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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.

Cohesin is a multiprotein complex made up of core subunits Smc1, Smc3, and Rad21, and either Stag1 or Stag2. Normal haematopoietic development relies on crucial functions of cohesin in cell division and regulation of gene expression via three-dimensional chromatin organization. Cohesin subunit STAG2 is frequently mutated in myeloid malignancies, but the individual contributions of Stag variants to haematopoiesis or malignancy are not fully understood. Zebrafish have four Stag paralogues (Stag1a, Stag1b, Stag2a, and Stag2b), allowing detailed genetic dissection of the contribution of Stag1-cohesin and Stag2-cohesin to development. Here we characterize for the first time the expression patterns and functions of zebrafish stag genes during embryogenesis. Using loss-of-function CRISPR-Cas9 zebrafish mutants, we show that stag1a and stag2b contribute to primitive embryonic haematopoiesis. Both stag1a and stag2b mutants present with erythropenia by 24 h post-fertilization. Homozygous loss of either paralogue alters the number of haematopoietic/vascular progenitors in the lateral plate mesoderm. The lateral plate mesoderm zone of scl-positive cells is expanded in stag1a mutants with concomitant loss of kidney progenitors, and the number of spi1-positive cells are increased, consistent with skewing toward primitive myelopoiesis. In contrast, stag2b mutants have reduced haematopoietic/vascular mesoderm and downregulation of primitive erythropoiesis. Our results suggest that Stag1 and Stag2 proteins cooperate to balance the production of primitive haematopoietic/vascular progenitors from mesoderm.

FUNDING This work was supported by the FCT – Fundação para a Ciência e a Tecnologia, I.P. (PTDC/BTM-SAL/29377/2017) and iNOVA4Health (UID/Multi/04462/2019). The Advanced Imaging Facility of the Instituto Gulbenkian de Ciência was supported by national Portuguese funding co-financed by the Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and FCT (PPBI-POCI-01- 0145-FEDER-022122). AT was funded by the FCT in the context of a program contract under “Norma Transitória” (DL57/2016 of 29 August, as amended by DL57/2017 of 19 July). During early embryogenesis, the hemogenic endothelium of the developing dorsal aorta is the main source of definitive hematopoietic stem cells (HSCs), which will generate all blood cell lineages of the adult organism. The hemogenic endothelial cells (HECs) of the dorsal aorta are known to arise from the splanchnic lateral plate mesoderm. However, the specific cell lineages and developmental paths that give rise to aortic HECs are still unclear. Over the past half a century, the scientific debate on the origin of aortic HECs and HSCs has largely focused on two potential and apparently alternative birthplaces, the extraembryonic yolk sac blood islands and the intraembryonic splanchnic mesoderm. However, as we argue, both yolk sac blood islands and aortic HECs may have a common hemangioblastic origin. Further insight into aortic HEC development is being gained from fate-mapping studies that address the identity of progenitor cell lineages, rather than their physical location within the developing embryo. In this perspective article, we discuss the current knowledge on the origin of aortic HECs with a particular focus on the evidence provided by studies in the avian embryo, a model that pioneered the field of developmental hematopoiesis. publishersversion published

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.

Positional information driving limb muscle patterning is contained in lateral plate mesoderm-derived tissues, such as tendon or muscle connective tissue but not in myogenic cells themselves. The long-standing consensus is that myogenic cells originate from the somitic mesoderm, while connective tissue fibroblasts originate from the lateral plate mesoderm. We challenged this model using cell and genetic lineage tracing experiments in birds and mice, respectively, and identified a subpopulation of myogenic cells at the muscle tips close to tendons originating from the lateral plate mesoderm and derived from connective tissue gene lineages. Analysis of single-cell RNA-sequencing data obtained from limb cells at successive developmental stages revealed a subpopulation of cells displaying a dual muscle and connective tissue signature, in addition to independent muscle and connective tissue populations. Active BMP signalling was detected in this junctional cell sub-population and at the tendon/muscle interface in developing limbs. BMP gain- and loss-of-function experiments performedin vivoandin vitroshowed that this signalling pathway regulated a fibroblast-to-myoblast conversion. We propose that localised BMP signalling converts a subset of lateral plate mesoderm-derived fibroblasts to a myogenic fate and establishes a boundary of fibroblast-derived myonuclei at the muscle/tendon interface to control the muscle pattern during limb development.

