Modeling Human Paraxial Mesoderm Development with Pluripotent Stem Cells.

Paraxial mesoderm in the early embryo is segmented into epithelial blocks called somites that establish the metameric organization of the vertebrate body plan. Somites are sequentially formed from head to tail in a rhythmic manner controlled by an oscillating gene regulatory network known as the segmentation clock. We know very little about this important process during human development due to limited access to human embryos and ethical concerns. To bypass these difficulties, model systems derived from human pluripotent stem cells have been established. Here, we detail three protocols modeling different aspects of human paraxial mesoderm development in vitro: a 2D cell monolayer system recapitulating dynamics of the human segmentation clock, a 3D organoid system called "somitoid" supporting the simultaneous formation of somite-like structures, and another organoid system called "segmentoid" reconstituting in vivo-like hallmarks of somitogenesis. Together, these complementary model systems provide an excellent platform to decode somitogenesis and advance human developmental biology.

Modeling of intramembranous ossification using human pluripotent stem cell-derived paraxial mesoderm derivatives.

Vertebrates form their skeletal tissues from three distinct origins (the neural crest, paraxial mesoderm, and lateral plate mesoderm) through two distinct modes of ossification (intramembranous and endochondral ossification). Since the paraxial mesoderm generates both intramembranous and endochondral bones, it is thought to give rise to both osteoprogenitors and osteo-chondroprogenitors. However, it remains unclear what directs the paraxial mesoderm-derived cells toward these different fates in distinct skeletal elements during human skeletal development. To answer this question, we need experimental systems that recapitulate paraxial mesoderm-mediated intramembranous and endochondral ossification processes. In this study, we aimed to develop a human pluripotent stem cell (hPSC)-based system that models the human intramembranous ossification process. We found that spheroid culture of the hPSC-derived paraxial mesoderm derivatives generates osteoprogenitors or osteo-chondroprogenitors depending on stimuli. The former induced intramembranous ossification, and the latter endochondral ossification, in mouse renal capsules. Transcriptional profiling supported the notion that bone signatures were enriched in the intramembranous bone-like tissues. Thus, we developed a system that recapitulates intramembranous ossification, and that enables the induction of two distinct modes of ossification by controlling the cell fate of the hPSC-derived paraxial mesoderm derivatives.

Dermomyotome-derived endothelial cells migrate to the dorsal aorta to support hematopoietic stem cell emergence.

Development of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from ventral aortic endothelium in all vertebrate animals studied. Work in zebrafish has demonstrated that arterial and venous endothelial precursors arise from distinct subsets of lateral plate mesoderm. Here, we profile the transcriptome of the earliest detectable endothelial cells (ECs) during zebrafish embryogenesis to demonstrate that tissue-specific EC programs initiate much earlier than previously appreciated, by the end of gastrulation. Classic studies in the chick embryo showed that paraxial mesoderm generates a subset of somite-derived endothelial cells (SDECs) that incorporate into the dorsal aorta to replace HSPCs as they exit the aorta and enter circulation. We describe a conserved program in the zebrafish, where a rare population of endothelial precursors delaminates from the dermomyotome to incorporate exclusively into the developing dorsal aorta. Although SDECs lack hematopoietic potential, they act as a local niche to support the emergence of HSPCs from neighboring hemogenic endothelium. Thus, at least three subsets of ECs contribute to the developing dorsal aorta: vascular ECs, hemogenic ECs, and SDECs. Taken together, our findings indicate that the distinct spatial origins of endothelial precursors dictate different cellular potentials within the developing dorsal aorta.

Single-cell chromatin accessibility profiling of cell-state-specific gene regulatory programs during mouse organogenesis.

In mammals, early organogenesis begins soon after gastrulation, accompanied by specification of various type of progenitor/precusor cells. In order to reveal dynamic chromatin landscape of precursor cells and decipher the underlying molecular mechanism driving early mouse organogenesis, we performed single-cell ATAC-seq of E8.5-E10.5 mouse embryos. We profiled a total of 101,599 single cells and identified 41 specific cell types at these stages. Besides, by performing integrated analysis of scATAC-seq and public scRNA-seq data, we identified the critical cis-regulatory elements and key transcription factors which drving development of spinal cord and somitogenesis. Furthermore, we intersected accessible peaks with human diseases/traits-related loci and found potential clinical associated single nucleotide variants (SNPs). Overall, our work provides a fundamental source for understanding cell fate determination and revealing the underlying mechanism during postimplantation embryonic development, and expand our knowledge of pathology for human developmental malformations.

