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Primordial germ cells (PGCs) are highly specialized cells that play a relevant role in the maintenance and evolution of the species, since they create new combinations of genetic information between the organisms. Amphibians are a class of amniote vertebrates that are divided into three subclasses, the anurans (frogs and toads), the urodeles (salamanders and newts), and the gymnophiones (caecilians). The study of PGCs in amphibians has been addressed in more detail in anurans while little is known about the biology of this cell lineage in urodeles. Studies in some urodeles species have suggested that PGCs are of mesodermal origin, specifying in the lateral plate mesoderm at the late gastrula stage. With classical experiments it shown that, there is an induction of mesoderm, therefore most likely urodeles PGCs develop from unspecialized mesodermal tissue that responds to extracellular signals. However, some fundamental biological processes of PGCs such as the analysis of their specification, arrival, and colonization to the gonads, and their maintenance and differentiation into mature and fertile gametes remain to be elucidated. Therefore, knowledge about the biology of PGCs is of great importance to ensure the perpetuation of urodeles amphibians, as some species are in danger of becoming extinct.

Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro., The authors form pre-epicardial cells (PECs) from hiPSC-derived lateral plate mesoderm on treating with BMP4, RA and VEGF, and co-culture these PECs with cardiomyocytes, inducing cardiomyocyte aggregation, proliferation and network formation with more mature structures and improved beating/contractility.

Background Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus. Results hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut and lateral plate mesoderm with different degrees of overlap. Conclusions Hand gene expression in the gut, peripheral nervous system and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes. This article is protected by copyright. All rights reserved.

Meis1 and Meis2 are homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Elimination of Meis genes after limb induction has shown their role in limb proximo-distal patterning; however, limb development in the complete absence of Meis function has not been studied. Here, we report that Meis1/2 inactivation in the lateral plate mesoderm of mouse embryos leads to limb agenesis. Meis and Tbx factors converge in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of Fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior specification results from an early role of Meis factors in establishing the limb antero-posterior prepattern required for Shh activation. Our results demonstrate roles for Meis transcription factors in early limb development and identify their involvement in previously undescribed interaction networks that regulate organogenesis., Double conditional knockout of Meis1/2 in the limb forming region of mouse embryos results in the complete absence of limb, while embryos developed with one functional Meis allele, so identifying the role of Meis in antero-posterior and proximo-distal patterning.

During early embryogenesis, the three germ layers, mesoderm, endoderm and ectoderm, must form in the correct positions for normal development to proceed. In the amphibian embryo, mesoderm is induced from cells of the marginal zone, at the equator of the spherical embryo, by signals emanating from the vegetal region. Later, during gastrulation, mesoderm is further patterned by signals from the dorsal organiser. Here, we review our current knowledge of mesoderm formation, highlighting the importance of the transforming growth factor-beta (TGF-β) family of extracellular signalling molecules in mesoderm induction and the role of maternal factors in activating these molecules. We also discuss mechanisms of limiting induction to the marginal zone by TGF-β inhibitors in the animal hemisphere, the role of FGF signalling in maintaining the mesoderm and the role of BMP and BMP antagonists in patterning mesoderm during gastrulation. Key Concepts Mesoderm is induced to form from marginal zone cells of the Xenopus embryo in response to signals secreted from the vegetal hemisphere. Mesoderm is induced by members of the Nodal/Activin sub-family of the transforming growth factor-beta (TGF-β) family of secreted signalling factors. Fibroblast growth factor (FGF) family members maintain mesoderm. Mesoderm induction is limited to the marginal zone of the embryo by TGF-β signalling inhibitors expressed in the animal region. Maternal transcription factors, such as VegT and β-catenin, activate expression of TGF-β genes. Mesoderm is further patterned during gastrulation through both activation and inhibition of BMP signalling. Keywords: Xenopus; embryo; mesoderm; TGF-β; Nodal

Nodal signaling is essential for mesoderm and endoderm formation, as well as neural plate induction and establishment of left-right asymmetry. However, the mechanisms controlling expression of Nodal pathway genes in these contexts are not fully known. Previously, we showed that Cdx1b induces expression of downstream Nodal signaling factors during early endoderm formation. In this study, we show that Cdx1b also regulates epithalamic asymmetry in zebrafish embryos by modulating expression of ndr2 and lft1. We first knocked down cdx1b with translation-blocking and splicing-blocking morpholinos (MOs). Most embryos injected with translation-blocking MOs showed absent ndr2, lft1 and pitx2c expression in the left dorsal diencephalon during segmentation and pharyngula stages accompanied by aberrant parapineal migration and habenular laterality at 72 ​h post fertilization (hpf). These defects were less frequent in embryos injected with splicing-blocking MO. To confirm the morphant phenotype, we next generated both zygotic (Z)cdx1b−/− and maternal zygotic (MZ)cdx1b−/− mutants by CRISPR-Cas9 mutagenesis. Expression of ndr2, lft1 and pitx2c was absent in the left dorsal diencephalon of a high proportion of MZcdx1b−/− mutants; however, aberrant dorsal diencephalic pitx2c expression patterns were observed at low frequency in Zcdx1b−/− mutant embryos. Correspondingly, dysregulated parapineal migration and habenular laterality were also observed in MZcdx1b−/− mutant embryos at 72 hpf. On the other hand, Kupffer’s vesicle cilia length and number, expression pattern of spaw in the lateral plate mesoderm and pitx2c in the gut as well as left-right patterning of various visceral organs were not altered in MZcdx1b−/− mutants compared to wild-type embryos. Chromatin immunoprecipitation revealed that Cdx1b directly regulates ndr2 and lft1 expression. Furthermore, injection of cdx1b-vivo MO1 but not cdx1b-vivo 4 ​mm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23–24 ​s stages. Together, our results suggest that Cdx1b regulates transcription of ndr2 and lft1 to maintain proper Nodal activity in the dorsal diencephalon and epithalamic asymmetry in zebrafish embryos.

Unidirectional fluid flow generated by motile cilia at the left-right organizer (LRO) breaks left-right (L-R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L-R symmetry breaking. The diversity of mechanisms for L-R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L-R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L-R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.

The discovery of mesoderm inducing signals helped usher in the era of molecular developmental biology, and today the mechanisms of mesoderm induction and patterning are still intensely studied. Mesoderm induction begins during gastrulation, but recent evidence in vertebrates shows that this process continues after gastrulation in a group of posteriorly localized cells called neuromesodermal progenitors (NMPs). NMPs reside within the post-gastrulation embryonic structure called the tailbud, where they make a lineage decision between ectoderm (spinal cord) and mesoderm. The majority of NMP-derived mesoderm generates somites, but also contributes to lateral mesoderm fates such as endothelium. The discovery of NMPs provides a new paradigm in which to study vertebrate mesoderm induction. This review will discuss mechanisms of mesoderm induction within NMPs, and how they have informed our understanding of mesoderm induction more broadly within vertebrates as well as animal species outside of the vertebrate lineage. Special focus will be given to the signaling networks underlying NMP-derived mesoderm induction and patterning, as well as emerging work on the significance of partial epithelial to mesenchymal states in coordinating cell fate and morphogenesis.

