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Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations. [ABSTRACT FROM AUTHOR]

The origin of paired appendages became one of the most important adaptations of vertebrates, allowing them to lead active lifestyles and explore a wide range of ecological niches. The basic form of paired appendages in evolution is the fins of fishes. The problem of paired appendages has attracted the attention of researchers for more than 150 years. During this time, a number of theories have been proposed, mainly based on morphological data, two of which, the Balfour‐Thacher‐Mivart lateral fold theory and Gegenbaur's gill arch theory, have not lost their relevance. So far, however, none of the proposed ideas has been supported by decisive evidence. The study of the evolutionary history of the appearance and development of paired appendages lies at the intersection of several disciplines and involves the synthesis of paleontological, morphological, embryological, and genetic data. In this review, we attempt to summarize and discuss the results accumulated in these fields and to analyze the theories put forward regarding the prerequisites and mechanisms that gave rise to paired fins and limbs in vertebrates. [ABSTRACT FROM AUTHOR]

Elongation of the posterior body axis is distinct from that of the anterior trunk and head. Early drivers of posterior elongation are the neural plate/tube and notochord, later followed by the presomitic mesoderm (PSM), together with the neural tube and notochord. In axolotl, posterior neural plate-derived PSM is pushed posteriorly by convergence and extension of the neural plate. The PSM does not go through the blastopore but turns anteriorly to join the gastrulated paraxial mesoderm. To gain a deeper understanding of the process of axial elongation, a detailed characterization of PSM morphogenesis, which precedes somite formation, and of other tissues (such as the epidermis, lateral plate mesoderm and endoderm) is needed. We investigated these issues with specific tissue labelling techniques (DiI injections and GFP+ tissue grafting) in combination with optical tissue clearing and 3D reconstructions. We defined a spatiotemporal order of PSM morphogenesis that is characterized by changes in collective cell behaviour. The PSM forms a cohesive tissue strand and largely retains this cohesiveness even after epidermis removal. We show that during embryogenesis, the PSM, as well as the lateral plate and endoderm move anteriorly, while the net movement of the axis is posterior. [ABSTRACT FROM AUTHOR]

Elongation of the posterior body axis is distinct from that of the anterior trunk and head. Early drivers of posterior elongation are the neural plate/tube and notochord, later followed by the presomitic mesoderm (PSM), together with the neural tube and notochord. In axolotl, posterior neural plate-derived PSM is pushed posteriorly by convergence and extension of the neural plate. The PSM does not go through the blastopore but turns anteriorly to join the gastrulated paraxial mesoderm. To gain a deeper understanding of the process of axial elongation, a detailed characterization of PSM morphogenesis, which precedes somite formation, and of other tissues (such as the epidermis, lateral plate mesoderm and endoderm) is needed. We investigated these issues with specific tissue labelling techniques (DiI injections and GFP+ tissue grafting) in combination with optical tissue clearing and 3D reconstructions. We defined a spatiotemporal order of PSM morphogenesis that is characterized by changes in collective cell behaviour. The PSM forms a cohesive tissue strand and largely retains this cohesiveness even after epidermis removal. We show that during embryogenesis, the PSM, as well as the lateral plate and endoderm move anteriorly, while the net movement of the axis is posterior.

The skeletal system derives from multiple embryonic sources whose derivatives must develop in coordination to produce an integrated whole. In particular, interactions across the lateral somitic frontier, where derivatives of the somites and lateral plate mesoderm come into contact, are important for proper development. Many questions remain about genetic control of this coordination, and embryological information is incomplete for some structures that incorporate the frontier, including the sternum. Hox genes act in both tissues as regulators of skeletal pattern. Here, we used conditional deletion to characterize the tissue-specific contributions of Hoxa5 to skeletal patterning. We found that most aspects of the Hoxa5 skeletal phenotype are attributable to its activity in one or the other tissue, indicating largely additive roles. However, multiple roles are identified at the junction of the T1 ribs and the anterior portion of the sternum, or presternum. The embryology of the presternum has not been well described in mouse. We present a model for presternum development, and show that it arises from multiple, paired LPM-derived primordia. We show evidence that HOXA5 expression marks the embryonic precursor of a recently identified lateral presternum structure that is variably present in therians.

Smooth muscle cells (SMCs) represent a major structural and functional component of many organs during embryonic development and adulthood. These cells are a crucial component of vertebrate structure and physiology, and an updated overview of the developmental and functional process of smooth muscle during organogenesis is desirable. Here, we describe the developmental origin of SMCs within different tissues by comparing their specification and differentiation with other organs, including the cardiovascular, respiratory and intestinal systems. We then discuss the instructive roles of smoothmuscle in the development of such organs through signaling and mechanical feedback mechanisms. By understanding SMC development, we hope to advance therapeutic approaches related to tissue regeneration and other smooth muscle-related diseases. [ABSTRACT FROM AUTHOR]

