Mesodermal Expression Research Articles

Initial WNT/β-Catenin or BMP Activation Modulates Inflammatory Response of Mesodermal Progenitors Derived from Human Induced Pluripotent Stem Cells.

Wound healing in adults largely depends on the functional state of multipotent mesenchymal stromal cells (MSCs). Human fetal tissues at the early stages of development are known to heal quickly with a full-quality restoration of the original structure. The differences in the molecular mechanisms that determine the functional activity of mesodermal cells in fetuses and adults remain virtually unknown. Using two independent human induced pluripotent stem cell (iPSC) lines, we examined the effects of the initial WNT and BMP activation on the differentiation of iPSCs via mesodermal progenitors into MSCs and highlighted the functions of these cells that are altered by the proinflammatory microenvironment. The WNT-induced mesoderm commitment of the iPSCs enhanced the expression of paraxial mesoderm (PM)-specific markers, while the BMP4-primed iPSCs exhibited increased levels of lateral mesoderm (LM)-specific genes. The inflammatory status and migration rate of the isogenic iPSC-derived mesoderm cells were assessed via gene expression analysis and scratch assay under the receptor-dependent activation of the proinflammatory IFN-γ or TNF-α signaling pathway. Reduced IDO1 and ICAM1 expression levels were detected in the WNT- and BMP-induced MSC progenitors compared to the isogenic MSCs in response to stimulation with IFN-γ and TNF-α. The WNT- and BMP-induced MSC progenitors exhibited a higher migration rate than isogenic MSCs upon IFN-γ exposure. The established isogenic cellular model will provide new opportunities to elucidate the mechanisms of regeneration and novel therapeutics for wound healing.

PEG 300 Promotes Mesodermal Differentiation in iPSC-Derived Embryoid Body Formation In Vitro.

Embryoid bodies (EB) are sensitive to changes in the culture conditions. Recent studies show that the addition of PEG 300 to culture medium affects cell growth and differentiation; however, its effect on the embryoid body is unclear. This study aims to understand the role of PEG 300 in the process of EB formation and germ layer differentiation. EBs formed more efficiently and differentiated toward the mesoderm when cultured in a medium supplemented with appropriate concentrations of PEG 300. The expression of T/Bry, a marker of mesodermal differentiation, increases in EBs in the PEG group, and the expression of TUBB3 generally decreases, showing a quantitative relationship with PEG. Furthermore, further differentiation of PEG-pretreated EB into vascular smooth muscle cells (VSMCs) by directional induction shows that PEG 300-pretreated induced VSMCs have higher expression of phenotypic markers and greater secretory and contractile functions. This study highlights the role of PEG 300 in the culture medium during EB differentiation, which can significantly enhance mesodermal gene expression and the efficiency of subsequent differentiation into smooth muscle cells and other target cells.

A Potential Role of fgf4, fgf24, and fgf17 in Pharyngeal Pouch Formation in Zebrafish.

In vertebrates, Fgf signaling is essential for the development of pharyngeal pouches, which controls facial skeletal development. Genetically, fgf3 and fgf8 are required for pouch formation in mice and zebrafish. However, loss-of-function phenotypes of fgf3 and fgf8 are milder than expected in mice and zebrafish, which suggests that an additional fgf gene(s) would be involved in pouch formation. Here, we analyzed the expression, regulation, and function of three fgfs, fgf4, fgf24, and fgf17, during pouch development in zebrafish. We find that they are expressed in the distinct regions of pharyngeal endoderm in pouch formation, with fgf4 and fgf17 also being expressed in the adjacent mesoderm, in addition to previously reported endodermal fgf3 and mesodermal fgf8 expression. The endodermal expression of fgf4, fgf24, and fgf17 and the mesodermal expression of fgf4 and fgf17 are positively regulated by Tbx1 but not by Fgf3, in pouch formation. Fgf8 is required to express the endodermal expression of fgf4 and fgf24. Interestingly, however, single mutant, all double mutant combinations, and triple mutant for fgf4, fgf24, and fgf17 do not show any defects in pouches and facial skeletons. Considering a high degree of genetic redundancy in the Fgf signaling components in craniofacial development in zebrafish, our result suggests that fgf4, fgf24, and fgf17 have a potential role for pouch formation, with a redundancy with other fgf gene(s).

ETV2 induces endothelial, but not hematopoietic, lineage specification in birds.