Come together over me: Cells that form the dermatocranium and chondrocranium in mice.

Most bone develops either by intramembranous ossification where bone forms within a soft connective tissue, or by endochondral ossification by way of a cartilage anlagen or model. Bones of the skull can form endochondrally or intramembranously or represent a combination of the two types of ossification. Contrary to the classical definition of intramembranous ossification, we have previously described a tight temporo-spatial relationship between cranial cartilages and dermal bone formation and proposed a mechanistic relationship between chondrocranial cartilage and dermal bone. Here, we further investigate this relationship through an analysis of how cells organize to form cranial cartilages and dermal bone. Using Wnt1-Cre2 and Mesp1-Cre transgenic mice, we determine the derivation of cells that comprise cranial cartilages from either cranial neural crest (CNC) or paraxial mesoderm (PM). We confirm a previously determined CNC-PM boundary that runs through the hypophyseal fenestra in the cartilaginous braincase floor and identify four additional CNC-PM boundaries in the chondrocranial lateral wall, including a boundary that runs along the basal and apical ends of the hypochiasmatic cartilage. Based on the knowledge that as osteoblasts differentiate from CNC- and PM-derived mesenchyme, the differentiating cells express the transcription factor genes RUNX2 and osterix (OSX), we created a new transgenic mouse line called R2Tom. R2Tom mice carry a tdTomato reporter gene joined with an evolutionarily well-conserved enhancer sequence of RUNX2. R2Tom mice crossed with Osx-GFP mice yield R2Tom;Osx-GFP double transgenic mice in which various stages of osteoblasts and their precursors are detected with different fluorescent reporters. We use the R2Tom;Osx-GFP mice, new data on the cell derivation of cranial cartilages, histology, immunohistochemistry, and detailed morphological observations combined with data from other investigators to summarize the differentiation of cranial mesenchyme as it forms condensations that become chondrocranial cartilages and associated dermal bones of the lateral cranial wall. These data advance our previous findings of a tendency of cranial cartilage and dermal bone development to vary jointly in a coordinated manner, promoting a role for cranial cartilages in intramembranous bone formation.

Peripheral nerve development in zebrafish requires muscle patterning by tcf15/paraxis.

The vertebrate peripheral nervous system (PNS) is an intricate network that conveys sensory and motor information throughout the body. During development, extracellular cues direct the migration of axons and glia through peripheral tissues. Currently, the suite of molecules that govern PNS axon-glial patterning is incompletely understood. To elucidate factors that are critical for peripheral nerve development, we characterized the novel zebrafish mutant, stl159, that exhibits abnormalities in PNS patterning. In these mutants, motor and sensory nerves that develop adjacent to axial muscle fail to extend normally, and neuromasts in the posterior lateral line system, as well as neural crest-derived melanocytes, are incorrectly positioned. The stl159 genetic lesion lies in the basic helix-loop-helix (bHLH) transcription factor tcf15, which has been previously implicated in proper development of axial muscles. We find that targeted loss of tcf15 via CRISPR-Cas9 genome editing results in the PNS patterning abnormalities observed in stl159 mutants. Because tcf15 is expressed in developing muscle prior to nerve extension, rather than in neurons or glia, we predict that tcf15 non-cell-autonomously promotes peripheral nerve patterning in zebrafish through regulation of extracellular patterning cues. Our work underscores the importance of muscle-derived factors in PNS development.

GDF5+ chondroprogenitors derived from human pluripotent stem cells preferentially form permanent chondrocytes.