Endothelial cells appear to emerge from diverse progenitors. However, to which extent their developmental origin contributes to define their cellular and molecular characteristics remains largely unknown. Here, we report that a subset of endothelial cells that emerge from the tailbud possess unique molecular characteristics that set them apart from stereotypical lateral plate mesoderm (LPM)-derived endothelial cells. Lineage tracing shows that these tailbud-derived endothelial cells arise at mid-somitogenesis stages, and surprisingly do not require Npas4l or Etsrp function, indicating that they have distinct spatiotemporal origins and are regulated by distinct molecular mechanisms. Microarray and single cell RNA-seq analyses reveal that somitogenesis- and neurogenesis-associated transcripts are over-represented in these tailbud-derived endothelial cells, suggesting that they possess a unique transcriptomic signature. Taken together, our results further reveal the diversity of endothelial cells with respect to their developmental origin and molecular properties, and provide compelling evidence that the molecular characteristics of endothelial cells may reflect their distinct developmental history.

Cohesin is a multiprotein complex made up of core subunits Smc1, Smc3 and Rad21, and either Stag1 or Stag2. Normal haematopoietic development relies on crucial functions of cohesin in cell division and regulation of gene expression via three-dimensional chromatin organisation. Cohesin subunit STAG2 is frequently mutated in myeloid malignancies, but the individual contributions of Stag variants to haematopoiesis or malignancy are not fully understood. Zebrafish have four Stag paralogues (Stag1a, Stag1b, Stag2a and Stag2b), allowing detailed genetic dissection of the contribution of Stag1-cohesin and Stag2-cohesin to development. Here we characterize for the first time the expression patterns and functions of zebrafish stag genes during embryogenesis. Using loss-of-function CRISPR-Cas9 zebrafish mutants, we show that stag1a and stag2b contribute to primitive embryonic haematopoiesis. Both stag1a and stag2b mutants present with erythropenia by 24 hours post-fertilisation. Homozygous loss of either paralog alters the number of haematopoietic/vascular progenitors in the lateral plate mesoderm. The lateral plate mesoderm zone of scl-positive cells is expanded in stag1a mutants with concomitant loss of kidney progenitors, and the number of spi1-positive cells are increased, consistent with skewing toward primitive myelopoiesis. In contrast, stag2b mutants have reduced haematopoietic/vascular mesoderm and downregulation of primitive erythropoiesis. Our results suggest that Stag1 and Stag2 proteins cooperate to balance the production of primitive haematopoietic/vascular progenitors from mesoderm.

Skeletal elements have a diverse range of shapes and sizes specialized to their various roles including protecting internal organs, locomotion, feeding, hearing, and vocalization. The precise positioning, size, and shape of skeletal elements is therefore critical for their function. During embryonic development, bone forms by endochondral or intramembranous ossification and can arise from the paraxial and lateral plate mesoderm or neural crest. This review describes inductive mechanisms to position and pattern bones within the developing embryo, compares and contrasts the intrinsic vs extrinsic mechanisms of endochondral and intramembranous skeletal development, and details known cellular processes that precisely determine skeletal shape and size. Key cellular mechanisms are employed at distinct stages of ossification, many of which occur in response to mechanical cues (eg, joint formation) or preempting future load‐bearing requirements. Rapid shape changes occur during cellular condensation and template establishment. Specialized cellular behaviors, such as chondrocyte hypertrophy in endochondral bone and secondary cartilage on intramembranous bones, also dramatically change template shape. Once ossification is complete, bone shape undergoes functional adaptation through (re)modeling. We also highlight how alterations in these cellular processes contribute to evolutionary change and how differences in the embryonic origin of bones can influence postnatal bone repair., Key Findings Compares and contrasts Endochondral and intramembranous bone developmentReviews embryonic origins of different bonesDescribes the cellular and molecular mechanisms of positioning skeletal elements.Describes mechanisms of skeletal growth with a focus on the generation of skeletal shape

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.

