. However, the timing of early cleavage and detailed morphogenetic events leading to the formation of the idiosyncratic mitraria larva of O. fusiformis remain largely unexplored.ResultsOwenia fusiformis undergoes equal spiral cleavage where the first quartet of animal micromeres are slightly larger than the vegetal macromeres. Cleavage results in a coeloblastula approximately 5 h post-fertilization (hpf) at 19 °C. Gastrulation occurs via invagination and completes 4 h later, with putative mesodermal precursors and the chaetoblasts appearing 10 hpf at the dorso-posterior side. Soon after, at 11 hpf, the apical tuft emerges, followed by the first neurons (as revealed by the expression of elav1 and synaptotagmin-1) in the apical organ and the prototroch by 13 hpf. Muscles connecting the chaetal sac to various larval tissues develop around 18 hpf and by the time the mitraria is fully formed at 22 hpf, there are FMRFamide+ neurons in the apical organ and prototroch, the latter forming a prototrochal ring. As the mitraria feeds, it grows in size and the prototroch expands through active proliferation. The larva becomes competent after ~ 3 weeks post-fertilization at 15 °C, when a conspicuous juvenile rudiment has formed ventrally.ConclusionsOwenia fusiformis embryogenesis is similar to that of other equal spiral cleaving annelids, supporting that equal cleavage is associated with the formation of a coeloblastula, gastrulation via invagination, and a feeding trochophore-like larva in Annelida. The nervous system of the mitraria larva forms earlier and is more elaborated than previously recognized and develops from anterior to posterior, which is likely an ancestral condition to Annelida. Altogether, our study identifies the major developmental events during O. fusiformis ontogeny, defining a conceptual framework for future investigations.

Cardiac Development: A Glimpse on Its Translational Contributions

Cardiac development is a complex developmental process that is initiated soon after gastrulation, as two sets of precardiac mesodermal precursors are symmetrically located and subsequently fused at the embryonic midline forming the cardiac straight tube. Thereafter, the cardiac straight tube invariably bends to the right, configuring the first sign of morphological left–right asymmetry and soon thereafter the atrial and ventricular chambers are formed, expanded and progressively septated. As a consequence of all these morphogenetic processes, the fetal heart acquired a four-chambered structure having distinct inlet and outlet connections and a specialized conduction system capable of directing the electrical impulse within the fully formed heart. Over the last decades, our understanding of the morphogenetic, cellular, and molecular pathways involved in cardiac development has exponentially grown. Multiples aspects of the initial discoveries during heart formation has served as guiding tools to understand the etiology of cardiac congenital anomalies and adult cardiac pathology, as well as to enlighten novels approaches to heal the damaged heart. In this review we provide an overview of the complex cellular and molecular pathways driving heart morphogenesis and how those discoveries have provided new roads into the genetic, clinical and therapeutic management of the diseased hearts.

Enhanced Canonical Wnt Signaling During Early Zebrafish Development Perturbs the Interaction of Cardiac Mesoderm and Pharyngeal Endoderm and Causes Thyroid Specification Defects.

Background: Congenital hypothyroidism due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the majority of cases. This situation reflects, in part, our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification. Methods: We treated zebrafish embryos during different developmental periods with small-molecule compounds known to manipulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. We used the antisense morpholino (MO) technique to create a zebrafish acardiac model. For thyroid rescue experiments, bone morphogenetic protein (BMP) pathway induction in zebrafish embryos was obtained by manipulation of heat-shock inducible transgenic lines. Results: Combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of MO-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development. Conclusion: Collectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.

3022 – GENERATION OF RETINOIC ACID-DEPENDENT HEMOGENIC ENDOTHELIAL PROGENITORS FROM HUMAN PLURIPOTENT STEM CELLS

The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a- mesodermal population that gives rise to a clonally multipotent definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes, and is functionally unresponsive to RA treatment. Thus, the specification of a RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNAseq analyses, we identified that two distinct KDR+CD235a- populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, CYP26A1, a RA degrading enzyme, was expressed in KDR+CD235a-CXCR4- mesoderm, and this was the only mesodermal population harboring definitive hematopoietic potential. In sharp contrast, KDR+CD235a-CXCR4+ mesoderm expressed ALDH1A2, a key enzyme in the synthesis of RA and lacked any hematopoietic potential. However, the stage-specific application of retinoic acid signaling to this CXCR4+ mesodermal population resulted in the robust specification of CD34+ HE with multi-lineage definitive erythroid, myeloid and lymphoid hematopoietic potential. Further, in vivo correlates to these functionally-distinct mesodermal precursors could be found within a scRNA-seq dataset of gastrulating murine embryos. Additional comparison to human embryos demonstrated that CD34+ cells specified with retinol from CXCR4+ mesoderm have fetal-like HOXA expression. Collectively, this represents the first ever characterization of stage-specific RA-dependent hPSC-derived definitive hematopoiesis, and its mesodermal progenitor. This novel insight into human hematopoietic development will ultimately provide the basis for the de novo specification of HSCs.