The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects.

To ensure the formation of a properly patterned embryo, multiple processes must operate harmoniously at sequential phases of development. This is implemented by mutual interactions between cells and tissues that together regulate the segregation and specification of cells, their growth and morphogenesis. The formation of the spinal cord and paraxial mesoderm derivatives exquisitely illustrate these processes. Following early gastrulation, while the vertebrate body elongates, a population of bipotent neuromesodermal progenitors resident in the posterior region of the embryo generate both neural and mesodermal lineages. At later stages, the somitic mesoderm regulates aspects of neural patterning and differentiation of both central and peripheral neural progenitors. Reciprocally, neural precursors influence the paraxial mesoderm to regulate somite-derived myogenesis and additional processes by distinct mechanisms. Central to this crosstalk is the activity of the axial notochord, which, via sonic hedgehog signaling, plays pivotal roles in neural, skeletal muscle and cartilage ontogeny. Here, we discuss the cellular and molecular basis underlying this complex developmental plan, with a focus on the logic of sonic hedgehog activities in the coordination of the neural-mesodermal axis.

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. Electronic supplementary material The online version of this article (10.1186/s13227-019-0128-3) contains supplementary material, which is available to authorized users.

Summary Lateral plate mesoderm (LPM) cells differentiate into various cell types including endothelial and hematopoietic cells. In zebrafish embryos, LPM cells migrate toward the midline along the ventral surfaces of somites during which their cell fate specification depends upon efficient integrin-mediated cell adhesion and migration. Herein, we present a protocol for analysis of integrin-mediated cell adhesion of LPM cells isolated from zebrafish embryos. This allows the study of the molecular mechanisms underlying integrin activation required for LPM cell fate specification. For complete details on the use and execution of this protocol, please refer to Rho et al. (2019)., Graphical abstract, Highlights • Zebrafish breeding and preparation of the LPM cells dissociated from the embryos • Protocol for LPM cell adhesion to the fibronectin-coated dish • Imaging the LPM cells adhering to the fibronectin-coated dish • Analysis of cell spreading and focal adhesion formations in the LPM cells, Lateral plate mesoderm (LPM) cells differentiate into various cell types including endothelial and hematopoietic cells. In zebrafish embryos, LPM cells migrate toward the midline along the ventral surfaces of somites during which their cell fate specification depends upon efficient integrin-mediated cell adhesion and migration. Herein, we present a protocol for analysis of integrin-mediated cell adhesion of LPM cells isolated from zebrafish embryos. This allows the study of the molecular mechanisms underlying integrin activation required for LPM cell fate specification.

Summary: The composition of cell surface glycoconjugates is set by controlled glycosyltransferase genes, whose function, in the context of cell fate decisions, is poorly understood. Using a human pluripotent cell (hPC)-based differentiation model, we have uncovered a critical function in the reduction of α1-2 fucosyltransferase (FUT1)—a glycosyltransferase catalyzing the synthesis of fucosylated glycoconjugates in α1-2 linkage—in promoting the acquisition of germ layer cell identity. We show that a FUT1 loss of function leads to the induction of lateral mesoderm gene expression, while a FUT1 gain of function prevents lateral mesoderm commitment and impairs endoderm and ectoderm fate specification, such that FUT1-overexpressing hESCs do not differentiate into cardiomyocyte lineages. Analysis of single-cell RNA-seq in mouse embryos (E5.5–E7.5) and embryo staining depict a spatiotemporal pattern of FUT1 activity, first in posterior and then in anterior-distal epiblast cells and later in the restricted region of the ectoderm and endoderm. Notably, fucosyltransferase-2 (FUT2), which acts similar to FUT1 and has been deemed indistinguishable from it, does not add α1-2 fucosyl residues to glycoconjugates instead of FUT1. According to molecular fate maps of known germ-layer markers, FUT1 is expressed in epiblast cells that acquired different layer potency than FUT2. These findings highlight an exclusive function of FUT1 and its synthesized α1-2 fucosylated glycoconjugates in deriving lineage specification and prompt a revision to the functionality of FUT1, in comparison to FUT2, during development.

In mammals, the pre-gastrula proximal epiblast gives rise to primordial germ cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work has shown that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here, we show that, in mice, OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and a deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs also occurs in mutant embryos. We propose that the OTX2-mediated repressive control of Oct4 and Nanog is the basis of the mechanism that determines epiblast contribution to germline and somatic lineage.

Transcriptional regulatory networks refine gene expression boundaries throughout embryonic development to define the precise dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that establish the boundary between the IM and its neighboring vessel progenitors are poorly understood. Here, we delineate new roles for the zinc finger transcription factor Osr1 in kidney and vessel progenitor development. Zebrafishosr1mutants display decreased IM formation and premature emergence of neighboring lateral vessel progenitors (LVPs). These phenotypes contrast with the increased IM and absent LVPs observed with loss of the bHLH transcription factor Hand2, and loss ofhand2partially suppresses theosr1mutant phenotypes.hand2andosr1are both expressed in the posterior lateral mesoderm, butosr1expression decreases dramatically prior to LVP emergence. Overexpressingosr1inhibits LVP development while enhancing IM formation. Together, our data demonstrate thatosr1modulates both the extent of IM formation and the temporal dynamics of LVP development, suggesting that a balance between levels ofosr1andhand2expression is essential to demarcate the dimensions of kidney and vessel progenitor territories.SUMMARY STATEMENTAnalysis of theosr1mutant phenotype reveals roles in determining the extent of intermediate mesoderm formation while inhibiting premature differentiation of neighboring vessel progenitors.

During vertebrate embryonic development complex morphogenetic events drive the formation of internal organs associated with the developing digestive tract. The foregut organs derive from hepatopancreatic precursor cells that originate bilaterally within the endoderm monolayer, and subsequently converge toward the midline where they coalesce to produce the gut tube from which the liver and pancreas form. The progenitor cells of these internal organs are influenced by the lateral plate mesoderm (LPM), which helps direct them towards their specific fates. However, it is not completely understood how the bilateral organ precursors move toward the embryonic midline and ultimately coalesce to form functional organs. Here we demonstrate that the zebrafish homeobox gene hoxb5b regulates morphogenesis of the foregut endoderm at the midline. At early segmentation stages, hoxb5b is expressed in the LPM adjacent to the developing foregut endoderm. By 24 hpf hoxb5b is expressed directly in the endoderm cells of the developing gut tube. When Hoxb5b function is disrupted, either by morpholino knockdown or sgRNA/Cas9 somatic disruption, the process of foregut morphogenesis is disrupted, resulting in a bifurcated foregut. By contrast, knockdown of the paralogous hoxb5a gene does not alter gut morphology. Further analysis has indicated that Hoxb5b knockdown specimens produce endocrine pancreas cell types, but liver cells are absent. Finally, cell transplantation experiments revealed that Hoxb5b function in the endoderm is not needed for proper coalescence of the foregut at the midline. Together, our findings imply that midline morphogenesis of foregut endoderm is guided by a hoxb5b-mediated mechanism that functions extrinsically, likely within the LPM. Loss of hoxb5b function prevents normal coalescence of endoderm cells at the midline and thus disrupts gut morphogenesis.