The dermis has disparate embryonic origins; abdominal dermis develops from lateral plate mesoderm, dorsal dermis from paraxial mesoderm and facial dermis from neural crest. However, the cell and molecular differences and their functional implications have not been described. We hypothesise that the embryonic origin of the dermis underpins regional characteristics of skin, including its response to wounding. We have compared abdomen, back and cheek, three anatomical sites representing the distinct embryonic tissues from which the dermis can arise, during homeostasis and wound repair using RNA sequencing, histology and fibroblast cultures. Our transcriptional analyses demonstrate differences between body sites that reflect their diverse origins. Moreover, we report histological and transcriptional variations during a wound response, including site differences in ECM composition, cell migration and proliferation, and re‐enactment of distinct developmental programmes. These findings reveal profound regional variation in the mechanisms of tissue repair. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

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.

Congenital tracheomalacia, resulting from incomplete tracheal cartilage development, is a relatively common birth defect that severely impairs breathing in neonates. Mutations in the Hedgehog (HH) pathway and downstream Gli transcription factors are associated with tracheomalacia in patients and mouse models; however, the underlying molecular mechanisms are unclear. Using multipleHH/Glimouse mutants including one that mimics Pallister-Hall Syndrome, we show that excessive Gli repressor activity prevents specification of tracheal chondrocytes. Lineage tracing experiments show that Sox9+ chondrocytes arise from HH-responsive splanchnic mesoderm in the fetal foregut that expresses the transcription factor Foxf1. Disrupted HH/Gli signaling results in 1) loss of Foxf1 which in turn is required to support Sox9+ chondrocyte progenitors and 2) a dramatic reduction inRspo2, a secreted ligand that potentiates Wnt signaling known to be required for chondrogenesis. These results reveal a HH-Foxf1-Rspo2 signaling axis that governs tracheal cartilage development and informs the etiology of tracheomalacia.SUMMARY STATEMENTThis work provides a mechanistic basis for tracheomalacia in patients with Hedgehog pathway mutations.

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.

The lateral plate mesoderm (LPM) forms the progenitor cells that constitute the heart and cardiovascular system, blood, kidneys, smooth muscle lineage and limb skeleton in the developing vertebrate embryo. Despite this central role in development and evolution, the LPM remains challenging to study and to delineate, owing to its lineage complexity and lack of a concise genetic definition. Here, we outline the processes that govern LPM specification, organization, its cell fates and the inferred evolutionary trajectories of LPM-derived tissues. Finally, we discuss the development of seemingly disparate organ systems that share a common LPM origin., Summary: The lateral plate mesoderm is the origin of several major cell types and organ systems in the vertebrate body plan. How this mesoderm territory emerges and partitions into its downstream fates provides clues about vertebrate development and evolution.

Normal body situs requires precise spatiotemporal expression of theNodal-Lefty-Pitx2cascade in the lateral plate mesoderm. The ultimate output of this patterning is establishment of the left-right axis, which provides vital cues for correct organ formation and function. Mutations, deletions and duplications inPITX2andFOXC1lead to the rare genetic disease Axenfeld-Rieger syndrome (ARS). While situs defects are not a recognised feature of ARS, partial penetrance of cardiac septal defects and valve incompetence is observed; both of these congenital heart defects (CHDs) also occur following disruption of left-right patterning. Here we investigated whetherfoxc1genes have a critical role in specifying organ situs. We demonstrate that CRISPR/Cas9 generated mutants for the zebrafish paralogsfoxc1aandfoxc1brecapitulate ARS phenotypes including craniofacial dysmorphism, hydrocephalus and intracranial haemorrhage. Furthermore,foxc1a-/-;foxc1b-/-mutant animals display cardiac and gut situs defects. ModellingFOXC1duplication by transient mRNA overexpression revealed that increasedfoxc1dosage also results in organ situs defects. Analysis of known left-right patterning genes revealed a loss in expression of theNODALantagonistlefty2in the lateral plate mesoderm. Consistently,LEFTY2mutations are known to cause human cardiac situs defects. Our data reveal a novel role for the forkhead-box transcription factorfoxc1in patterning of the left-right axis, and provide a plausible mechanism for the incidence of congenital heart defects in Axenfeld-Rieger syndrome patients.Author SummaryThis manuscript investigates the functional consequences of abrogating the activity of Foxc1 (Forkhead Box C1). We demonstrate that loss of zebrafishfoxc1aandfoxc1bresults in phenotypes that resemble human patients with deletions in theFOXC1locus. Notably, such phenotypes include alterations to the morphology of the heart. Investigations into the mechanisms underlying this phenotype led to the discovery that Foxc1 functions as a regulator of left-right patterning. Most components of left-right specification function normally infoxc1a/bmutants, but there is a pronounced loss oflefty2, a known inhibitor of Nodal signaling. This supports a model in which Foxc1 regulates situs of the heart via the regulation of Lefty2.