It has been established in the mouse model that during embryogenesis joint cartilage is generated from a specialized progenitor cell type, distinct from that responsible for the formation of growth plate cartilage. We recently found that mesodermal progeny of human pluripotent stem cells gave rise to two types of chondrogenic mesenchymal cells in culture: SOX9+ and GDF5+ cells. The fast-growing SOX9+ cells formed in vitro cartilage that expressed chondrocyte hypertrophy markers and readily underwent mineralization after ectopic transplantation. In contrast, the slowly growing GDF5+ cells derived from SOX9+ cells formed cartilage that tended to express low to undetectable levels of chondrocyte hypertrophy markers, but expressed PRG4, a marker of embryonic articular chondrocytes. The GDF5+-derived cartilage remained largely unmineralized in vivo. Interestingly, chondrocytes derived from the GDF5+ cells seemed to elicit these activities via non-cell-autonomous mechanisms. Genome-wide transcriptomic analyses suggested that GDF5+ cells might contain a teno/ligamento-genic potential, whereas SOX9+ cells resembled neural crest-like progeny-derived chondroprogenitors. Thus, human pluripotent stem cell-derived GDF5+ cells specified to generate permanent-like cartilage seem to emerge coincidentally with the commitment of the SOX9+ progeny to the tendon/ligament lineage.

Paraxial mesoderm organoids model development of human somites.

During the development of the vertebrate embryo, segmented structures called somites are periodically formed from the presomitic mesoderm (PSM) and give rise to the vertebral column. While somite formation has been studied in several animal models, it is less clear how well this process is conserved in humans. Recent progress has made it possible to study aspects of human paraxial mesoderm (PM) development such as the human segmentation clock in vitro using human pluripotent stem cells (hPSCs); however, somite formation has not been observed in these monolayer cultures. Here, we describe the generation of human PM organoids from hPSCs (termed Somitoids), which recapitulate the molecular, morphological, and functional features of PM development, including formation of somite-like structures in vitro. Using a quantitative image-based screen, we identify critical parameters such as initial cell number and signaling modulations that reproducibly yielded formation of somite-like structures in our organoid system. In addition, using single-cell RNA-sequencing and 3D imaging, we show that PM organoids both transcriptionally and morphologically resemble their in vivo counterparts and can be differentiated into somite derivatives. Our organoid system is reproducible and scalable, allowing for the systematic and quantitative analysis of human spine development and disease in vitro.

Humans are part of a group of animals called vertebrates, which are all the animals with backbones. Broadly, all vertebrates have a similar body shape with a head at one end and a left and right side that are similar to each other. Although this is not very obvious in humans, vertebrate bodies are derived from pairs of segments arranged from the head to the tail. Each of these segments or somites originates early in embryonic development. Cells from each somite then divide, grow and specialize to form bones such as the vertebrae of the vertebral column, muscles, skin, and other tissues that make up each segment. Studying different animals during embryonic development has provided insights into how somites form and grow, but it is technically difficult to do and only provides an approximate model of how somites develop in humans. Being able to make and study somites using human cells in the lab would help scientists learn more about how somite formation in humans is regulated. Budjan et al. grew human stem cells in the lab as three-dimensional structures called organoids, and used chemical signals similar to the ones produced in the embryo during development to make the cells form somites. Various combinations of signals were tested to find the best way to trigger somite formation. Once the somites formed, Budjan et al. measured them and studied their structure and the genes they used. They found that these lab-grown somites have the same size and structure as natural somites and use many of the same genes. This new organoid model provides a way to study human somite formation and development in the lab for the first time. This can provide insights into the development and evolution of humans and other animals that could then help scientists understand diseases such as the development of abnormal spinal curvature that affects around 1 in 10,000 newborns.

BMP signaling is enhanced intracellularly by FHL3 controlling WNT-dependent spatiotemporal emergence of the neural crest.

The spatiotemporal coordination of multiple morphogens is essential for embryonic patterning yet poorly understood. During neural crest (NC) formation, dynamic bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and WNT signals cooperate by acting on mesoderm and ectoderm. Here, we show that Fhl3, a scaffold LIM domain protein, modulates BMP gradient interpretation during NC induction. During gastrulation, low BMP signaling neuralizes the neural border (NB) ectoderm, while Fhl3 enhances Smad1 intracellular response in underlying paraxial mesoderm, triggering the high WNT8 signals needed to pattern the NB. During neurulation, fhl3 activation in NC ectoderm promotes simultaneous high BMP and BMP-dependent WNT activity required for specification. Mechanistically, Fhl3 interacts with Smad1 and promotes Smad1 binding to wnt8 promoter in a BMP-dependent manner. Consequently, differential Fhl3 expression in adjacent cells ensures a finely tuned coordination of BMP and WNT signaling at several stages of NC development, starting by positioning the NC-inducing mesoderm center under competent NB ectoderm.