Visceral organs, such as the lungs, stomach and liver, are derived from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding splanchnic mesoderm (SM). While DE patterning is fairly well studied, the paracrine signaling controlling SM regionalization and how this is coordinated with epithelial identity is obscure. Here, we use single cell transcriptomics to generate a high-resolution cell state map of the embryonic mouse foregut. This identifies a diversity of SM cell types that develop in close register with the organ-specific epithelium. We infer a spatiotemporal signaling network of endoderm-mesoderm interactions that orchestrate foregut organogenesis. We validate key predictions with mouse genetics, showing the importance of endoderm-derived signals in mesoderm patterning. Finally, leveraging these signaling interactions, we generate different SM subtypes from human pluripotent stem cells (hPSCs), which previously have been elusive. The single cell data can be explored at: https://research.cchmc.org/ZornLab-singlecell., The fetal murine foregut develops into visceral organs via interactions between the mesoderm and endoderm, but how is unclear. Here, the authors use single cell RNAseq to show a diversity in organ specific splanchnic mesoderm cell-types, infer a signalling network governing organogenesis and use this to differentiate human pluripotent stem cells.

Proper left-right (LR) axis establishment is critical for organogenesis in vertebrates. Previously, we reported that zinc finger transcription factors zinc finger transcription factor 1 (znfl1s) are expressed in the tailbud and axial mesoderm in zebrafish. However, a role of znfl1s in LR axis development has not been demonstrated. Here, we discovered that the knockdown of znfl1s using morpholino (MO) in whole embryos or dorsal forerunner cells (DFCs) interrupted LR asymmetry and normal development of the heart, liver, and pancreas. Whole-embryo knockdown of znfl1s by MO or clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) resulted in the absent expression of nodal gene spaw and Nodal signaling-related genes lft1, lft2, and pitx2c in the left lateral plate mesoderm (LPM), and Spaw, Lft1, Lft2, and Pitx2c play important roles in LR axis development in zebrafish. However, specific knockdown of znfl1s in DFCs resulted in random expression of spaw, lft1, lft2, and pitx2c. Knockdown of znfl1s led to abnormal cilia formation by the downregulation of fgfr1a and foxj1a expression. The expression of spaw, lft1, lft2, and pitx2c was partially rescued by the overexpression of fgfr1a mRNA in znfl1s morphants. Taken together, our results suggest that znfl1s regulate laterality development in zebrafish embryos through controlling the expression of fgfr1a.

The murine pancreas buds from the ventral embryonic endoderm at approximately 8.75 dpc and a second pancreas bud emerges from the dorsal endoderm by 9.0 dpc. Although it is clear that secreted signals from adjacent mesoderm-derived sources are required for both the appropriate emergence and further refinement of the pancreatic endoderm, neither the exact signals nor the requisite tissue sources have been defined in mammalian systems. Herein we use DiI fate mapping of cultured murine embryos to identify the embryonic sources of both the early inductive and later condensed pancreatic mesenchyme. Despite being capable of supporting pancreas induction from dorsal endoderm in co-culture experiments, we find that in the context of the developing embryo, the dorsal aortae as well as the paraxial, intermediate, and lateral mesoderm derivatives only transiently associate with the dorsal pancreas bud, producing descendants that are decidedly anterior to the pancreas bud. Unlike these other mesoderm derivatives, the axial (notochord) descendants maintain association with the dorsal pre-pancreatic endoderm and early pancreas bud. This fate mapping data points to the notochord as the likely inductive source in vivo while also revealing dynamic morphogenetic movements displayed by individual mesodermal subtypes. Because none of the mesoderm examined above produced the pancreatic mesenchyme that condenses around the induced bud to support exocrine and endocrine differentiation, we also sought to identify the mesodermal origins of this mesenchyme. We identify a portion of the coelomic mesoderm that contributes to the condensed pancreatic mesenchyme. In conclusion, we identify a portion of the notochord as a likely source of the signals required to induce and maintain the early dorsal pancreas bud, demonstrate that the coelomic mesothelium contributes to the dorsal and ventral pancreatic mesenchyme, and provide insight into the dynamic morphological rearrangements of mesoderm-derived tissues during early organogenesis stages of mammalian development.

Isl1 is required for two processes during hindlimb development: initiation of the processes directing hindlimb development in the lateral plate mesoderm and configuring posterior hindlimb field in the nascent hindlimb buds. During these processes, Isl1 expression is restricted to the posterior mesenchyme of hindlimb buds. How this dynamic change in Isl1 expression is regulated remains unknown. We found that two evolutionarily conserved sequences, located 3' to the Isl1 gene, regulate LacZ transgene expression in the hindlimb-forming region in mouse embryos. Both sequences contain GATA binding motifs, and expression pattern analysis identified that Gata6 is expressed in the flank and the anterior portion of nascent hindlimb buds. Recent studies have shown that conditional inactivation of Gata6 in mice causes hindlimb-specific pre-axial polydactyly, indicating a role of Gata6 in anterior-posterior patterning of hindlimbs. We studied whether Gata6 restricts Isl1 in the nascent hindlimb bud through the cis-regulatory modules. In vitro experiments demonstrate that GATA6 binds to the conserved GATA motifs in the cis-regulatory modules. GATA6 repressed expression of a luciferase reporter that contains the cis-regulatory modules by synergizing with Zfpm2. Analyses of Gata6 mutant embryos showed that ISL1 levels are higher in the anterior of nascent hindlimb buds than in wild type. Moreover, we detected a greater number of Isl1-transcribing cells in the anterior of nascent hindlimb buds in Gata6 mutants. Our results support a model in which Gata6 contributes to repression of Isl1 expression in the anterior of nascent hindlimb buds.