SummaryVertebrates show conserved left-right (L-R) asymmetry of internal organs controlled by Nodal-Pitx2/Lefty signaling [1-3]. Modifications in L-R asymmetry occur in mutants [4] and rarely in humans [5], but little is known about natural L-R changes during evolution. Here we describe changes in L-R asymmetry inAstyanax mexicanus, a teleost with ancestral surface (surface fish) and derived cave (cavefish) morphs [6]. In teleosts, Nodal-Pitx2 signaling is activated in the left lateral plate mesoderm (LPM), the cardiac tube jogs to the left and loops to the right (D-looping), and the liver and pancreas form on opposite sides of the midline. Surface fish show conventional L-R patterning, but cavefish can show Nodal-Pitx2 expression in the right LPM or bilaterally, left (L)-looping hearts, and reversed liver and pancreas asymmetry, and these reversals have no effect on survival. The Lefty1 Nodal antagonist is expressed along the surface fish and cavefish midlines, but expression of the Lefty2 antagonist is absent in the LPM of most cavefish embryos, suggesting a role forlefty2(lft2) in changing organ asymmetry. Although CRISPR-Cas9lft2editing affected D-looping in surface fish, the cavefishlft2gene showed no coding mutations, and was expressed normally during cavefish gastrulation, suggesting downregulation by regulatory changes. Reciprocal hybridization, the fertilization of cavefish eggs with surface fish sperm andvice versa, indicated that the change in cavefish L-R asymmetry is a maternal genetic effect. Our studies reveal natural changes in internal organ asymmetry during evolution and introduceA. mexicanusas a new model to study the underlying mechanisms.

Development of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from ventral aortic endothelium in all vertebrate animals studied. Work in zebrafish has demonstrated that arterial and venous endothelial precursors arise from distinct subsets of lateral plate mesoderm. Earlier studies in the chick showed that paraxial mesoderm generates another subset of endothelial cells that incorporate into the dorsal aorta to replace HSPCs as they exit the aorta and enter circulation. Here we show that a similar process occurs in the zebrafish, where a population of endothelial precursors delaminates from the somitic dermomyotome to incorporate exclusively into the developing dorsal aorta. Whereas somite-derived endothelial cells (SDECs) lack hematopoietic potential, they act as local niche to support the emergence of HSPCs from neighboring hemogenic endothelium. Thus, at least three subsets of endothelial cells (ECs) contribute to the developing dorsal aorta: vascular ECs, hemogenic ECs, and SDECs. Taken together, our findings indicate that the distinct spatial origins of endothelial precursors dictate different cellular potentials within the developing dorsal aorta.

ATF3 provides cardiac and hematopoietic progenitors with metabolic adaptive capacity for protection of differentiation potential., The cardiac and hematopoietic progenitors (CPs and HPs, respectively) in the mesoderm ultimately form a well-organized circulation system, but mechanisms that reconcile their development remain elusive. We found that activating transcription factor 3 (atf3) was highly expressed in the CPs, HPs, and mesoderm, in zebrafish. The atf3−/− mutants exhibited atrial dilated cardiomyopathy and a high ratio of immature myeloid cells. These manifestations were primarily caused by the blockade of differentiation of both CPs and HPs within the anterior lateral plate mesoderm. Mechanistically, Atf3 targets cebpγ to repress slc2a1a-mediated glucose utilization. The high rate of glucose metabolism in atf3−/− mutants inhibited the differentiation of progenitors by changing the redox state. Therefore, atf3 could provide CPs and HPs with metabolic adaptive capacity to changes in glucose levels. Our study provides new insights into the role of atf3 in the coordination of differentiation of CPs and HPs by regulating glucose metabolism.

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues., Highlighted Article: Analysis of conditional and temporal knockout mice reveals roles of Ebf3 in the differentiation of lateral plate mesenchymal cells towards pre-osteoblasts during sternum ossification at the boundary of the lateral and somitic mesoderm.