In this study, we elucidate a single cell resolution fate map in the zebrafish in a sub-section of the anterior Lateral Plate Mesoderm (aLPM) at 18 hpf. Our results show that this tissue is not organized into segregated regions but gives rise to intermingled pericardial sac, peritoneum, pharyngeal arch and cardiac precursors. We further report upon asymmetrical contributions of lateral aLPM-derived heart precursors—specifically that twice as many heart precursors arise from the right side versus the left side of the embryo. Cell tracking analyses and large-scale cell labeling of the lateral aLPM corroborate these differences and show that the observed asymmetries are dependent upon Tbx5a expression. Previously, it was shown that cardiac looping was affected in Tbx5a knock-down and knock-out zebrafish (Garrity et al., 2002; Parrie et al., 2013); our present data also implicate tbx5a function in cell specification, establishment and maintenance of cardiac left-right asymmetry. • Fate mapping of anterior lateral plate mesoderm elucidates organ contributions. • aLPM precursors are not regionalized by eventual fate in 18 hpf zebrafish embryos. • Cardiac precursors are overrepresented on the right side of the embryo. • Cell tracking reveals migration variances between left versus right side precursors. • Left-Right asymmetry is dependent upon tbx5a function. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors. [Display omitted] • Established a primary 3D limb culture system that maintains early limb progenitor cells • Used a reprogramming approach to identify factors imbuing a limb progenitor state • Together, Prdm16, Zbtb16, and Lin28a can convert non-limb cells to limb progenitors • Trajectory analysis identifies Lin41 as facilitating reprogramming by the other factors Atsuta et al. identified a 3D limb culture protocol that can maintain limb progenitors in vitro and screened for factors that can reprogram non-limb mesenchyme to a limb progenitor state. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscle in the trunk derives from the somites, paired segments of paraxial mesoderm. Whereas axial musculature develops within the somite, appendicular muscle develops following migration of muscle precursors into lateral plate mesoderm. The development of muscles bridging axial and appendicular systems appears mixed. Results: We examine development of three migratory muscle precursor‐derived muscles in zebrafish: the sternohyoideus (SH), pectoral fin (PF), and posterior hypaxial (PHM) muscles. We show there is an anterior to posterior gradient to the developmental gene expression and maturation of these three muscles. SH muscle precursors exhibit a long delay between migration and differentiation, PF muscle precursors exhibit a moderate delay in differentiation, and PHM muscle precursors show virtually no delay between migration and differentiation. Using lineage tracing, we show that lateral plate contribution to the PHM muscle is minor, unlike its known extensive contribution to the PF muscle and absence in the ventral extension of axial musculature. Conclusions: We propose that PHM development is intermediate between a migratory muscle mode and an axial muscle mode of development, wherein the PHM differentiates after a very short migration of its precursors and becomes more anterior primarily by elongation of differentiated muscle fibers. Key Findings: The posterior hypaxial muscle straddles the axial and appendicular systems in teleosts.The posterior hypaxial muscle is neither a simple ventral extension of axial myotome nor a classic MMP‐derived muscle like the pectoral fin muscles.The development of the posterior hypaxial muscle is intermediate to axial and appendicular muscle development. [ABSTRACT FROM AUTHOR]

• Progenitors of the dorsal mesentery and gonad arise from ventro-medial lateral plate mesoderm (LPM). • Ventromedial LPM repetitively undergoes epithelial to mesenchymal transitioning (EMT) during embryogenesis. • Transition of BMP4 expression medio-lateral axis to dorso-medial axis plays key roles in the gonad and mesentery formation. • Sonic hedgehog, secreted from endoderm, maintains BMP4 expression to establish dorso-ventral patterning in medial LPM. • Ovarian surface epithelium, a progeny of coelomic epithelium, causes EMT in a strictly regulated manner. Dorsal mesentery and gonad (ovary and testis) are formed in distinct regions of the body and have different characteristics. Recent studies using chicken embryos showed that progenitors of these two organs are derived from the coelomic lining region, a ventral part of the medial lateral plate mesoderm (M-LPM). Furthermore, both types of progenitors develop in a similar manner, concomitant with morphological changes termed the epithelial-to-mesenchymal transition (EMT). EMT processes in both dorsal mesentery and gonad formation are regulated by BMP signaling. Interestingly, EMT-based morphogenetic events occur repetitively at M-LPM specification before dorsal mesenteric and gonadal formation, at ovary formation later in embryogenesis, and even during adult ovary repair. We review recent findings related to EMT-based morphogenesis and the governing molecular mechanisms, mainly in early dorsal mesenteric and gonadal formation, as well as in their anlages and derivatives. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Intestinal smooth muscle cells (iSMCs) are a crucial component of the adult gastrointestinal tract and support intestinal differentiation, peristalsis and epithelial homeostasis during development. Despite these crucial roles, the origin of iSMCs and the mechanisms responsible for their differentiation and function remain largely unknown in vertebrates. Here, we demonstrate that iSMCs arise from the lateral plate mesoderm (LPM) in a stepwise process. Combining pharmacological and genetic approaches, we show that TGFß/Alk5 signaling drives the LPM ventral migration and commitment to an iSMC fate. The Alk5-dependent induction of zeb1a and foxo1a is required for this morphogenetic process: zeb1a is responsible for driving LPM migration around the gut, whereas foxo1a regulates LPM predisposition to iSMC differentiation. We further show that TGFß, zeb1a and foxo1a are tightly linked together by miR-145. In iSMC-committed cells, TGFß induces the expression of miR-145, which in turn is able to downregulate zeb1a and foxo1a. The absence of miR-145 results in only a slight reduction in the number of iSMCs, which still express mesenchymal genes but fail to contract. Together, our data uncover a cascade of molecular events that govern distinct morphogenetic steps during the emergence and differentiation of vertebrate iSMCs. [ABSTRACT FROM AUTHOR]

Formation of epithelia through mesenchymal-epithelial transition (MET) is essential for embryonic development and for many physiological and pathological processes. This study investigates MET in vivo in the chick embryo lateral mesoderm, where a multilayered mesenchyme transforms into two parallel epithelial sheets that constitute the coelomic lining of the embryonic body cavity. Prior to MET initiation, mesenchymal cells exhibit non-polarized distribution of multiple polarity markers, albeit not aPKC. We identified an epithelializing wave that sweeps across the lateral mesoderm, the wavefront of which is characterized by the accumulation of basal fibronectin and a network of 3D rosettes composed of polarized, wedge-shaped cells surrounding a central focus of apical markers, now including aPKC. Initiation of the MET process is dependent on extracellular matrix-integrin signaling acting through focal adhesion kinase and talin, whereas progression through the rosette phase requires aPKC function. We present a stepwise model for MET, comprising polarization, 3D-rosette, and epithelialization stages. [Display omitted] • MET occurs as a coordinated wave that spreads across the lateral mesoderm • The wavefront of the MET wave is composed of 3D-rosette transitional structures • ECM-integrin signaling is required for the initiation of MET • aPKC is required for transition through the rosette phase Abboud Asleh et al. find that mesenchymal-epithelial transition (MET) in the chick embryo lateral mesoderm is characterized by a morphogenetic wave of 3D-rosette structures that sweeps across the embryo, and they identify roles for ECM-integrin signaling and atypical protein kinase C activity at specific points in the MET process. [ABSTRACT FROM AUTHOR]