The field of hematopoietic and vascular developmental research owes its origin to the chick embryo. Many key concepts, such as the hematopoietic stem cell, hemangioblast and hemogenic endothelium, were first proposed in this model organism. Genetically tractable models have gradually replaced the chick in the past two decades. However, advances in comparative genomics, transcriptomics and promoteromics promise a re-emergence of the chick embryo as a powerful model for hematopoietic/vascular research. This review summarizes the current status of our understanding of early blood/vascular development in the chick, focusing primarily on the processes of primitive hematopoiesis and early vascular network formation in the extraembryonic and lateral plate mesoderm territories. Emphasis is given to ontological and molecular association between the blood and endothelial cells and to the evolutionary relationship between the hemangioblasts, common precursors for the blood and endothelial lineages, and the coelomic epithelial lining cells. Links between early blood/vascular development and later definitive hematopoiesis are also discussed. Finally, potential applications of the chick model for comparative and omics-level studies of the blood/vascular system are highlighted.

Elucidating how body parts from different primordia are integrated during development is essential for understanding the nature of morphological evolution. In tetrapod evolution, while the position of the hindlimb has diversified along with the vertebral formula, the mechanism responsible for this coordination has not been well understood. However, this synchronization suggests the presence of an evolutionarily conserved developmental mechanism that coordinates the positioning of the hindlimb skeleton derived from the lateral plate mesoderm with that of the sacral vertebrae derived from the somites. Here we show that GDF11 secreted from the posterior axial mesoderm is a key factor in the integration of sacral vertebrae and hindlimb positioning by inducing Hox gene expression in two different primordia. Manipulating the onset of GDF11 activity altered the position of the hindlimb in chicken embryos, indicating that the onset of Gdf11 expression is responsible for the coordinated positioning of the sacral vertebrae and hindlimbs. Through comparative analysis with other vertebrate embryos, we also show that each tetrapod species has a unique onset timing of Gdf11 expression, which is tightly correlated with the anteroposterior levels of the hindlimb bud. We conclude that the evolutionary diversity of hindlimb positioning resulted from heterochronic shifts in Gdf11 expression, which led to coordinated shifts in the sacral-hindlimb unit along the anteroposterior axis.

Canonical Wnt/β-catenin (Wnt) signaling plays multiple conserved roles during fate specification of cardiac progenitors in developing vertebrate embryos. Although lineage analysis in ascidians and mice has indicated there is a close relationship between the cardiac second heart field (SHF) and pharyngeal muscle (PM) progenitors, the signals underlying directional fate decisions of the cells within the cardio-pharyngeal muscle field in vertebrates are not yet understood. Here, we examined the temporal requirements of Wnt signaling in cardiac and PM development. In contrast to a previous report in chicken embryos that suggested Wnt inhibits PM development during somitogenesis, we find that in zebrafish embryos Wnt signaling is sufficient to repress PM development during anterior-posterior patterning. Importantly, the temporal sensitivity of dorso-anterior PMs to increased Wnt signaling largely overlaps with when Wnt signaling promotes specification of the adjacent cardiac progenitors. Furthermore, we find that excess early Wnt signaling can cell autonomously promote expansion of the first heart field (FHF) progenitors at the expense of PM and SHF within the anterior lateral plate mesoderm (ALPM). Our study provides insight into an antagonistic developmental mechanism that balances the sizes of the adjacent cardiac and PM progenitor fields in early vertebrate embryos.

Retinoic acid (RA) signaling is a central pathway regulating anterior-posterior patterning of the embryo through its targets, the Hox genes. RA is produced by two sequential oxidations from vitamin A (retinol) and this biosynthesis has to be regulated temporally, spatially and quantitatively. Mining Xenopus embryonic expression databases identified a novel component of the RA metabolic network, ADHFe1. Using Xenopus laevis embryos as our experimental system we determined the temporal and spatial pattern of AdhFe1 expression. Gain- and loss-of-function of ADHFe1 were induced to study its function and the regulation of the AdhFe1 gene by RA was studied. Expression analysis localized the ADHFe1 protein to the late Spemann's organizer, the trunk organizer. Subsequently, ADHFe1 can be detected in the prechordal mesoderm, the notochord and the lateral plate mesoderm. Manipulation of ADHFe1 levels affects the normal Hox gene expression. The effects of ADHFe1 manipulation can by rescued by increasing the levels of RA or its biosynthesis. Expression of the AdhFe1 gene is regulated by RA itself. ADHFe1 is an enzyme active already during gastrula stages and the protein is still present during neurula stages. ADHFe1 regulates the expression of the Hox genes during the early patterning of the trunk. The effect of ADHFe1 on Hox expression is mediated through regulation of RA levels. ADHFe1 probably reduces retinaldehyde to retinol thereby restricting the availability of retinaldehyde, the substrate needed by retinaldehyde dehydrogenases to produce RA making it a novel regulator of RA concentrations in the embryo and RA homeostasis.

The early mammalian embryo is characterized by the presence of three germ layers-the outer ectoderm, middle mesoderm and inner endoderm. The mesoderm is organized into paraxial, intermediate and lateral plate mesoderm. The musculature, vasculature and heart of the adult body are the major derivatives of mesoderm. Tracing back the developmental process to generate these specialized tissues has sparked much interest in the field of regenerative medicine focusing on generating specialized tissues to treat patients with degenerative diseases. Several Long Non-Coding RNAs (lncRNAs) have been identified as regulators of development, proliferation and differentiation of various tissues of mesodermal origin. A better understanding of lncRNAs that can regulate the development of these tissues will open potential avenues for their therapeutic utility and enhance our knowledge about disease progression and development. In this review, we aim to summarize the functions and mechanisms of lncRNAs regulating the early mesoderm differentiation, development and homeostasis of skeletal muscle and cardiovascular system with an emphasis on their therapeutic potential.

The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. At the initiation of tracheal development, endoderm begins to express Nkx2.1, and then mesoderm expresses the Tbx4 gene. Loss of β-catenin in fetal mouse mesoderm causes loss of Tbx4+ tracheal mesoderm and tracheal cartilage agenesis. The mesenchymal Tbx4 expression relies on endodermal Wnt activation and Wnt ligand secretion but is independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generates tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation is required along with WNT to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair., 呼吸器の発生をつかさどるメカニズムの解明 --発生現象の発見に基づくES細胞から気管組織の作出へ--. 京都大学プレスリリース. 2020-09-03.

The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. Loss of β-catenin in fetal mouse mesoderm caused loss of Tbx4+tracheal mesoderm and tracheal cartilage agenesis. The Tbx4 expression relied on endodermal Wnt activity and its downstream Wnt ligand but independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Repopulatingin vivomodel, activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generated tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation was required along with Wnt to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.