In zebrafish, cilia movement within the Kupffer's vesicle (KV) generates a fluid flow responsible for accumulating nodal signals exclusively in the left lateral plate mesoderm, thereby initiating left-right patterning (LRP). Defects in LRP cause devastating congenital disorders including congenital heart malformations due to organ mis-positioning. We identified the miR-103/107 family to be involved in regulating LRP. Depletion of miR-103/107 in zebrafish embryos resulted in malpositioned and malformed visceral organs and hearts due to disturbed LRP gene expression, indicating early defects in LRP. Additionally, loss of miR-103/107 affected KV morphogenesis and cilia formation without disturbing endoderm development. Human fibroblasts depleted of miR-103a/107 often failed to extend cilia or developed shorter cilia, indicating functional conservation between species. We identified arl6, araf and foxH1 as direct targets of miR-103/107 providing a mechanistic link to cilia development and nodal signal titration. We describe a new microRNA family controlling KV development and hence influencing establishment of internal organ asymmetry.

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. How the flow signal is processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal drives LPM Nodal cascade induction. Here, we identify the mechanism of dand5 downregulation, finding that its posttranscriptional repression is a central process in symmetry breaking. Specifically, the RNA binding protein Bicc1 interacts with a proximal element in the 3’-UTR of dand5 to repress translation in a dicer1-dependent manner. The bicc1/dicer1 module acts downstream of flow, as LRO ciliation was not affected upon its loss. Loss of bicc1 or dicer1 was rescued by parallel knockdown of dand5, placing both genes in the process of flow sensing.

Background Fgf10 is expressed in various tissues and organs, such as the limb bud, heart, inner ear, and head mesenchyme. Previous studies identified Fgf10 enhancers for the inner ear and heart. However, Fgf10 enhancers for other tissues have not been identified. Results By using primary culture chick embryo lateral plate mesoderm cells, we compared activities of deletion constructs of the Fgf10 promoter region, cloned into a promoter-less luciferase reporter vector. We identified a 0.34-kb proximal promoter that can activate luciferase expression. Then, we cloned 11 evolutionarily conserved sequences located within or outside of the Fgf10 gene into the 0.34-kb promoter-luciferase vector, and tested their activities in vitro using primary cultured cells. Two sequences showed the highest activities. By using the Tol2 system and electroporation into chick embryos, activities of the 0.34-kb promoter with and without the two sequences were tested in vivo. No activities were detected in limb buds. However, the 0.34-kb promoter exhibited activities in the dorsal midline of the brain, while Fgf10 is detected in broader region in the brain. The two noncoding sequences negatively acted on the 0.34-kb promoter in the brain. Conclusions The proximal 0.34-kb promoter has activities to drive expression in restricted areas of the brain. Developmental Dynamics 247:1253-1263, 2018. © 2018 Wiley Periodicals, Inc.

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.

BACKGROUND Spatiotemporal perturbation of signaling pathways in vivo remains challenging and requires precise transgenic control of signaling effectors. Fibroblast growth factor (FGF) signaling guides multiple developmental processes, including body axis formation and cell fate patterning. In zebrafish, mutants and chemical perturbations affecting FGF signaling have uncovered key developmental processes; however, these approaches cause embryo-wide perturbations, rendering assessment of cell-autonomous vs. non-autonomous requirements for FGF signaling in individual processes difficult. RESULTS Here, we created the novel transgenic line fgfr1-dn-cargo, encoding dominant-negative Fgfr1a with fluorescent tag under combined Cre/lox and heatshock control to perturb FGF signaling spatiotemporally. Validating efficient perturbation of FGF signaling by fgfr1-dn-cargo primed with ubiquitous CreERT2, we established that primed, heatshock-induced fgfr1-dn-cargo behaves similarly to pulsed treatment with the FGFR inhibitor SU5402. Priming fgfr1-dn-cargo with CreERT2 in the lateral plate mesoderm triggered selective cardiac and pectoral fin phenotypes without drastic impact on overall embryo patterning. Harnessing lateral plate mesoderm-specific FGF inhibition, we recapitulated the cell-autonomous and temporal requirement for FGF signaling in pectoral fin outgrowth, as previously inferred from pan-embryonic FGF inhibition. CONCLUSIONS As a paradigm for rapid Cre/lox-mediated signaling perturbations, our results establish fgfr1-dn-cargo as a genetic tool to define the spatiotemporal requirements for FGF signaling in zebrafish. Developmental Dynamics 247:1146-1159, 2018. © 2018 Wiley Periodicals, Inc.