Evolutionary transitions are frequently associated with novel anatomical structures, 1 but the origins of the structures themselves are often poorly known. We use developmental, genetic, and paleontological data to demonstrate that the therian sternum was assembled from pre-existing elements. Imaging of the perinatal mouse reveals two paired sternal elements, both composed primarily of cells with lateral plate mesoderm origin. Location, articulations, and development identify them as homologs of the interclavicle and the sternal bands of synapsid outgroups. The interclavicle, not previously recognized in therians, 2 articulates with the clavicle and differs from the sternal bands in both embryonic HOX expression and pattern of skeletal maturation. The sternal bands articulate with the ribs in two styles, most clearly differentiated by their association with sternebrae. Evolutionary trait mapping indicates that the interclavicle and sternal bands were independent elements throughout most of synapsid history. The differentiation of rib articulation styles and the subdivision of the sternal bands into sternebrae were key innovations likely associated with transitions in locomotor and respiratory mechanics. 3,4 Fusion of the interclavicle and the anterior sternal bands to form a presternum anterior to the first sternebra was a historically recent innovation unique to therians. Subsequent disassembly of the radically reduced sternum of mysticete cetaceans was element specific, reflecting the constraints that conserved developmental programs exert on composite structures. [Display omitted] • The therian sternum is a composite that includes the ancestral interclavicle • Each element has an independent developmental program and evolutionary history • The stepwise addition and loss of elements can be traced in the fossil record • Retained ancestral elements highlight the conservatism of tissue interactions Brent et al. demonstrate that the therian sternum is composed of multiple elements, each with a unique developmental program and evolutionary history. The presence of the ancestral interclavicle in the sternum of living taxa highlights the conservatism of tissue-tissue interactions in vertebrate morphogenesis. [ABSTRACT FROM AUTHOR]

Haematopoietic stem cells ( HSCs) emerge from the haemogenic endothelium ( HE) localised in the ventral wall of the embryonic dorsal aorta ( DA). The HE generates HSCs through a process known as the endothelial to haematopoietic transition ( EHT), which has been visualised in live embryos and is currently under intense study. However, EHT is the culmination of multiple programming events, which are as yet poorly understood, that take place before the specification of HE. A number of haematopoietic precursor cells have been described before the emergence of definitive HSCs, but only one haematovascular progenitor, the definitive haemangioblast ( DH), gives rise to the DA, HE and HSCs. DHs emerge in the lateral plate mesoderm ( LPM) and have a distinct origin and genetic programme compared to other, previously described haematovascular progenitors. Although DHs have so far only been established in Xenopus embryos, evidence for their existence in the LPM of mouse and chicken embryos is discussed here. We also review the current knowledge of the origins, lineage relationships, genetic programming and differentiation of the DHs that leads to the generation of HSCs. Importantly, we discuss the significance of the gene regulatory network ( GRN) that controls the programming of DHs, a better understanding of which may aid in the establishment of protocols for the de novo generation of HSCs in vitro. [ABSTRACT FROM AUTHOR]

Summary Positioning organs in the body often requires the movement of multiple tissues, yet the molecular and cellular mechanisms coordinating such movements are largely unknown. Here, we show that bidirectional signaling between EphrinB1 and EphB3b coordinates the movements of the hepatic endoderm and adjacent lateral plate mesoderm (LPM), resulting in asymmetric positioning of the zebrafish liver. EphrinB1 in hepatoblasts regulates directional migration and mediates interactions with the LPM, where EphB3b controls polarity and movement of the LPM. EphB3b in the LPM concomitantly repels hepatoblasts to move leftward into the liver bud. Cellular protrusions controlled by Eph/Ephrin signaling mediate hepatoblast motility and long-distance cell-cell contacts with the LPM beyond immediate tissue interfaces. Mechanistically, intracellular EphrinB1 domains mediate EphB3b-independent hepatoblast extension formation, while EpB3b interactions cause their destabilization. We propose that bidirectional short- and long-distance cell interactions between epithelial and mesenchyme-like tissues coordinate liver bud formation and laterality via cell repulsion. [ABSTRACT FROM AUTHOR]

Transplantation of vascular smooth muscle cells (VSMCs) is a promising cellular therapy to promote angiogenesis and wound healing. However, VSMCs are derived from diverse embryonic sources which may influence their role in the development of vascular disease and in its therapeutic modulation. Despite progress in understanding the mechanisms of VSMC differentiation, there remains a shortage of robust methods for generating lineage-specific VSMCs from pluripotent and adult stem/progenitor cells in serum-free conditions. Here we describe a method for differentiating pluripotent stem cells, such as embryonic and induced pluripotent stem cells, as well as skin-derived precursors, into lateral plate-derived VSMCs including ‘coronary-like’ VSMCs and neural crest-derived VSMC, respectively. We believe this approach will have broad applications in modeling origin-specific disease vulnerability and in developing personalized cell-based vascular grafts for regenerative medicine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The neck acquired flexibility through modifications of the head-trunk interface in vertebrate evolution. Although developmental programs for the neck musculoskeletal system have attracted the attention of evolutionary developmental biologists, how the heart, shoulder and surrounding tissues are modified during development has remained unclear. Here we show, through observation of the lateral plate mesoderm at cranial somite levels in chicken-quail chimeras, that the deep part of the lateral body wall is moved concomitant with the caudal transposition of the heart, resulting in the infolding of the expanded cervical lateral body wall into the thorax. Judging from the brachial plexus pattern, an equivalent infolding also appears to take place in mammalian and turtle embryos. In mammals, this infolding process is particularly important because it separates the diaphragm from the shoulder muscle mass. In turtles, the expansion of the cervical lateral body wall affects morphogenesis of the shoulder. Our findings highlight the cellular expansion in developing amniote necks that incidentally brought about the novel adaptive traits. [ABSTRACT FROM AUTHOR]

Tbx5 plays a pivotal role in vertebrate forelimb initiation, and loss-of-function experiments result in deformed or absent forelimbs in all taxa studied to date. Combining single-cell fate mapping and three-dimensional cell tracking in the zebrafish, we describe a Tbx5a-dependent cell convergence pattern that is both asymmetric and topological within the fin-field lateral plate mesoderm during early fin bud initiation. We further demonstrate that a mesodermal Fgf24 convergence cue controlled by Tbx5a underlies this asymmetric convergent motility. Partial reduction in Tbx5a or Fgf24 levels disrupts the normal fin-field cell motility gradient and results in anteriorly biased perturbations of fin-field cell convergence and truncations in the pectoral fin skeleton, resembling aspects of the forelimb skeletal defects that define individuals with Holt-Oram syndrome. This study provides a quantitative reference model for fin-field cell motility during vertebrate fin bud initiation and suggests that a pre-pattern of anteroposterior fate specification is already present in the fin-field before or during migration because perturbations to these early cell movements result in the alteration of specific fates. [ABSTRACT FROM AUTHOR]