Visceral organs, such as the lungs, stomach, liver and pancreas, derive from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) epithelium and the surrounding splanchnic mesoderm (SM) 1,2 . This foregut patterning, which occurs between embryonic day (E) 8.5 and E9.5 in the mouse embryo, equivalent to 17-23 days of human gestation, defines the landscape of the thoracic cavity and disruptions in this process can lead to severe congenital defects. While patterning of the endoderm lineages has been fairly well studies, the SM which is known to provide many paracrine factors required for organogeneis is virtually unstudied 1,2 . In particular we lack a comprehensive understanding of the molecular nature of SM regional identity, the mechanisms by which SM signaling boundaries are established, the role of the epithelium in SM patterning and how SM and DE lineages are dynamically coordinated during organogenesis. Here we used single cell transcriptomics to generate a high-resolution expression map of the embryonic mouse foregut. This uncovered an unexpected diversity in SM progenitors that developed in close register with the organ-specific epithelium. These data allowed us to infer a spatial and temporal signaling roadmap of the combinatorial endoderm-mesoderm interactions that orchestrate foregut organogenesis. We validated key predictions with mouse genetics, showing importance of epithelial signaling in mesoderm patterning. Finally, we leveraged the signaling road map to generate different SM subtypes from human pluripotent stem cells (hPSCs), which previously have been elusive.

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo., Numerous tissues are derived from the lateral plate mesoderm (LPM) but how this is specified is unclear. Here, the authors identify a pan-LPM reporter activity found in the zebrafish draculin (drl) gene that also shows transgenic activity in LPM-corresponding territories of several chordates, including chicken, axolotl, lamprey, Ciona, and amphioxus.

Powered flight was fundamental to the establishment and radiation of birds. However, flight has been lost multiple times throughout avian evolution. Convergent losses of flight within the ratites (flightless paleognaths, including the emu and ostrich) often coincide with reduced wings. Although there is a wealth of anatomical knowledge for several ratites, the genetic mechanisms causing these changes remain debated. Here, we use a multidisciplinary approach employing embryological, genetic, and genomic techniques to interrogate the mechanisms underlying forelimb heterochrony in emu embryos. We show that the initiation of limb formation, an epithelial to mesenchymal transition (EMT) in the lateral plate mesoderm (LPM) and myoblast migration into the LPM, occur at equivalent stages in the emu and chick. However, the emu forelimb fails to subsequently proliferate. The unique emu forelimb expression of Nkx2.5, previously associated with diminished wing development, initiates after this stage (concomitant with myoblast migration into the LPM) and is therefore unlikely to cause this developmental delay. In contrast, RNA sequencing of limb tissue reveals significantly lower Fgf10 expression in the emu forelimb. Artificially increasing Fgf10 expression in the emu LPM induces ectodermal Fgf8 expression and a limb bud. Analyzing open chromatin reveals differentially active regulatory elements near Fgf10 and Sall-1 in the emu wing, and the Sall-1 enhancer activity is dependent on a likely Fgf-mediated Ets transcription factor-binding site. Taken together, our results suggest that regulatory changes result in lower expression of Fgf10 and a concomitant failure to express genes required for limb proliferation in the early emu wing bud.

In vertebrates, two pairs of buds that give rise to the fore- and hindlimbs form at discrete positions along the rostral-caudal axis of the body. The mechanism responsible for the positioning of the limb buds is still largely unknown. Here we show a novel function for Cut homeobox transcription factor 2 (Cux2), the ortholog of Drosophila cut, in refining the forelimb field during chick development. Cux2 is expressed in the forelimb field before the emergence of the limb buds. Knocking down the expression of Cux2 using small interfering RNA (siRNA) resulted in a caudal shift of the forelimb bud, whereas misexpression of Cux2 or the constitutively active Cux2-VP16 caused a rostral shift of the forelimb bud or reduction of the forelimb field along the anterior-posterior axis. Further functional analyses revealed that expression of Hoxb genes and retinaldehyde dehydrogenase 2 (Raldh2), which are involved in limb positioning, are directly activated by Cux2 in the lateral plate mesoderm. Our data suggest that Cux2 in the lateral plate mesoderm refines the forelimb field via regulation of Raldh2 and Hoxb genes in chicken embryos., Summary: Cux2 in the lateral plate mesoderm refines the forelimb field via regulation of Raldh2 and Hoxb genes in chicken embryos.

Summary Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the antero-posterior axis remains largely unknown. Hox genes have long been suspected to control limb position; however, supporting evidences are mostly correlative and their role in this process is unclear. Here, we show that limb position is determined early in development through the action of Hox genes. Dynamic lineage analysis revealed that, during gastrulation, the forelimb, interlimb, and hindlimb fields are progressively generated and concomitantly patterned by the collinear activation of Hox genes in a two-step process. First, the sequential activation of Hoxb genes controls the relative position of their own collinear domains of expression in the forming lateral plate mesoderm, as demonstrated by functional perturbations during gastrulation. Then, within these collinear domains, we show that Hoxb4 anteriorly and Hox9 genes posteriorly, respectively, activate and repress the expression of the forelimb initiation gene Tbx5 and instruct the definitive position of the forelimb. Furthermore, by comparing the dynamics of Hoxb genes activation during zebra finch, chicken, and ostrich gastrulation, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether, our results that characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb positioning in avians show a direct and early role for Hox genes in this process., Graphical Abstract, Highlights • Forelimb, interlimb, and hindlimb domains are sequentially laid during gastrulation • Temporal collinear activation controls Hox sequential expression in these domains • Hox domains differentially instruct (Hox4) or repress (Hox9) limb formation • Changes in collinear activation underlie bird natural variation in limb position, How limbs reproducibly form along the vertebrate body remains largely unknown. Whereas Hox genes have long been suspected to regulate limb position, their role in this process is unclear. Here, Moreau et al. show a direct and early role for Hox genes in the regulation and natural variation of the forelimb position in birds.

Intermediate mesoderm (IM) is the strip of tissue lying between the paraxial mesoderm (PAM) and the lateral plate mesoderm that gives rise to the kidneys and gonads. Chick fate mapping studies suggest that IM is specified shortly after cells leave the primitive streak and that these cells do not require external signals to express IM-specific genes (James and Schultheiss, 2003). Surgical manipulations of the chick embryo, however, revealed that PAM-specific signals are required for IM differentiation into pronephros – the first kidney (Mauch et al., 2000). Here, we use a genetic approach in mice to examine the dependency of IM on proper PAM formation. In Tbx6 null mutant embryos, which form 7–9 improperly patterned anterior somites, IM formation is severely compromised, while in Tbx6 hypomorphic embryos, where somites form but are improperly patterned along the axis, the impact to IM formation is lessened. These results suggest that IM and its derivatives, the kidneys and the gonads, are directly or indirectly dependent on proper PAM formation. This has implications for humans harboring Tbx6 mutations which are known to have somite-derived defects including congenital scoliosis (Sparrow et al., 2013; Wu et al., 2015).