The epithalamic region of fishes shows prominent left-right asymmetries that are executed by nodal signaling upstream of the asymmetry-determining transcription factor pitx2. Previous reports have identified that nodal controls the left-sided pitx2 expression in the lateral plate mesoderm through an enhancer present in the last intron of this gene. However, whether similar regulation occurs also in the case of epithalamic asymmetry is currently unresolved. Here, we address some of the cis-regulatory information that control asymmetric pitx2 expression in epithalamus by presenting a Tg(pitx2:EGFP) 116-17 transgenic medaka model, which expresses enhanced green fluorescent protein (EGFP) under control of an intronic enhancer. We show that this transgene recapitulates epithalamic expression of the endogenous pitx2 and that it responds to nodal signaling inhibition. Further, we identify that three foxh1-binding sites present in this enhancer modulate expression of the transgene and that the second site is absolutely necessary for the left-sided epithalamic expression while the other two sites may have subtler regulative roles. We provide evidence that left-sided epithalamic pitx2 expression is controlled through an enhancer present in the last intron of this gene and that the regulatory logic underlying asymmetric pitx2 expression is shared between epithalamic and lateral plate mesoderm regions.

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.

BACKGROUND: HOX proteins act during development to regulate musculoskeletal morphology. HOXA5 patterns skeletal structures surrounding the cervical-thoracic transition including the vertebrae, ribs, sternum and forelimb girdle. However, the tissue types in which it acts to pattern the skeleton, and the ultimate fates of embryonic cells that activate Hoxa5 expression, are unknown. METHODS: A detailed characterization of HOXA5 expression by immunofluorescence was combined with Cre/LoxP genetic lineage tracing to map the fate of Hoxa5 expressing cells in axial musculoskeletal tissues and in their precursors, the somites and lateral plate mesoderm. RESULTS: HOXA5 protein expression is dynamic and spatially restricted in derivatives of both the lateral plate mesoderm and somites, including a subset of the lateral sclerotome, suggesting a local role in regulating early skeletal patterning. HOXA5 expression persists from somite stages through late development in differentiating skeletal and connective tissues, pointing to a continuous and direct role in skeletal patterning. In contrast, HOXA5 expression is excluded from the skeletal muscle and muscle satellite cell lineages. Furthermore, the descendants of Hoxa5-expressing cells, even after HOXA5 expression has extinguished, never contribute to these lineages. CONCLUSIONS: Together, these findings suggest cell autonomous roles for HOXA5 in skeletal development, as well as non-cell autonomous functions in muscle through expression in surrounding connective tissues. They also support the notion that different Hox genes display diverse tissue specificities and locations to achieve their patterning activity.

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.

Summary The identification of cell surface proteins on stem cells or stem cell derivatives is a key strategy for the functional characterization, isolation, and understanding of stem cell population dynamics. Here, using an integrated mass spectrometry- and microarray-based approach, we analyzed the surface proteome and transcriptome of cardiac progenitor cells (CPCs) generated from the stage-specific differentiation of mouse and human pluripotent stem cells. Through bioinformatics analysis, we have identified and characterized FZD4 as a marker for lateral plate mesoderm. Additionally, we utilized FZD4, in conjunction with FLK1 and PDGFRA, to further purify CPCs and increase cardiomyocyte (CM) enrichment in both mouse and human systems. Moreover, we have shown that NORRIN presented to FZD4 further increases CM output via proliferation through the canonical WNT pathway. Taken together, these findings demonstrate a role for FZD4 in mammalian cardiac development., Highlights • Identified and characterized FZD4 as a new marker for lateral plate mesoderm • FZD4, in conjunction with FLK1 and PDGFRA, increases cardiomyocyte enrichment • FZD4 is expressed in the human system and shows enrichment in cardiomyocytes • NORRIN addition shows increase in cardiomyocyte output from FZD4 progenitor cells, In this article, Zandstra and colleagues have identified FZD4 as a new marker for lateral plate mesoderm. Using FZD4, they showed an increase in cardiac progenitor cell purity and a subsequent increase in cardiomyocyte induction in both the mouse and human systems. These findings demonstrate a role for FZD4 in mammalian cardiac development.