Cardiovascular system develops from the lateral plate mesoderm. Its three primary cell lineages (hematopoietic, endothelial, and muscular) are specified by the sequential actions of conserved transcriptional factors. ETV2, a master regulator of mammalian hemangioblast development, however, is absent in the chicken genome and acts downstream of NPAS4L in zebrafish. Here, we investigated the epistatic relationship between NPAS4L and ETV2 in avian hemangioblast development. We showed that ETV2 is deleted in all 363 avian genomes analyzed. Mouse ETV2 induced LMO2, but not NPAS4L or SCL, expression in chicken mesoderm. Squamate (lizards, geckos, and snakes) genomes contain both NPAS4L and ETV2 In Madagascar ground gecko, both genes were expressed in developing hemangioblasts. Gecko ETV2 induced only LMO2 in chicken mesoderm. We propose that both NPAS4L and ETV2 were present in ancestral amniote, with ETV2 acting downstream of NPAS4L in endothelial lineage specification. ETV2 may have acted as a pioneer factor by promoting chromatin accessibility of endothelial-specific genes and, in parallel with NPAS4L loss in ancestral mammals, has gained similar function in regulating blood-specific genes.

CDX2 dose-dependently influences the gene regulatory network underlying human extraembryonic mesoderm development.

Loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT), heterozygous (CDX2-Het), and homozygous null CDX2 (CDX2-KO) genotypes, differentiating these cells in a 2D gastruloid model, and subjecting these cells to single-nucleus RNA and ATAC sequencing, we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile, suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo, we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo.

Sall4 regulates posterior trunk mesoderm development by promoting mesodermal gene expression and repressing neural genes in the mesoderm.

The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.

Ultrastructure of the lamprey head mesoderm reveals evolution of the vertebrate head

The cranial muscle is a critical component in the vertebrate head for a predatory lifestyle. However, its evolutionary origin and possible segmental nature during embryogenesis have been controversial. In jawed vertebrates, the presence of pre-otic segments similar to trunk somites has been claimed based on developmental observations. However, evaluating such arguments has been hampered by the paucity of research on jawless vertebrates. Here, we discovered different cellular arrangements in the head mesoderm in lamprey embryos (Lethenteron camtschaticum) using serial block-face scanning electron and laser scanning microscopies. These cell populations were morphologically and molecularly different from somites. Furthermore, genetic comparison among deuterostomes revealed that mesodermal gene expression domains were segregated antero-posteriorly in vertebrates, whereas such segregation was not recognized in invertebrate deuterostome embryos. These findings indicate that the vertebrate head mesoderm evolved from the anteroposterior repatterning of an ancient mesoderm and developmentally diversified before the split of jawless and jawed vertebrates.

Inka1b expression in the head mesoderm is dispensable for facial cartilage development

Inka box actin regulator 1 (Inka1) is a novel protein identified in Xenopus and is found in vertebrates. While Inka1 is required for facial skeletal development in Xenopus and zebrafish, it is dispensable in mice despite its conserved expression in the cranial neural crest, indicating that Inka1 function in facial skeletal development is not conserved among vertebrates. Zebrafish bears two paralogs of inka1 (inka1a and inka1b) in the genome, with the biological roles of inka1b barely known. Here, we analyzed the expression and function of inka1b during facial skeletal development in zebrafish. inka1b was expressed sequentially in the head mesoderm adjacent to the pharyngeal pouches essential for facial skeletal development at the stage of arch segmentation. However, a loss-of-function mutation in inka1b displayed normal head development, including the pouches and facial cartilages. The normal head of inka1b mutant fish was unlikely a result of the genetic redundancy of inka1b with inka1a, given the distinct expression of inka1a and inka1b in the cranial neural crest and head mesoderm, respectively, during craniofacial development. Our findings suggest that the inka1b expression in the head mesoderm might not be essential for head development in zebrafish.

A mosaic of conserved and novel modes of gene expression and morphogenesis in mesoderm and muscle formation of a larval bivalve