The vertebrate limb with its complex anatomy develops from a small bud of undifferentiated mesoderm cells encased in ectoderm. The bud has its own intrinsic polarity and can develop autonomously into a limb without reference to the rest of the embryo. In this review, recent advances are integrated with classical embryology, carried out mainly in chick embryos, to present an overview of how the embryo makes a limb bud. We will focus on how mesoderm cells in precise locations in the embryo become determined to form a limb and express the key transcription factors Tbx4 (leg/hindlimb) or Tbx5 (wing/forelimb). These Tbx transcription factors have equivalent functions in the control of bud formation by initiating a signalling cascade involving Wnts and fibroblast growth factors ( FGFs) and by regulating recruitment of mesenchymal cells from the coelomic epithelium into the bud. The mesoderm that will form limb buds and the polarity of the buds is determined with respect to both antero-posterior and dorso-ventral axes of the body. The position in which a bud develops along the antero-posterior axis of the body will also determine its identity - wing/forelimb or leg/hindlimb. Hox gene activity, under the influence of retinoic acid signalling, is directly linked with the initiation of Tbx5 gene expression in the region along the antero-posterior axis of the body that will form wings/forelimbs and determines antero-posterior polarity of the buds. In contrast, Tbx4 expression in the regions that will form legs/hindlimbs is regulated by the homeoprotein Pitx1 and there is no evidence that Hox genes determine antero-posterior polarity of the buds. Bone morphogenetic protein ( BMP) signalling determines the region along the dorso-ventral axis of the body in which both wings/forelimbs and legs/hindlimbs develop and dorso-ventral polarity of the buds. The polarity of the buds leads to the establishment of signalling regions - the dorsal and ventral ectoderm, producing Wnts and BMPs, respectively, the apical ectodermal ridge producing fibroblast growth factors and the polarizing region, Sonic hedgehog (Shh). These signals are the same in both wings/forelimbs and legs/hindlimbs and control growth and pattern formation by providing the mesoderm cells of the limb bud as it develops with positional information. The precise anatomy of the limb depends on the mesoderm cells in the developing bud interpreting positional information according to their identity - determined by Pitx1 in hindlimbs - and genotype. The competence to form a limb extends along the entire antero-posterior axis of the trunk - with Hox gene activity inhibiting the formation of forelimbs in the interlimb region - and also along the dorso-ventral axis. [ABSTRACT FROM AUTHOR]

Introduction: In 1931, Simpson et al. coined the term "peritoneal carcinomatosis" to describe the regional spread of ovarian tumors as localized or extended with involvement of the peritoneal serous membrane and neighboring anatomical structures. Research into the origin of peritoneal carcinomatosis is based on two phases in a woman's life: Embryo development: During week 3, the bilaminar disc becomes a trilaminar disc called the mesoderm. Inside the lateral plate mesoderm, the coelomic cavity is divided into 2 layers: the parietal (somatic) mesoderm, which gives rise to the parietal peritoneum and pleural surfaces; and the visceral (splanchnic) mesoderm, which gives rise to the visceral peritoneum, visceral surface of the pleura, gonadal stroma, and the muscular layer of the hollow viscera and its mesenteries. Tumor spread: Transcoelomic metastasis and metaplasia of pluripotent stem cells in the peritoneum was involved in the pathogenesis of ovarian cancer. This involvement takes the form of a synchronous malignant transformation at multiple foci and may cause intraperitoneal field cancerization. Pluripotent stem cells play a role both in the development of the embryonic peritoneum and in the spread of transcoelomic tumors. Consequently, knowledge of the origin of these cells (embryonic or current) could be extremely useful. The many markers that act during the embryonic period can affect descendants, that is, cells are already marked before specification and differentiation are activated. Thus, programmed activation could be attributed to genetic and epigenetic changes. [ABSTRACT FROM AUTHOR]

In vertebrates, paired appendages (limbs and fins) are derived from the somatic mesoderm subsequent to the separation of the lateral plate mesoderm into somatic and splanchnic layers. This is less clear for teleosts, however, because the developmental processes of separation into two layers and of extension over the yolk have rarely been studied. During teleost evolution, the position of pelvic fins has generally shifted rostrally ( Rosen; Nelson, 1982, 1994), although at the early embryonic stage the presumptive pelvic fin cells are initially located near the future anus region - the anterior border of hoxc10a expression in the spinal cord - regardless of their final destination. Our previous studies in zebrafish (abdominal pelvic fins) and Nile tilapia (thoracic pelvic fins) showed that the presumptive pelvic fin cells shift their position with respect to the body trunk after its protrusion from the yolk surface. Furthermore, in Nile tilapia, presumptive pelvic fin cells migrate anteriorly on the yolk surface. Here, we examined the embryonic development of the lateral plate mesoderm at histological levels in the pufferfish Takifugu niphobles, which belongs to the highly derived teleost order Tetraodontiformes, and lacks pelvic fins. Our results show that, in T. niphobles, the lateral plate mesoderm bulges out as two separate layers of cells alongside the body trunk prior to its further extension to cover the yolk sphere. Once the lateral plate mesoderm extends laterally, it rapidly covers the surface of the yolk. Furthermore, cells located near the anterior border of hoxc10a expression in the spinal cord reach the anterior-most region of the yolk surface. In light of our previous and current studies, we propose that anterior migration of presumptive pelvic fin cells might be required for them to reach the thoracic or more anterior positions as is seen in other highly derived teleost groups. [ABSTRACT FROM AUTHOR]

Species-specific symmetry-breaking events at the left–right organizer (LRO) drive an evolutionarily-conserved cascade of gene expression in the lateral plate mesoderm that is required for the asymmetric positioning of organs within the body cavity. The mechanisms underlying the transfer of the left and right laterality information from the LRO to the lateral plate mesoderm are poorly understood. Here, we investigate the role of Claudin-10, a tight junction protein, in facilitating the transfer of left–right identity from the LRO to the lateral plate mesoderm. Claudin-10 is asymmetrically expressed on the right side of the chick LRO, Hensen’s node. Gain- and loss-of-function studies demonstrated that right-sided expression of Claudin-10 is essential for normal rightward heart tube looping, the first morphological asymmetry during organogenesis. Manipulation of Claudin-10 expression did not perturb asymmetric gene expression at Hensen’s node, but did disrupt asymmetric gene expression in the lateral plate mesoderm. Bilateral expression of Claudin-10 at Hensen’s node prevented expression of Nodal , Lefty-2 and Pitx2c in the left lateral plate mesoderm, while morpholino knockdown of Claudin-10 inhibited expression of Snail1 in the right lateral plate mesoderm. We also determined that amino acids that are predicted to affect ion selectivity and protein interactions that bridge Claudin-10 to the actin cytoskeleton were essential for its left–right patterning function. Collectively, our data demonstrate a novel role for Claudin-10 during the transmission of laterality information from Hensen’s node to both the left and right sides of the embryo and demonstrate that tight junctions have a critical role during the relay of left–right patterning cues from Hensen’s node to the lateral plate mesoderm. [ABSTRACT FROM AUTHOR]