Now published in Current Biology doi: 10.1016/j.cub.2018.11.009; International audience; Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the anteroposterior axis remains largely unknown. Hox genes have long been suspected to control limb position, however supporting evidences are mostly correlative and their role in this process remains unclear. Here we show that Hox genes determine the avian forelimb position in a two-step process: first, their sequential collinear activation during gastrulation controls the relative position of their own successive expression domains along the body axis. Then, within these collinear domains, Hox genes differentially activate or repress the genetic cascade responsible for forelimb initiation. Furthermore, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether our results which characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb position in avians, show a direct and early role for Hox genes in this process.

Precise spatiotemporal expression of the Nodal-Lefty-Pitx2 cascade in the lateral plate mesoderm establishes the left&ndash, right axis, which provides vital cues for correct organ formation and function. Mutations of one cascade constituent PITX2 and, separately, the Forkhead transcription factor FOXC1 independently cause a multi-system disorder known as Axenfeld&ndash, Rieger syndrome (ARS). Since cardiac involvement is an established ARS phenotype and because disrupted left&ndash, right patterning can cause congenital heart defects, we investigated in zebrafish whether foxc1 contributes to organ laterality or situs. We demonstrate that CRISPR/Cas9-generated foxc1a and foxc1b mutants exhibit abnormal cardiac looping and that the prevalence of cardiac situs defects is increased in foxc1a&minus, /&minus, foxc1b&minus, homozygotes. Similarly, double homozygotes exhibit isomerism of the liver and pancreas, which are key features of abnormal gut situs. Placement of the asymmetric visceral organs relative to the midline was also perturbed by mRNA overexpression of foxc1a and foxc1b. In addition, an analysis of the left&ndash, right patterning components, identified in the lateral plate mesoderm of foxc1 mutants, reduced or abolished the expression of the NODAL antagonist lefty2. Together, these data reveal a novel contribution from foxc1 to left&ndash, right patterning, demonstrating that this role is sensitive to foxc1 gene dosage, and provide a plausible mechanism for the incidence of congenital heart defects in Axenfeld&ndash, Rieger syndrome patients.

Vertebrates display a vast array of morphological adaptations to ecological niches. These involve multiple large-scale changes of the appendages, such as evolving limbs from fins, wings (birds, bats), and even the loss of appendages (caecilians, lizards, snakes) during the transition from water to land. Reversals also exist, such as whales evolving back to having fins. This tremendous diversity of appendages leads to development of vertebrate appendage being a model for understanding the evolution and development of appendage morphologies. The development of appendages from buds is conserved among vertebrate. Appendage growth depends on cell proliferation and specializations along proximal-distal (PD), anterior-posterior (AP) and dorsal-ventral (DV) axes. Cell proliferation in lateral plate mesoderm relates to the initiation of buds. The growth of ectodermal cells forms an apical ectodermal ridge along the bud and secreted fibroblast growth factors (Fgfs) control outgrowth of the bud. Gene expression in a specific time and region and levels of expression determines if a bud becomes a fin, wing or limb. Several genes and regulation signal pathways have evolved during transition of fins to alternative types of limbs. Fgfs, Wnts, Shh, RA and Bmps signaling regulate the genes Tbx5 , Tbx4 , Fgf8 , and Bmp and the Hox gene family, respectively, which are essential for appendage development and evolution. These genes associate with chondrocyte proliferation and differentiation. T-box , fgfs and wnts in lateral plate mesoderm are essential for lateral bud initiation, which relates to the transition of ancestral median fins to paired fins. The origin of appendages for the emergence onto land was depended on regions and levels of expression for such as Bmp4 , Hoxd13 and hand2 , which bmps and shh signals regulate, respectively. Hox13 is expressed on the distal margin of fins and limbs buds while the larger-scale region of expression in the limb bud constitutes the basis of the development of the PD axis in terrestrial vertebrates. Loss of Hand2 leads to the deficiency of hind-limbs in whales. High levels of expression of Bmps in the forelimb of bats lead the forelimb to be longer than the hind-one. Furthermore, not only signaling regulation, but also transcription factors, regions of genome regulation and some long non-coding RNAs (lncRNAs) play regulatory roles in gene expression during appendage development.

Aquaporin 8 (Aqp8) is a transmembrane protein that is selectively permeated by water and some small solutes, and physiologically contributes to acid-base equilibrium in the gastrointestinal tract. Here, we described the characterization and spatiotemporal expression pattern of zebrafish aqp8 (zaqp8) gene family, including zaqp8a.1, zaqp8a.2, and zaqp8b, during the early developmental stages. The expression of zaqp8a.1 started first in the lateral plate mesoderm at the 12-somite stage (ss) and then expanded sequentially to the dorsal aorta, intersegmental blood vessels and then to the dorsal longitudinal anastomotic vessel at 24 h post fertilization (hpf). At 28 hpf, expression of zaqp8a.1 was also detected in the embryonic heart tube. Four days post fertilization (dpf), strong zaqp8a.1 expression was detected in the gastrointestinal tract and liver. By 72 hpf, the expression of zaqp8a.2 was first detected in the primitive gut region but not detected in the liver. The expression of zaqp8b was first detected in the intermediate mesoderm at 10 ss. From 24 hpf to 6 dpf, the proximal convoluted segment of the embryonic kidney was marked by zaqp8b expression Overall, these differential expression patterns of aqp8a.1, aqp8a.2, and aqp8b suggest that they possibly play distinct roles throughout the embryonic development by controlling or maintaining organ-specific cellular water homeostasis. Our study provides new evidence that organogenesis requires differential roles of Aqp8 proteins in zebrafish.

Nodal activity in the left lateral plate mesoderm is a conserved sign of irreversible left-right asymmetry at early somite stages of the vertebrate embryo. An earlier, paraxial nodal domain accompanies the emergence and initial extension of the notochord and is either left-sided, as in the chick and pig, or symmetrical, as in the mouse and rabbit; intriguingly, this interspecific dichotomy is mirrored by divergent morphological features of the posterior notochord (also known as the left-right organizer), which is ventrally exposed to the yolk sac cavity and carries motile cilia in the latter 2 species only. By introducing the cattle embryo as a new model organism for early left-right patterning, we present data to establish 2 groups of mammals characterized by both the morphology of the left-right organizer and the dynamics of paraxial nodal expression: presence and absence of a ventrally open surface of the early (plate-like) posterior notochord correlates with a symmetrical (in mice and rabbits) versus an asymmetrical (in pigs and cattle) paraxial nodal expression domain next to the notochordal plate. High-resolution histological analysis reveals that the latter domain defines in all 4 mammals a novel ‘parachordal' axial mesoderm compartment, the topography of which changes according to the specific regression of the similarly novel subchordal mesoderm during the initial phases of notochord development. In conclusion, the mammalian axial mesoderm compartment (1) shares critical conserved features despite the marked differences in early notochord morphology and early left-right patterning and (2) provides a dynamic topographical framework for nodal activity as part of the mammalian left-right organizer.