The mesoderm gives rise to several key morphological features of bilaterian animals including endoskeletal elements and the musculature. A number of regulatory genes involved in mesoderm and/or muscle formation (e.g., Brachyury (Bra), even-skipped (eve), Mox, myosin II heavy chain (mhc)) have been identified chiefly from chordates and the ecdysozoans Drosophila and Caenorhabditis elegans, but data for non-model protostomes, especially those belonging to the ecdysozoan sister clade, Lophotrochozoa (e.g., flatworms, annelids, mollusks), are only beginning to emerge. Within the lophotrochozoans, Mollusca constitutes the most speciose and diverse phylum. Interestingly, however, information on the morphological and molecular underpinnings of key ontogenetic processes such as mesoderm formation and myogenesis remains scarce even for prominent molluscan sublineages such as the bivalves. Here, we investigated myogenesis and developmental expression of Bra, eve, Mox, and mhc in the quagga mussel Dreissena rostriformis, an invasive freshwater bivalve and an emerging model in invertebrate evodevo. We found that all four genes are expressed during mesoderm formation, but some show additional, individual sites of expression during ontogeny. While Mox and mhc are involved in early myogenesis, eve is also expressed in the embryonic shell field and Bra is additionally present in the foregut. Comparative analysis suggests that Mox has an ancestral role in mesoderm and possibly muscle formation in bilaterians, while Bra and eve are conserved regulators of mesoderm development of nephrozoans (protostomes and deuterostomes). The fully developed Dreissena veliger larva shows a highly complex muscular architecture, supporting a muscular ground pattern of autobranch bivalve larvae that includes at least a velum muscle ring, three or four pairs of velum retractors, one or two pairs of larval retractors, two pairs of foot retractors, a pedal plexus, possibly two pairs of mantle retractors, and the muscles of the pallial line, as well as an anterior and a posterior adductor. As is typical for their molluscan kin, remodelling and loss of prominent larval features such as the velum musculature and various retractor systems appear to be also common in bivalves.

The H3K27M mutation alters stem cell growth, epigenetic regulation, and differentiation potential

BackgroundNeurodevelopmental disorders increase brain tumor risk, suggesting that normal brain development may have protective properties. Mutations in epigenetic regulators are common in pediatric brain tumors, highlighting a potentially central role for disrupted epigenetic regulation of normal brain development in tumorigenesis. For example, lysine 27 to methionine mutation (H3K27M) in the H3F3A gene occurs frequently in Diffuse Intrinsic Pontine Gliomas (DIPGs), the most aggressive pediatric glioma. As H3K27M mutation is necessary but insufficient to cause DIPGs, it is accompanied by additional mutations in tumors. However, how H3K27M alone increases vulnerability to DIPG tumorigenesis remains unclear.ResultsHere, we used human embryonic stem cell models with this mutation, in the absence of other DIPG contributory mutations, to investigate how H3K27M alters cellular proliferation and differentiation. We found that H3K27M increased stem cell proliferation and stem cell properties. It interfered with differentiation, promoting anomalous mesodermal and ectodermal gene expression during both multi-lineage and germ layer-specific cell specification, and blocking normal differentiation into neuroectoderm. H3K27M mutant clones exhibited transcriptomic diversity relative to the more homogeneous wildtype population, suggesting reduced fidelity of gene regulation, with aberrant expression of genes involved in stem cell regulation, differentiation, and tumorigenesis. These phenomena were associated with global loss of H3K27me3 and concordant loss of DNA methylation at specific genes in H3K27M-expressing cells.ConclusionsTogether, these data suggest that H3K27M mutation disrupts normal differentiation, maintaining a partially differentiated state with elevated clonogenicity during early development. This disrupted response to early developmental cues could promote tissue properties that enable acquisition of additional mutations that cooperate with H3K27M mutation in genesis of DMG/DIPG. Therefore, this work demonstrates for the first time that H3K27M mutation confers vulnerability to gliomagenesis through persistent clonogenicity and aberrant differentiation and defines associated alterations of histone and DNA methylation.

CD1d expression demarcates CDX4+ hemogenic mesoderm with definitive hematopoietic potential

To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4hi expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications.

Reconstruction of dynamic regulatory networks reveals signaling-induced topology changes associated with germ layer specification.

SummaryElucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. Unfortunately, existing computational gene regulatory network (GRN) reconstruction methods are imprecise, computationally burdensome, and fail to reveal dynamic regulatory topologies. Here, we present Epoch, a reconstruction tool that uses single-cell transcriptomics to accurately infer dynamic networks. We apply Epoch to identify the dynamic networks underpinning directed differentiation of mouse embryonic stem cells (ESCs) guided by multiple signaling pathways, and we demonstrate that modulating these pathways drives topological changes that bias cell fate potential. We also find that Peg3 rewires the pluripotency network to favor mesoderm specification. By integrating signaling pathways with GRNs, we trace how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively. Finally, we identify regulatory circuits of patterning and axis formation that distinguish in vitro and in vivo mesoderm specification.

A 37 kb region upstream of brachyury comprising a notochord enhancer is essential for notochord and tail development.

ABSTRACTThe node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor brachyury (T) is essential for both formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T that is essential for notochord function and tailbud outgrowth. Within that region, we discovered a T-binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord, allowing proper trunk and tail development.