Embryoid bodies have a number of similarities with cells in gastrulation, which provides useful biological information about embryonic stem cell differentiation. Extensive research has been done to study the control of embryoid body-mediated embryonic stem cell differentiation in various research fields. Recently, microengineering technology has been used to control the size of embryoid bodies and to direct lineage specific differentiation of embryonic stem cells. However, the underlying biology of developmental events in the embryoid bodies of different sizes has not been well elucidated. In this study, embryoid bodies with different sizes were generated within microfabricated PEG microwell arrays, and a series of gene and molecular expressions related to early developmental events was investigated to further elucidate the size-mediated differentiation. The gene and molecular expression profile suggested preferential visceral endoderm formation in 450 μm embryoid bodies and preferential lateral plate mesoderm formation in 150 μm embryoid bodies. These aggregates resulted in higher cardiac differentiation in 450 μm embryoid bodies and higher endothelial differentiation in 150 μm embryoid bodies, respectively. Our findings may provide further insight for understanding embryoid body size-mediated developmental progress.[Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The position of the pelvic fins among teleost fishes has tended to shift rostrally during evolution. This positional shift seems to have led to the diversification of feeding behavior and allowed adaptation to new environments. To understand the developmental basis of this shift in pelvic fin position among teleosts, we investigated the embryonic development of the lateral plate mesoderm, which gives rise to the pelvic fins, at histological levels in the medaka Oryzias latipes (abdominal pelvic fins) and Nile tilapia Oreochromis niloticus (thoracic pelvic fins). Our histological analyses revealed that the lateral plate mesodermal cells expand not only ventrally but also rostrally to cover the yolk during embryogenesis of both medaka and Nile tilapia. In medaka, we also found that the lateral plate mesoderm completely covered the yolk prior to the initiation of the pelvic fin buds, whereas in Nile tilapia the pelvic fin buds appeared in the body wall from the lateral plate mesoderm at the thoracic level when the lateral plate mesodermal cells only covered one-third of the yolk. We discuss the relevance of such differences in the rate of the lateral plate mesoderm expansion on the yolk surface and the position of the pelvic fins. [ABSTRACT FROM AUTHOR]

Abstract: Asymmetric fluid flow in the node and Nodal signaling in the left lateral plate mesoderm (LPM) drive left–right patterning of the mammalian body plan. However, the mechanisms linking fluid flow to asymmetric gene expression in the LPM remain unclear. Here we show that the small GTPase Rab23, known for its role in Hedgehog signaling, plays a separate role in Nodal signaling and left–right patterning in the mouse embryo. Rab23 is not required for initial symmetry breaking in the node, but it is required for expression of Nodal and Nodal target genes in the LPM. Microinjection of Nodal protein and transfection of Nodal cDNA in the embryo indicate that Rab23 is required for the production of functional Nodal signals, rather than the response to them. Using gain- and loss-of function approaches, we show that Rab23 plays a similar role in zebrafish, where it is required in the teleost equivalent of the mouse node, Kupffer׳s vesicle. Collectively, these data suggest that Rab23 is an essential component of the mechanism that transmits asymmetric patterning information from the node to the LPM. [Copyright &y& Elsevier]

Normal heart development requires appropriate levels of retinoic acid (RA) signaling. RA levels in embryos are dampened by Cyp26 enzymes, which metabolize RA into easily degraded derivatives. Loss of Cyp26 function in humans is associated with numerous developmental syndromes that include cardiovascular defects. Although previous studies have shown that Cyp26-deficient vertebrate models also have cardiovascular defects, the mechanisms underlying these defects are not understood. Here, we found that in zebrafish, two Cyp26 enzymes, Cyp26a1 and Cyp26c1, are expressed in the anterior lateral platemesoderm (ALPM) and predominantly overlap with vascular progenitors (VPs). Although singular knockdown of Cyp26a1 or Cyp26c1 does not overtly affect cardiovascular development, double Cyp26a1 and Cyp26c1 (referred to here as Cyp26)-deficient embryos have increased atrial cells and reduced cranial vasculature cells. Examining the ALPM using lineage tracing indicated that in Cyp26-deficient embryos the myocardial progenitor field contains excess atrial progenitors and is shifted anteriorly into a region that normally solely gives rise to VPs. Although Cyp26 expression partially overlaps with VPs in the ALPM, we found that Cyp26 enzymes largely act cell non-autonomously to promote appropriate cardiovascular development. Our results suggest that localized expression of Cyp26 enzymes cell non-autonomously defines the boundaries between the cardiac and VP fields within the ALPM through regulating RA levels, which ensures a proper balance of myocardial and endothelial lineages. Our study provides novel insight into the earliest consequences of Cyp26 deficiency that underlie cardiovascular malformations in vertebrate embryos. [ABSTRACT FROM AUTHOR]

Background: Neuronal guidance cues influence endothelial cell (EC) behavior to shape the embryonic vascular system. The repulsive neuronal guidance cue, Semaphorin 3E (Sema3E), is critical for creating avascular zones that instruct and subsequently pattern the first embryonic vessels, the paired dorsal aortae (DA). Sema3E−/− embryos develop highly branched plexus-like vessels during vasculogenesis, instead of smooth paired vessels. Unexpectedly, despite these severe DA patterning defects, mutant mice are viable throughout adulthood. Results: Examination of Sema3E−/− mice reveals that the plexus-like DA resolve into single, unbranched vessels between embryonic day (E) E8.25 and E8.75. Although fusion of Sema3E−/− DA occurs slightly earlier than in heterozygotes, the DA are otherwise indistinguishable, suggesting a complete 'rescue' in their development. Resolution of the DA null plexuses occurs by remodeling rather than by means of changes in cell proliferation or death. Conclusions: Normalization of Sema3E−/− DA patterning defects demonstrates resilience of embryonic vascular patterning programs. Additional repulsive guidance cues within the lateral plate mesoderm likely re-establish avascular zones lost in Sema3E−/− embryos and guide resolution of mutant plexus into branchless, parallel aortae. Our observations explain how Sema3E−/− mice survive throughout development and into adulthood, despite severe initial vascular defects. Developmental Dynamics 242:570-580, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Limb regeneration involves re-establishing a limb development program from cells within adult tissues. Identifying molecular handles that provide insight into the relationship between cell differentiation status and cell lineage is an important step to study limb blastema cell formation. Here, using single cell PCR, focusing on newly isolated Twist1 sequences, we molecularly profile axolotl limb blastema cells using several progenitor cell markers. We link their molecular expression profile to their embryonic lineage via cell tracking experiments. We use in situ hybridization to determine the spatial localization and extent of overlap of different markers and cell types. Finally, we show by single cell PCR that the mature axolotl limb harbors a small but significant population of Twist1 + cells. [Copyright &y& Elsevier]