The limbs are a significant evolutionary innovation that enabled vertebrates to diversify and colonise new environments. Tetrapods have two pairs of limbs, forelimbs in the upper body and hindlimbs in the lower body. The morphologies of the forelimbs and hindlimbs are distinct, reflecting their specific locomotory functions although they share many common signalling networks that regulate their development. The paired appendages in vertebrates form at fixed positions along the rostral-caudal axis and this occurs as a consequence of earlier subdivision of the lateral plate mesoderm (LPM) into regions with distinct limb forming potential. In this review, we discuss the molecular mechanisms that confer a broad region of the flank with limb-forming potential and its subsequent refinement into distinct forelimb-forming, hindlimb-forming and interlimb territories.

The Brachyury gene encodes a transcription factor that is conserved across all animals. In non-chordate metazoans, brachyury is primarily expressed in ectoderm regions that are added to the endodermal gut during development, and often form a ring around the site of endoderm internalization in the gastrula, the blastopore. In chordates, this brachyury ring is conserved, but the gene has taken on a new role in the formation of the mesoderm. In this phylum, a novel type of mesoderm that develops into notochord and somites has been added to the ancestral lateral plate mesoderm. Brachyury contributes to a shift in cell fate from neural ectoderm to posterior notochord and somites during a major lineage segregation event that in Xenopus and in the zebrafish takes place in the early gastrula. In the absence of this brachyury function, impaired formation of posterior mesoderm indirectly affects the gastrulation movements of peak involution and convergent extension. These movements are confined to specific regions and stages, leaving open the question why brachyury expression in an extensive, coherent ring, before, during and after gastrulation, is conserved in the two species whose gastrulation modes differ considerably, and also in many other metazoan gastrulae of diverse structure.

The TGF-beta signal Nodal is the major effector of left-right axis development. In mice, Nodal forms heterodimers with Gdf1 and is restricted to the left side by Cerl2/Dand5. Nodal expression then propagates up the lateral plate mesoderm (LPM) by autoinduction, while Lefty1 inhibition maintains it on the left. Studies in zebrafish have suggested some parallels, but also differences, between the modes of left-right patterning in mouse and zebrafish. To address these discrepancies, we generated single and double zebrafish mutants for spaw (the Nodal ortholog), dand5 (the Cerl2 ortholog) and lefty1, and performed biochemical and activity assays with Spaw and Vg1/Gdf3 (the Gdf1 ortholog). Contrary to previous findings, spaw mutants failed to initiate spaw expression in the LPM, and asymmetric heart looping was absent, similar to mouse Nodal mutants. In blastoderm assays, Vg1 and Spaw were interdependent for inducing target gene expression, and contrary to previous results, formed heterodimers. Loss of Dand5 or Lefty1 caused bilateral spaw expression, similar to mouse mutants, and premature expression and propagation of spaw in the LPM. Finally, Lefty1 inhibition of Nodal activity could be replaced by uniform exposure to a Nodal signaling inhibitor. Collectively, these results indicate that Dand5 activity biases Spaw-Vg1 heterodimer activity to the left, Spaw around Kupffer's vesicle induces the expression of spaw in the LPM, and global Nodal inhibition maintains the left bias of Spaw activity, demonstrating conservation between zebrafish and mouse mechanisms of left-right patterning.

In vertebrates, head and trunk muscles develop from different mesodermal populations and are regulated by distinct genetic networks. Neck muscles at the head-trunk interface remain poorly defined due to their complex morphogenesis and dual mesodermal origins. Here, we use genetically modified mice to establish a 3D model that integrates regulatory genes, cell populations and morphogenetic events that define this transition zone. We show that the evolutionary conserved cucullaris-derived muscles originate from posterior cardiopharyngeal mesoderm, not lateral plate mesoderm, and we define new boundaries for neural crest and mesodermal contributions to neck connective tissue. Furthermore, lineage studies and functional analysis ofTbx1-andPax3-null mice reveal a unique genetic program for somitic neck muscles that is distinct from that of somitic trunk muscles. Our findings unveil the embryological and developmental requirements underlying tetrapod neck myogenesis and provide a blueprint to investigate how muscle subsets are selectively affected in some human myopathies.

Pellino proteins are associated with immune and stress responses through their effects on NF-κB signaling and B-cell development, and through their role as a scaffold in TLR/IL-1R signaling pathways. However, their function during embryonic development is unclear. Here, we report the developmental expression patterns and functions of peli1b, which encodes a zebrafish ortholog of human Pellino1. Maternal peli1b transcripts were present in zebrafish embryos at the 1-cell stage and zygotic transcripts appeared in the shield area at 6 hours post fertilization (hpf), particularly in the neural plate of the dorsal region. peli1b transcripts were concentrated in the somites, lens, myogenic cells, lateral plate mesoderm, and presomitic mesoderm at 12 hpf, but expression shifted to the telencephalon, diencephalon, hindbrain, and rhombomeres (r1–7) at 24 hpf. Distribution of peli1b transcripts was further restricted to the telencephalon, diencephalon, hindbrain, eyes, and pectoral fins at 48 hpf. Knock-down of peli1b with a peli1b antisense morpholino resulted in significant developmental defects and a reduction in size of the telencephalon, diencephalon, rhombomeres (r1–7), and spinal cord at 24 hpf. When peli1b-knock-down embryos were analyzed for zic3, a marker associated with the central nervous system, we found lower levels of zic3 transcripts in the shield area at 6 hpf and in the posterior diencephalon, dorsal neural plate, midbrain, and hindbrain at 14 hpf. Finally, the ERK3/4 inhibitor SB203580 also induced a significant reduction in the level of zic3 transcripts in the neural plate at 6 hpf and in the posterior diencephalon, dorsal neural plate, midbrain, hindbrain, segmental plate, dorsal spinal cord, and dorsal posterior neural plate at 14 hpf. It is thus likely that the association between Peli1b and brain development in zebrafish embryos occurs via ERK3/4 pathways.

The formation of somites, coelomic sacs, splanchnic mesoderm, fat bodies, circular system, gonads, and musculature in the embryo of the primitive moth, Neomicropteryx nipponensis Issiki, is described. The following paired somites are formed: the labral, antennal, intercalary, mandibular, maxillary, labial, three thoracic, and 11 abdominal. Small but distinct coelomic cavities appear in all these somites. Labral somites differentiate into the labral muscles, stomodaeal muscles, and dorsal dilator muscles of the pharynx. Antennal somites differentiate into the antennal muscles, aorta, and the ventral dilator muscles of the pharynx. Intercalary somites are short-lived, disintegrating to liberate many free cells into the yolk. The suboesophageal body is not formed. Mandibular somites differentiate into the mandibular flexor and extensor muscles. Maxillary and labial somites differentiate into the splanchnic mesoderm, fat bodies, and into muscles of the maxillolabial region. Three pairs of thoracic and ten pairs of abdominal somites split into the splanchric and somatic mesoderm. The 11th abdominal somites merge into the proctodaeal mesoderm, and differentiate into the musculature of the hindgut. The heart is formed by the fusion of the cardioblasts derived from the first thoracic to the tenth abdominal segment. The aorta arises from the antennal median mesoderm. Blood cells are derived from the median mesodermal cells of the maxillary to the tenth abdominal segment. Germ cells appear at the mediodorsal corner of each somite in the fifth abdominal segment. They become enclosed with a mesodermal sheath to form a pair of rudimentary gonads in this segment. Major muscles in the head, thorax, and abdomen of the fully grown embryo are described.