Downregulation of Grem1 expression in the distal limb mesoderm is a necessary precondition for phalanx development.

The phalanges are the final skeletal elements to form in the vertebrate limb and their identity is regulated by signaling at the phalanx forming region (PFR) located at the tip of the developing digit ray. Here, we seek to explore the relationship between PFR activity and phalanx morphogenesis, which define the most distal limb skeletal elements, and signals associated with termination of limb outgrowth. As Grem1 is extinguished in the distal chick limb mesoderm, the chondrogenesis marker Aggrecan is up-regulated in the metatarsals and phalanges. Fate mapping confirms that subridge mesoderm cells contribute to the metatarsal and phalanges when subridge Grem1 is down-regulated. Grem1 overexpression specifically blocks chick phalanx development by inhibiting PFR activity. PFR activity and digit development are also disrupted following overexpression of a Gli3 repressor, which results in Grem1 expression in the distal limb and downregulation of Bmpr1b. Based on expression and fate mapping studies, we propose that downregulation of Grem1 in the distal limb marks the transition from metatarsal to phalanx development. This suggests that downregulation of Grem1 in the distal limb mesoderm is necessary for phalanx development. Grem1 downregulation allows for full PFR activity and phalanx progenitor cell commitment to digit fate.

Foxd4l1.1 Negatively Regulates Chordin Transcription in Neuroectoderm of Xenopus Gastrula.

Inhibition of the bone morphogenetic proteins (BMPs) is the primary step toward neuroectoderm formation in vertebrates. In this process, the Spemann organizer of the dorsal mesoderm plays a decisive role by secreting several extracellular BMP inhibitors such as Chordin (Chrd). Chrd physically interacts with BMP proteins and inhibits BMP signaling, which triggers the expression of neural-specific transcription factors (TFs), including Foxd4l1.1. Thus, Chrd induces in a BMP-inhibited manner and promotes neuroectoderm formation. However, the regulatory feedback mechanism of Foxd4l1.1 on mesodermal genes expression during germ-layer specification has not been fully elucidated. In this study, we investigated the regulatory mechanism of Foxd4l1.1 on chrd (a mesodermal gene). We demonstrate that Foxd4l1.1 inhibits chrd expression during neuroectoderm formation in two ways: First, Foxd4l1.1 directly binds to FRE (Foxd4l1.1 response elements) within the chrd promoter region to inhibit transcription. Second, Foxd4l1.1 physically interacts with Smad2 and Smad3, and this interaction blocks Smad2 and Smad3 binding to activin response elements (AREs) within the chrd promoter. Site-directed mutagenesis of FRE within the chrd(-2250) promoter completely abolished repressor activity of the Foxd4l1.1. RT-PCR and reporter gene assay results indicate that Foxd4l1.1 strongly inhibits mesoderm- and ectoderm-specific marker genes to maintain neural fate. Altogether, these results suggest that Foxd4l1.1 negatively regulates chrd transcription by dual mechanism. Thus, our study demonstrates the existence of precise reciprocal regulation of chrd transcription during neuroectoderm and mesoderm germ-layer specification in Xenopus embryos.

Formal proof of the requirement of MESP1 and MESP2 in mesoderm specification and their transcriptional control via specific enhancers in mice.

MESP1 and MESP2 are transcriptional factors involved in mesoderm specification, somite boundary formation and somite polarity regulation. However, Mesp quadruple mutant zebrafish displayed only abnormal somite polarity without mesoderm specification defects. In order to re-evaluate Mesp1/Mesp2 mutants in mice, Mesp1 and Mesp2 single knockouts (KOs), and a Mesp1/Mesp2 double KO were established using genome-editing techniques without introducing selection markers commonly used before. The Mesp1/Mesp2 double KO embryos exhibited markedly severe mesoderm formation defects that were similar to the previously reported Mesp1/Mesp2 double KO embryos, indicating species differences in the function of MESP family proteins. However, the Mesp1 KO did not display any phenotype, including heart formation defects, which have been reported previously. We noted upregulation of Mesp2 in the Mesp1 KO embryos, suggesting that MESP2 rescues the loss of MESP1 in mesoderm specification. We also found that Mesp1 and Mesp2 expression in the early mesoderm is regulated by the cooperation of two independent enhancers containing T-box- and TCF/Lef-binding sites. Deletion of both enhancers caused the downregulation of both genes, resulting in heart formation defects. This study suggests dose-dependent roles of MESP1 and MESP2 in early mesoderm formation.