Muscles of the vertebrate neck include the cucullaris and hypobranchials. Although a functional neck first evolved in the lobe-finned fishes (Sarcopterygii) with the separation of the pectoral/shoulder girdle from the skull, the neck muscles themselves have a much earlier origin among the vertebrates. For example, lampreys possess hypobranchial muscles, and may also possess the cucullaris. Recent research in chick has established that these two muscles groups have different origins, the hypobranchial muscles having a somitic origin but the cucullaris muscle deriving from anterior lateral plate mesoderm associated with somites 1-3. Additionally, the cucullaris utilizes genetic pathways more similar to the head than the trunk musculature. Although the latter results are from experiments in the chick, cucullaris homologues occur in a variety of more basal vertebrates such as the sharks and zebrafish. Data are urgently needed from these taxa to determine whether the cucullaris in these groups also derives from lateral plate mesoderm or from the anterior somites, and whether the former or the latter represent the basal vertebrate condition. Other lateral plate mesoderm derivatives include the appendicular skeleton (fins, limbs and supporting girdles). If the cucullaris is a definitive lateral plate-derived structure it may have evolved in conjunction with the shoulder/limb skeleton in vertebrates and thereby provided a greater degree of flexibility to the heads of predatory vertebrates. [ABSTRACT FROM AUTHOR]

129/SvEv mice with a loss-of-function mutation in the heterotrimeric G protein α-subunit gene Gnai3 have fusions of ribs and lumbar vertebrae, indicating a requirement for Gαi (the "inhibitory" class of a-subunits) in somite derivatives. Mice with mutations of Gnai1 or Gnai2 have neither defect, but loss of both Gnai3 and one of the other two genes increases the number and severity of rib fusions without affecting the lumbar fusions. No myotome defects are observed in Gnai3/Gnai1 double-mutant embryos, and crosses with a conditional allele of Gnai2 indicate that Gai is specifically required in cartilage precursors. Penetrance and expressivity of the rib fusion phenotype is altered in mice with a mixed C57BL/6 × 129/SvEv genetic background. These phenotypes reveal a previously unknown role for G protein-coupled signaling pathways in development of the axial skeleton. [ABSTRACT FROM AUTHOR]

Abstract: Possession of paired appendages is regarded as a novelty that defines crown gnathostomes and allows sophisticated behavioral and locomotive patterns. During embryonic development, initiation of limb buds in the lateral plate mesoderm involves several steps. First, the lateral plate mesoderm is regionalized into the cardiac mesoderm (CM) and the posterior lateral plate mesoderm (PLPM). Second, in the PLPM, Hox genes are expressed in a collinear manner to establish positional values along the anterior–posterior axis. The developing PLPM splits into somatic and splanchnic layers. In the presumptive limb field of the somatic layer, expression of limb initiation genes appears. To gain insight into the evolutionary sequence leading to the emergence of paired appendages in ancestral vertebrates, we examined the embryonic development of the ventral mesoderm in the cephalochordate amphioxus Branchiostoma floridae and of the lateral plate mesoderm in the agnathan lamprey Lethenteron japonicum, and studied the expression patterns of cognates of genes known to be expressed in these mesodermal layers during amniote development. We observed that, although the amphioxus ventral mesoderm posterior to the pharynx was not regionalized into CM and posterior ventral mesoderm, the lateral plate mesoderm of lampreys was regionalized into CM and PLPM, as in gnathostomes. We also found nested expression of two Hox genes (LjHox5i and LjHox6w) in the PLPM of lamprey embryos. However, histological examination showed that the PLPM of lampreys was not separated into somatic and splanchnic layers. These findings provide insight into the sequential evolutionary changes that occurred in the ancestral lateral plate mesoderm leading to the emergence of paired appendages. [Copyright &y& Elsevier]

Abstract: Given that the lateral plate mesoderm (LPM) gives rise to the cardiovascular system, identifying the cascade of signalling events that subdivides the LPM into distinct regions during development is an important question. Retinoic acid (RA) is known to be necessary for establishing the expression boundaries of important transcription factors that demarcate distinct regions along the anterior posterior axis of the LPM. Here, we demonstrate that fibroblast growth factor (Fgf) signalling is also necessary for regulating the expression domains of the same transcription factors (nkx2.5, foxf1, hand1 and sall3) by restricting the RA responsive LPM domains. When Fgf signalling is inhibited in neurula stage embryos, the more posterior LPM expression domains are lost, while the more anterior domains are extended further posterior. The domain changes are maintained throughout development as Fgf inhibition results in similar domain changes in late stage embryos. We also demonstrate that Fgf signalling is necessary for both the initiation of heart specification, and for maintaining heart specification until overt differentiation occurs. Fgf signalling is also necessary to restrict vascular patterning and create a vascular free domain in the posterior end of the LPM that correlates with the expression of hand1. Finally, we show cross talk between the RA and Fgf signalling pathways in the patterning of the LPM. We suggest that this tissue wide patterning event, active during the neurula stage, is an initial step in regional specification of the LPM, and this process is an essential early event in LPM patterning. [Copyright &y& Elsevier]