Cardiogenesis is a tightly controlled biological process required for formation of a functional heart. The transcription factor Foxc1 not only plays a crucial role in outflow tract development in mice, but is also involved in cardiac structure formation and normal function in humans. However, the molecular mechanisms by which Foxc1 controls cardiac development remain poorly understood. Previously, we reported that zebrafish embryos deficient in foxc1a, an ortholog of mammalian Foxc1, display pericardial edemas and die 9–10 days postfertilization. To further investigate Foxc1a's role in zebrafish cardiogenesis and identify its downstream target genes during early heart development, we comprehensively analyzed the cardiovascular phenotype of foxc1a-null zebrafish embryos. Our results confirmed that foxc1a-null mutants exhibit disrupted cardiac morphology, structure, and function. Performing transcriptome analysis on the foxc1a mutants, we found that the expression of the cardiac progenitor marker gene nkx2.5 was significantly decreased, but the expression of germ layer–patterning genes was unaffected. Dual-fluorescence in situ hybridization assays revealed that foxc1a and nkx2.5 are co-expressed in the anterior lateral plate mesoderm at the somite stage. Chromatin immunoprecipitation and promoter truncation assays disclosed that Foxc1a regulates nkx2.5 expression via direct binding to two noncanonical binding sites in the proximal nkx2.5 promoter. Moreover, functional rescue experiments revealed that developmental stage–specific nkx2.5 overexpression partially rescues the cardiac defects of the foxc1a-null embryos. Taken together, our results indicate that during zebrafish cardiogenesis, Foxc1a is active directly upstream of nkx2.5.

In humans, germline mutations in Trpm6 cause autosomal dominant hypomagnesemia with secondary hypocalcemia disorder. Loss of Trpm6 in mice also perturbs cellular magnesium homeostasis but additionally results in early embryonic lethality and neural tube closure defects. To define the mechanisms by which TRPM6 influences neural tube closure, we functionally characterized the role of TRPM6 during early embryogenesis in Xenopus laevis. The expression of Xenopus TRPM6 (XTRPM6) is elevated at the onset of gastrulation and is concentrated in the lateral mesoderm and ectoderm at the neurula stage. Loss of XTRPM6 produced gastrulation and neural tube closure defects. Unlike XTRPM6′s close homologue XTRPM7, whose loss interferes with mediolateral intercalation, depletion of XTRPM6 but not XTRPM7 disrupted radial intercalation cell movements. A zinc-influx assay demonstrated that TRPM6 has the potential to constitute functional channels in the absence of TRPM7. The results of our study indicate that XTRPM6 regulates radial intercalation with little or no contribution from XTRPM7 in the region lateral to the neural plate, whereas XTRPM7 is mainly involved in regulating mediolateral intercalation in the medial region of the neural plate. We conclude that both TRPM6 and TRPM7 channels function cooperatively but have distinct and essential roles during neural tube closure.

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.

Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart defects. Heart lectin-associated matrix protein-1 (hLAMP-1) is a matrix protein expressed within Hensen's node at Hamburger-Hamilton (HH) stage 4, in the lateral mesoderm by HH stages 5-6 and enhanced within the left pre-cardiac field at HH stage 7. At HH stages 15-16, hLAMP-1 expression is observed in the atrioventricular canal and the outflow tract. Also, the role of hLAMP-1 in induction of mesenchyme formation in chick heart has been well documented. To further elucidate the role of this molecule in heart development, we examined its expression patterns during HH stages 8-14 in the chick. In this regard, we immunostained sections of the heart during HH stages 8-14 with antibodies specific to hLAMP-1. Our results showed prominent expression of hLAMP-1-positive particles in the extracellular matrix associated with the pre-cardiac mesoderm, the endoderm, ectoderm as well as neuroectoderm at HH stages 8-9. After formation of the linear heart tube at HH stage 10, the expression of hLAMP-1-stained particles disappears in those regions of original contact between the endoderm and heart forming fields due to rupture of the dorsal mesocardium while their expression becomes confined to the arterial and venous poles of the heart tube. This expression pattern is maintained until HH stage 14. This expression pattern suggests that hLAMP-1 may be involved in the formation of the endocardial tube.