In vitro systems: A new window to the segmentation clock.

Segmental organization of the vertebrate body plan is established by the segmentation clock, a molecular oscillator that controls the periodicity of somite formation. Given the dynamic nature of the segmentation clock, in vivo studies in vertebrate embryos pose technical challenges. As an alternative, simpler models of the segmentation clock based on primary explants and pluripotent stem cells have recently been developed. These ex vivo and in vitro systems enable more quantitative analysis of oscillatory properties and expand the experimental repertoire applicable to the segmentation clock. Crucially, by eliminating the need for model organisms, in vitro models allow us to study the segmentation clock in new species, including our own. The human oscillator was recently recapitulated using induced pluripotent stem cells, providing a window into human development. Certainly, a combination of in vivo and in vitro work holds the most promising potential to unravel the mechanisms behind vertebrate segmentation.

Development of pluripotent stem cell-based human tenocytes.

Human pluripotent stem cells (PSCs) are used as a platform for therapeutic purposes such as cell transplantation therapy and drug discovery. Another motivation for studying PSCs is to understand human embryogenesis and development. All cell types that make up the body tissues develop through defined trajectories during embryogenesis. For example, paraxial mesoderm is considered to differentiate into several cell types including skeletal muscle cells, chondrocytes, osteocytes, dermal fibroblasts, and tenocytes. Tenocytes are fibroblast cells that constitute the tendon. The step-wise narrowing fate decisions of paraxial mesoderm in the embryo have been modeled in vitro using PSCs; however, deriving tenocytes from human-induced PSCs and their application in cell therapy have long been challenging. PSC-derived tenocytes can be used for a source of cell transplantation to treat a damaged or ruptured tendon due to injury, disorder, or aging. In this review, we discuss the latest research findings on the use of PSCs for studying the biology of tenocyte development and their application in therapeutic settings.

Five transcriptional factors reprogram fibroblast into myogenic lineage cells via paraxial mesoderm stage.

It is hard to supply satellite cells as a cell source for therapy of muscle degenerative disease since the sampling of muscle tissue is very invasive to a patient with muscular disease. Direct conversion allows us to get specific cell types by transduction of defined transcriptional factors. To induce myogenic direct conversion, we transduced five transcriptional factors including Pax3, Sox2, Klf4, c-Myc, and Esrrb into mouse embryonic fibroblasts. We found that the transduction of the five transcriptional factors induced myogenic direct conversion of fibroblast. We revealed that the transduced cells with the five transcriptional factors were converted to myogenic lineage cells through a paraxial mesoderm-like stage. The expression level of myogenic-related genes of the transduced cells gradually increased as the passage increased. The induced myogenic lineage cells differentiated into muscle fibers in virto and in vivo. The current study revealed that the five transcription factors generated myogenic lineage cells from fibroblast via a paraxial mesoderm stage. The induced myogenic lineage cells may have a potential being applied as cell source for degenerative muscle disease.

Neural tube development depends on notochord-derived sonic hedgehog released into the sclerotome.

Sonic hedgehog (Shh), produced in the notochord and floor plate, is necessary for both neural and mesodermal development. To reach the myotome, Shh has to traverse the sclerotome and a reduction of sclerotomal Shh affects myotome differentiation. By investigating loss and gain of Shh function, and floor-plate deletions, we report that sclerotomal Shh is also necessary for neural tube development. Reducing the amount of Shh in the sclerotome using a membrane-tethered hedgehog-interacting protein or Patched1, but not dominant active Patched, decreased the number of Olig2+ motoneuron progenitors and Hb9+ motoneurons without a significant effect on cell survival or proliferation. These effects were a specific and direct consequence of Shh reduction in the mesoderm. In addition, grafting notochords in a basal but not apical location, vis-à-vis the tube, profoundly affected motoneuron development, suggesting that initial ligand presentation occurs at the basal side of epithelia corresponding to the sclerotome-neural tube interface. Collectively, our results reveal that the sclerotome is a potential site of a Shh gradient that coordinates the development of mesodermal and neural progenitors.