Abstract: Limbed vertebrates have functionally integrated postcranial axial and appendicular systems derived from two distinct populations of embryonic mesoderm. The axial skeletal elements arise from the paraxial somites, the appendicular skeleton and sternum arise from the somatic lateral plate mesoderm, and all of the muscles for both systems arise from the somites. Recent studies in amniotes demonstrate that the scapula has a mixed mesodermal origin. Here we determine the relative contribution of somitic and lateral plate mesoderm to the avian scapula from quail-chick chimeras. We generate 3D reconstructions of the grafted tissue in the host revealing a very different distribution of somitic cells in the scapula than previously reported. This novel 3D visualization of the cryptic border between somitic and lateral plate populations reveals the dynamics of musculoskeletal morphogenesis and demonstrates the importance of 3D visualization of chimera data. Reconstructions of chimeras make clear three significant contrasts with existing models of scapular development. First, the majority of the avian scapula is lateral plate derived and the somitic contribution to the scapular blade is significantly smaller than in previous models. Second, the segmentation of the somitic component of the blade is partially lost; and third, there are striking differences in growth rates between different tissues derived from the same somites that contribute to the structures of the cervical thoracic transition, including the scapula. These data call for the reassessment of theories on the development, homology, and evolution of the vertebrate scapula. [Copyright &y& Elsevier]

Background: Mammals as a rule have seven cervical vertebrae, except for sloths and manatees. Bateson proposed that the change in the number of cervical vertebrae in sloths is due to homeotic transformations. A recent hypothesis proposes that the number of cervical vertebrae in sloths is unchanged and that instead the derived pattern is due to abnormal primaxial/abaxial patterning. Results: We test the detailed predictions derived from both hypotheses for the skeletal patterns in sloths and manatees for both hypotheses. We find strong support for Bateson's homeosis hypothesis. The observed vertebral and rib patterns cannot be explained by changes in primaxial/abaxial patterning. Vertebral patterns in sloths and manatees are similar to those in mice and humans with abnormal numbers of cervical vertebrae: incomplete and asymmetric homeotic transformations are common and associated with skeletal abnormalities. In sloths the homeotic vertebral shift involves a large part of the vertebral column. As such, similarity is greatest with mice mutant for genes upstream of Hox. Conclusions: We found no skeletal abnormalities in specimens of sister taxa with a normal number of cervical vertebrae. However, we always found such abnormalities in conspecifics with an abnormal number, as in many of the investigated dugongs. These findings strongly support the hypothesis that the evolutionary constraints on changes of the number of cervical vertebrae in mammals is due to deleterious pleitropic effects. We hypothesize that in sloths and manatees low metabolic and activity rates severely reduce the usual stabilizing selection, allowing the breaking of the pleiotropic constraints. This probably also applies to dugongs, although to a lesser extent. [ABSTRACT FROM AUTHOR]

Abstract: The lateral plate mesoderm (LPM) lines the body cavities, gives rise to the heart and circulatory system and is responsible for patterning the underlying endoderm. We describe gene expression domains within the lateral plate mesoderm of the neurula stage Xenopus embryo that demonstrate a marked anterior posterior pattern in that tissue. FoxF1 and Nkx-2.5 are expressed in the anterior LPM, Hand1 in the middle and Xsal-1 in the posterior LPM. Since retinoic acid is known to pattern many tissues during development, and RALDH2, the enzyme primarily responsible for retinoic acid synthesis, is expressed in the anterior and dorsal LPM, we hypothesized that retinoic acid is necessary for correct patterning of the LPM. Exposure to exogenous retinoic acid during neurulation led to an expansion of the anterior and middle expression domains and a reduction of the posterior domain whereas exposure to a retinoic acid antagonist resulted in smaller anterior and middle expression domains. Furthermore, inhibition of RALDH2, which should decrease endogenous RA levels, caused a reduction of anterior domains indicating that endogenous RA is necessary for regulating their size. After altering retinoic acid signaling in a temporally restricted window, the displaced anterior–posterior pattern is maintained until gut looping, as demonstrated by permanently altered Hand1, FoxF1, xHoxC-10, and Pitx2 expression domains. We conclude that the broad expression domains of key transcription factors demonstrate a novel anterior–posterior pattern within the LPM and that retinoic acid can regulate the size of these domains in a coordinated manner. [Copyright &y& Elsevier]

Abstract: We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm. [Copyright &y& Elsevier]

Abstract: Transient asymmetric Nodal signaling in the left lateral plate mesoderm (L LPM) during tailbud/early somitogenesis stages is associated in all vertebrates examined with the development of stereotypical left–right (L–R) organ asymmetry. In Xenopus, asymmetric expression of Nodal-related 1 (Xnr1) begins in the posterior L LPM shortly after the initiation of bilateral perinotochordal expression in the posterior tailbud. The L LPM expression domain rapidly shifts forward to cover much of the flank of the embryo before being progressively downregulated, also in a posterior-to-anterior direction. The mechanisms underlying the initiation and propagation of Nodal/Xnr1 expression in the L LPM, and its transient nature, are not well understood. Removing the posterior tailbud domain prevents Xnr1 expression in the L LPM, consistent with the idea that normal embryos respond to a posteriorly derived asymmetrically acting positive inductive signal. The forward propagation of asymmetric Xnr1 expression occurs LPM-autonomously via planar tissue communication. The shifting is prevented by Nodal signaling inhibitors, implicating an underlying requirement for Xnr1-to-Xnr1 induction. It is also unclear how asymmetric Nodal signals are modulated during L–R patterning. Small LPM grafts overexpressing Xnr1 placed into the R LPM of tailbud embryos induced the expression of the normally L-sided genes Xnr1, Xlefty, and XPitx2, and inverted body situs, demonstrating the late-stage plasticity of the LPM. Orthogonal Xnr1 signaling from the LPM strongly induced Xlefty expression in the midline, consistent with recent findings in the mouse and demonstrating for the first time in another species conservation in the mechanism that induces and maintains the midline barrier. Our findings suggest that there is long-range contralateral communication between L and R LPM, involving Xlefty in the midline, over a substantial period of tailbud embryogenesis, and therefore lend further insight into how, and for how long, the midline maintains a L versus R status in the LPM. [Copyright &y& Elsevier]

Abstract: Nodal-related genes have been implicated in mesendoderm induction, establishment of embryonic axes, neural patterning and left-right development among vertebrates. Here we report the isolation of three Nodal-related genes in medaka (Oryzias latipes). Based on sequence analysis and in accordance to zebrafish orthologues we named the isolated genes Ndr1, Ndr2 and Spaw. Gene expression analysis throughout medaka development confirmed this assignment. Ndr1 and Ndr2 are detectable during gastrulation whereas Ndr2 and Spaw are expressed asymmetrically during somitogenesis. In accordance with its zebrafish orthologue, Spaw is expressed as the first asymmetric marker in the left lateral plate mesoderm (LPM) and Ndr2 displays asymmetric expression domains in the brain and the LPM. In general, the spatial distribution of Nodal transcripts resembles those reported for zebrafish, in case of Ndr2, however, we report a novel left-right asymmetry in the posterior paraxial mesoderm flanking the Kupffer’s vesicle. [Copyright &y& Elsevier]