Hippo pathway regulates cell proliferation and differentiation. Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz) are effectors of Hippo pathway. The function of Yap/Taz in embryonic liver development has yet to be reported. Here yap1 and taz were found expressed in liver and other digestive organs in zebrafish embryos, and knockout of yap1 or taz did not lead to visible defects during embryogenesis. Interestingly, Taz was significantly increased in yap1 mutants, which may account for their normal development, albeit losing Yap1. However, yap1−/−; taz+/- embryos exhibited smaller digestive organs, and more than half of them showed bilateral livers and pancreas and non-looped intestines. Further analysis revealed that the disrupted gene function in yap1−/−; taz+/- embryos did not disturb liver bud formation, but instead impaired cell proliferation in liver and movement of the neighboring lateral plate mesoderm (LPM). Overexpression of wild type yap1 or taz could rescue the defective liver phenotypes in yap1−/−; taz+/- embryos, indicating that Yap1 cooperate with Taz to regulate the liver development. In addition, Yap1 was found to function in a Tead-dependent manner in the liver development. Our results suggest that Yap1/Taz regulate LPM movement and promote cell proliferation to ensure proper liver development in zebrafish.

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolated a pan-LPM enhancer in the zebrafishdraculin(drl) gene that provides specific LPM reporter activity from early gastrulation.In totolive imaging and lineage tracing ofdrl-based reporters captured the dynamic LPM emergence as lineage-restricted mesendoderm field. Thedrlpan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncovered specificdrlreporter activity in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey,Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.

TBX5 is essential for limb and heart development. Mutations in TBX5 are associated with Holt-Oram syndrome in humans. Due to the teleost specific genome duplication, zebrafish have two copies of TBX5: tbx5a and tbx5b. Both of these genes are expressed in regions of the lateral plate mesoderm and retina. In this study, we perform comparative RNA sequencing analysis on zebrafish embryos during the stages of lateral plate mesoderm migration. This work shows that knockdown of the Tbx5 paralogues results in altered gene expression in many tissues outside of the lateral plate mesoderm, especially in the somitic mesoderm and the intermediate mesoderm. Specifically, knockdown of tbx5b results in changes in somite size, in the differentiation of vasculature progenitors and in later patterning of trunk blood vessels.

Mesodermal populations can be generated in vitro from mouse embryonic stem cells (mESCs) using three-dimensional (3-D) aggregates called embryoid bodies or two-dimensional (2-D) monolayer culture systems. Here, we investigated whether Brachyury-expressing mesodermal cells generated using 3-D or 2-D culture systems are equivalent or, instead, have different properties. Using a Brachyury-GFP/E2-Crimson reporter mESC line, we isolated Brachyury-GFP+ mesoderm cells using flow-activated cell sorting and compared their gene expression profiles and ex vivo differentiation patterns. Quantitative real-time polymerase chain reaction analysis showed significant up-regulation of Cdx2, Foxf1 and Hoxb1 in the Brachyury-GFP+ cells isolated from the 3-D system compared with those isolated from the 2-D system. Furthermore, using an ex vivo mouse kidney rudiment assay, we found that, irrespective of their source, Brachyury-GFP+ cells failed to integrate into developing nephrons, which are derived from the intermediate mesoderm. However, Brachyury-GFP+ cells isolated under 3-D conditions appeared to differentiate into endothelial-like cells within the kidney rudiments, whereas the Brachyury-GFP+ isolated from the 2-D conditions only did so to a limited degree. The high expression of Foxf1 in the 3-D Brachyury-GFP+ cells combined with their tendency to differentiate into endothelial-like cells suggests that these mesodermal cells may represent lateral plate mesoderm., Summary: Brachyury-expressing mesoderm populations were generated from an E2-Crimson-expressing mESC reporter line using 3-D and 2-D culture systems and their differentiation potential was assessed following incorporation into mouse kidney rudiments.

Mesodermal populations can be generatedin vitrofrom mouse embryonic stem cells (mESCs) using three-dimensional (3-D) aggregates called embryoid bodies or two-dimensional (2-D) monolayer culture systems. Here, we investigated whetherBrachyury-expressing mesodermal cells generated using 3-D or 2-D culture systems are equivalent, or instead, have different properties. Using aBrachyury-GFP/E2-Crimson reporter mESC line, we isolatedBrachyury-GFP+mesoderm cells using flow-activated cell sorting and compared their gene expression profiles andex vivodifferentiation patterns. Quantitative RT-PCR analysis showed significant up-regulation ofCdx2,Foxf1andHoxb1in theBrachyury-GFP+cells isolated from the 3-D system compared with those isolated from the 2-D system. Furthermore, using an ex vivomouse kidney rudiment assay, we found that irrespective of their source,Brachyury-GFP+cells failed to integrate into developing nephrons, which are derived from the intermediate mesoderm. However,Brachyury-GFP+cells isolated under 3-D conditions appeared to differentiate into endothelial-like cells within the kidney rudiments, whereas theBrachyury-GFP+isolated from the 2-D conditions only did so to a limited degree. The high expression ofFoxf1in the 3-DBrachyury-GFP+cells combined with their tendency to differentiate into endothelial-like cells suggests these mesodermal cells may represent lateral plate mesoderm.

Vertebrate embryonic patterning depends on signaling from Nodal, a TGFβ superfamily member. There are three Nodal orthologs in zebrafish; southpaw directs left-right asymmetries, while squint and cyclops function earlier to pattern mesendoderm. TGFβ member Vg1 is implicated in mesoderm formation but the role of the zebrafish ortholog, Growth differentiation factor 3 (Gdf3), has not been fully explored. We show that zygotic expression of gdf3 is dispensable for embryonic development, while maternally deposited gdf3 is required for mesendoderm formation and dorsal-ventral patterning. We further show that Gdf3 can affect left-right patterning at multiple stages, including proper development of regional cell morphology in Kupffer’s vesicle and the establishment of southpaw expression in the lateral plate mesoderm. Collectively, our data indicate that gdf3 is critical for robust Nodal signaling at multiple stages in zebrafish embryonic development.