In ovo technique for cell injection in the CPM followed by bead implantation in the BA2 of chicken embryos.

The major advantage of chicken embryos model is their accessibility for microsurgical manipulations and the dissection of tissues for ex vivo explant culture. Branchial arches are embryonic structure located next to the top of developing heart. Each arch is made of surface ectoderm, endoderm, myogenic mesoderm cells and cranial neural crest cells. The myogenic mesoderm originates from cranial paraxial mesoderm (CPM), which is transiently migrated to branchial arches (BAs). The first branchial arch (BA1) mesoderm contributes to formation of mastication muscles. The second branchial arch (BA2) mesoderm gives rise to facial expression muscles. This article focuses on cell injection in the CPM and bead implantation (gain of function approaches) in the BA2. In order to follow the migration of mesoderm progenitor cells from CPM to BA2, we injected quail cells in the CPM of stage HH10-11 embryos, followed by implantation of SDF-1 bead at stage HH15-16. Later the attraction of quail cells (CXCR4+) towards the SDF-1 source has been observed, using whole-mount immunostaining of a specific quail antibody (QCPN) at stage HH19-22. •Our method, which involves bead implantation followed by quail cell injection, provides useful tools for tracing migratory mesodermal cells in vivo.•The proposed method does not require any commercial kits and can be used for various developmental process.•It does not employ any complicated methods such as genetically engineered permanent cell labeling, multiplicity of fluorescent markers or clonal analysis.

Exposure-based assessment of chemical teratogenicity using morphogenetic aggregates of human embryonic stem cells.

Pluripotent stem cells recapitulate many aspects of embryogenesis in vitro. Here, we established a novel culture system to differentiate human embryonic stem cell aggregates (HESCA), and evaluated its utility for teratogenicity assessment. Culture of HESCA with modulators of developmental signals induced morphogenetic and molecular changes associated with differentiation of the paraxial mesoderm and neuroectoderm. To examine impact of teratogenic exposures on HESCA differentiation, 18 compounds were tested, for which adequate information on in vivo plasma concentrations is available. HESCA treated with each compound were examined for gross morphology and transcript levels of 15 embryogenesis regulator genes. Significant alterations in the transcript levels were observed for 94% (15/16) of the teratogenic exposures within 5-fold margin, whereas no alteration was observed for 92% (11/12) of the non-teratogenic exposures. Our study demonstrates that transcriptional changes in HESCA serve as predictive indicator of teratogenicity in a manner comparable to in vivo exposure levels.

Gene profiling of head mesoderm in early zebrafish development: insights into the evolution of cranial mesoderm.

BACKGROUND: The evolution of the head was one of the key events that marked the transition from invertebrates to vertebrates. With the emergence of structures such as eyes and jaws, vertebrates evolved an active and predatory life style and radiated into diversity of large-bodied animals. These organs are moved by cranial muscles that derive embryologically from head mesoderm. Compared with other embryonic components of the head, such as placodes and cranial neural crest cells, our understanding of cranial mesoderm is limited and is restricted to few species. RESULTS: Here, we report the expression patterns of key genes in zebrafish head mesoderm at very early developmental stages. Apart from a basic anterior-posterior axis marked by a combination of pitx2 and tbx1 expression, we find that most gene expression patterns are poorly conserved between zebrafish and chick, suggesting fewer developmental constraints imposed than in trunk mesoderm. Interestingly, the gene expression patterns clearly show the early establishment of medial-lateral compartmentalisation in zebrafish head mesoderm, comprising a wide medial zone flanked by two narrower strips. CONCLUSIONS: In zebrafish head mesoderm, there is no clear molecular regionalisation along the anteroposterior axis as previously reported in chick embryos. In contrast, the medial-lateral regionalisation is formed at early developmental stages. These patterns correspond to the distinction between paraxial mesoderm and lateral plate mesoderm in the trunk, suggesting a common groundplan for patterning head and trunk mesoderm. By comparison of these expression patterns to that of amphioxus homologues, we argue for an evolutionary link between zebrafish head mesoderm and amphioxus anteriormost somites.