Signaling by members of TGF-β superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus, two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus neurulae. Left-side injection caused heart-specific left–right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left–right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than neurula embryos. Injection of inhibitors into either side of neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1, antivin, and pitx2. SPC1 enzyme (Furin) was injected into the left or right LPM of mid-neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left–right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left–right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the neurulae is necessary for proper left–right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left–right axis determination of the heart and gut in Xenopus embryos. [ABSTRACT FROM AUTHOR]

Abstract: The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation. [Copyright &y& Elsevier]

Abstract: The nodal and nodal-related genes play fundamental roles during deuterostome left–right axis formation. Several of these genes show left-sided expression in the lateral plate mesoderm and brain region. We have isolated the nodal-related gene, CyNodal, from Cynops pyrrhogaster. CyNodal mRNA is detected at the marginal zone and left side of several tissues. The left-sideness of CyNodal mRNA expression is highly conserved throughout vertebrate evolution. However, CyNodal mRNA expression shows little variation from the Xenopus nodal-related gene 1, in that CyNodal gene expression in the left lateral plate mesoderm shifts from posterior to anterior at least twice. [Copyright &y& Elsevier]

In all vertebrates, invariant left/right (L/R) positioning and organization of the internal viscera is controlled by a conserved pathway. Nodal, a member of the TGFβ superfamily is a critical upstream component responsible for initiating L/R axis determination. Asymmetric Nodal expression in the node preceeds and foreshadows morphological L/R asymmetry. Here we address the mechanism of Nodal activation in the left LPM by studying the function of a novel enhancer element, the AIE. We show this element is exclusively active in cells of the left lateral plate mesoderm (LPM) and is not itself responding to Nodal asymmetry. To test the hypothesis that this element may initiate asymmetric Nodal expression in the LPM, we deleted it from the mouse germ line. Mice homozygous for the AIE deletion (NodalΔaie/Δaie) show no defects. However, we find that the AIE contributes to regulating the level of asymmetric Nodal activity; analysis of transheterozygous embryos (NodalΔaie/null) shows reduced Nodal expression in the left LPM associated with a low penetrance of L/R defects. Our findings point to the existence of two independent pathways that control Nodal expression in the left LPM. [Copyright &y& Elsevier]

Development of the visceral mesoderm is a critical process in the organogenesis of the gut. Elucidation of function and regulation of genes involved in the development of visceral mesoderm is therefore essential for an understanding of gut organogenesis. One of the genes specifically expressed in the lateral plate mesoderm, and later in its derivative, the visceral mesoderm, is the Fox gene FoxF1. Its function is critical for Xenopus gut development, and embryos injected with FoxF1 morpholino display abnormal gut development. In the absence of FoxF1 function, the lateral plate mesoderm, and later the visceral mesoderm, does not proliferate and differentiate properly. Region- and stage-specific markers of visceral mesoderm differentiation, such as Xbap and a-smooth muscle actin, are not activated. The gut does not elongate and coil. These experiments provide support for the function of FoxF1 in the development of visceral mesoderm and the organogenesis of the gut. At the molecular level, FoxF1 is a downstream target of BMP4 signaling. BMP4 can activate FoxF1 transcription in animal caps and overexpression of FoxF1 can rescue twinning phenotypes, which results from the elimination of BMP4 signaling. The cis-regulatory elements of FoxF1 are located within a 2 kb DNA fragment upstream of the coding region. These sequences can drive correct temporal-spatial expression of a GFP reporter gene in transgenic Xenopus tadpoles. These sequences represent a unique tool, which can be used to specifically alter gene expression in the lateral plate mesoderm. [ABSTRACT FROM AUTHOR]

Patterning events along the anterior–posterior (AP) axis of vertebrate embryos result in the distribution of muscle and bone forming a highly effective functional system. A key aspect of regionalized AP patterning results from variation in the migratory pattern of somite cells along the dorsal–ventral (DV) axis of the body. This occurs as somite cell populations expand around the axis or migrate away from the dorsal midline and cross into the lateral plate. The fate of somitic cells has been intensely studied and many details have been reported about inductive signaling from other tissues that influence somite cell fate and behavior. We are interested in understanding the specific differences between somites in particular AP regions and how these differences contribute to the global pattern of the organism. Using orthotopic transplants of segmental plate between quail and chick embryos, we have mapped the interface of the somitic and lateral plate mesoderm during the formation of the body wall in cervical and thoracic regions. This interface does not change dramatically in the mid-cervical region, but undergoes extensive changes in the thoracic region. Based on this regional mapping and consistent with the extensive literature, we suggest a revised method of classifying regions of the body wall that relies on embryonic cell lineages rather than adult functional criteria. [Copyright &y& Elsevier]

Retinoids (vitamin A and its derivatives) play essential roles during vertebrate development. Vitamin A deprivation leads to severe congenital malformations affecting many tissues, including diverse neural crest cell populations and the heart. The vitamin A signal is transduced by the retinoic acid receptors (RARα, RARβ, and RARγ). However, these receptors exhibit considerable functional redundancy, as judged by the mild phenotype of RAR single null mutants relative to the defects evoked by loss of multiple RARs. To circumvent this redundancy, the endogenous RARγ2 allele was replaced with a ligand-binding RARγ mutant (RARγE305) by gene targeting in mouse embryonic stem (ES) cells. Chimeric embryos derived from hemizygous RARγE305 ES cells displayed several defects similar to those observed in certain RAR double null mutants, including hypoplasia or absence of the caudal pharyngeal arches and myocardial deficiencies. The latter defects were not due to abnormal cardiac specification as affected hearts still expressed chamber-specific markers in an appropriate manner. Chimeras also displayed cardiac looping anomalies, which were associated with a reduction of Pitx2. This work suggests a role for RAR signaling in late looping morphogenesis and illustrates the utility of using a dominant-negative gene substitution approach to circumvent the functional redundancy inherent to the RAR family. [Copyright &y& Elsevier]