Individual and combined effects of BPA, BPS and BPAF on the cardiomyocyte differentiation of embryonic stem cells

Exposure to many kinds of bisphenols (BPs) is common, and the effects of BP mixtures may differ from those of individual BPs. Therefore, evaluating combined exposure effects is necessary. Our study evaluated the individual and combined exposure effects of bisphenol A (BPA), bisphenol S (BPS) and bisphenol AF (BPAF) on embryonic development using an embryonic stem cell test (EST) and a concentration additive (CA) model at relatively high doses to uncover the interaction model of the three BPs. Environmentally relevant concentrations were then used to evaluate the possible effects of the individual and combined BPs at actual human exposure levels. Exposure to relatively high-dose BPA, BPS and BPAF inhibited embryonic stem cell differentiation into cardiomyocytes and exhibited weak embryo toxicity. Individually, BPA, BPS and BPAF inhibited endoderm, mesoderm and ectoderm marker expression but enhanced pluripotency marker expression. Combined exposure to BPs had an additive effect on cardiomyocyte differentiation and embryonic stem cell proliferation based on the CA model. Environmentally relevant individual or combined BP doses (10 ng/ml individual BPA, BPS and BPAF doses or 1 ng/ml and 10 ng/ml BP mixture doses) failed to cause oxidative stress, DNA damage or apoptosis changes in stem cell differentiation. The cardiomyocyte differentiation ratio also did not change significantly. Individual and combined exposure to environmentally relevant BP doses led to a significant increase in collagen expression. BPAF and the combination of BPs increased the type 1 collagen level, while the combination also increased the type 3 collagen level, which may be related to p38 pathway activation. The p38 pathway inhibitor SB203580 inhibited the increase in collagen during cardiomyocyte differentiation caused by low-dose BPs. These results suggest that relatively high-dose BPs in combination have an additive effect on cardiomyocyte differentiation. Low-dose BPs individually and in combination may affect cardiomyocyte collagen through the p38 pathway.

Altering metabolite distribution at Xenopus cleavage stages affects left-right gene expression asymmetries.

The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.

Generation and trapping of a mesoderm biased state of human pluripotency

We postulate that exit from pluripotency involves intermediates that retain pluripotency while simultaneously exhibiting lineage-bias. Using a MIXL1 reporter, we explore mesoderm lineage-bias within the human pluripotent stem cell compartment. We identify a substate, which at the single cell level coexpresses pluripotent and mesodermal gene expression programmes. Functionally these cells initiate stem cell cultures and exhibit mesodermal bias in differentiation assays. By promoting mesodermal identity through manipulation of WNT signalling while preventing exit from pluripotency using lysophosphatidic acid, we ‘trap’ and maintain cells in a lineage-biased stem cell state through multiple passages. These cells correspond to a normal state on the differentiation trajectory, the plasticity of which is evidenced by their reacquisition of an unbiased state upon removal of differentiation cues. The use of ‘cross-antagonistic’ signalling to trap pluripotent stem cell intermediates with different lineage-bias may have general applicability in the efficient production of cells for regenerative medicine.

The Cdx transcription factors and retinoic acid play parallel roles in antero-posterior position of the pectoral fin field during gastrulation

The molecular regulators that determine the precise position of the vertebrate limb along the anterio-posterior axis have not been identified. One model suggests that a combination of hox genes in the lateral plate mesoderm (LPM) promotes formation of the limb field, however redundancy among duplicated paralogs has made this model difficult to confirm. In this study, we identify an optimal window during mid-gastrulation stages when transient mis-regulation of retinoic acid signaling or the caudal related transcription factor, Cdx4, both known regulators of hox genes, can alter the position of the pectoral fin field. We show that increased levels of either RA or Cdx4 during mid-gastrulation are sufficient to rostrally shift the position of the pectoral fin field at the expense of surrounding gene expression in the anterior lateral plate mesoderm (aLPM). Alternatively, embryos deficient for both Cdx4 and Cdx1a (Cdx-deficient) form pectoral fins that are shifted towards the posterior and reveal an additional effect on size of the pectoral fin buds. Prior to formation of the pectoral fin buds, the fin field in Cdx-deficient embryos is visibly expanded into the posterior LPM (pLPM) region at the expense of surrounding gene expression. The effects on gene expression immediately post-gastrulation and during somitogenesis support a model where RA and Cdx4 act in parallel to regulate the position of the pectoral fin. Our transient method is a potentially useful model for studying the mechanisms of limb positioning along the AP axis.