Paraxial Mesoderm Is the Major Source of Lymphatic Endothelium.

Endothelial cells (ECs), which line blood and lymphatic vessels, are generally described to come from the lateral plate mesoderm despite experimental evidence for a broader source of origin, including the paraxial mesoderm (PXM). Current dogma suggests that following specification from mesoderm, local environmental cues establish the distinct molecular and functional characteristics of ECs in different vascular beds. Here we present evidence to challenge this view, showing that lymphatic EC fate is imprinted during transition through the PXM lineage. We show that PXM-derived cells form the lymphatic endothelium of multiple organs and tissues, with a more restricted contribution to blood vessel endothelium. By deleting Prox1 specifically in PXM-derived cells, we show that this lineage is indispensable for lymphatic vessel development. Collectively, our data establish lineage history as a critical determinant of EC specialization, a finding with broad implications for our understanding of vascular development and heterogeneity.

Transcriptionally dynamic progenitor populations organised around a stable niche drive axial patterning.

The elongating mouse anteroposterior axis is supplied by progenitors with distinct tissue fates. It is not known whether these progenitors confer anteroposterior pattern to the embryo. We have analysed the progenitor population transcriptomes in the mouse primitive streak and tail bud throughout axial elongation. Transcriptomic signatures distinguish three known progenitor types (neuromesodermal, lateral/paraxial mesoderm and notochord progenitors; NMPs, LPMPs and NotoPs). Both NMP and LPMP transcriptomes change extensively over time. In particular, NMPs upregulate Wnt, Fgf and Notch signalling components, and many Hox genes as progenitors transit from production of the trunk to the tail and expand in number. In contrast, the transcriptome of NotoPs is stable throughout axial elongation and they are required for normal axis elongation. These results suggest that NotoPs act as a progenitor niche whereas anteroposterior patterning originates within NMPs and LPMPs.

Falcotentorial Location of Dural Arteriovenous Fistulas Derived from the Neural Crest as a Risk Factor for Aggressive Clinical Course.

The topographical distribution of dural arteriovenous fistulas (DAVFs) was analyzed based on the embryological anatomy of the dural membrane. Sixty-six consecutive cases of intracranial and spinal DAVFs were analyzed based on the angiography, and each shunt point was identified according to the embryological bony structures. The area of dural membranes was categorized into three different groups: a ventral group located on the endochondral bone (VE group), a dorsal group on the membranous bone (DM group), and a falcotentorial group (FT group) in the falx cerebri, tentorium cerebelli, falx cerebelli, and diaphragma sellae. The FT group was derived from the neural crest and designated when the dural membrane was formed only with the dura propria (meningeal layer of the dura mater) and not from the endosteal dura. Olfactory groove, falx, tent of the cerebellum, and nerve sleeve of spinal cord were categorized in the FT group, which presented later in life and which had a male predominance, more aggressive clinical presentations, and significant cortical and spinal venous reflux. The FT group was formed only with the dura propria that was considered as an independent risk factor for aggressive clinical course and hemorrhage of DAVFs.

Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells.

Somites (SMs) comprise a transient stem cell population that gives rise to multiple cell types, including dermatome (D), myotome (MYO), sclerotome (SCL) and syndetome (SYN) cells. Although several groups have reported induction protocols for MYO and SCL from pluripotent stem cells, no studies have demonstrated the induction of SYN and D from SMs. Here, we report systematic induction of these cells from human induced pluripotent stem cells (iPSCs) under chemically defined conditions. We also successfully induced cells with differentiation capacities similar to those of multipotent mesenchymal stromal cells (MSC-like cells) from SMs. To evaluate the usefulness of these protocols, we conducted disease modeling of fibrodysplasia ossificans progressiva (FOP), an inherited disease that is characterized by heterotopic endochondral ossification in soft tissues after birth. Importantly, FOP-iPSC-derived MSC-like cells showed enhanced chondrogenesis, whereas FOP-iPSC-derived SCL did not, possibly recapitulating normal embryonic skeletogenesis in FOP and cell-type specificity of FOP phenotypes. These results demonstrate the usefulness of multipotent SMs for disease modeling and future cell-based therapies.