Retinoic acid signaling is essential for formation of the heart tube in Xenopus

Retinoic acid is clearly important for the development of the heart. In this paper, we provide evidence that retinoic acid is essential for multiple aspects of cardiogenesis in Xenopus by examining embryos that have been exposed to retinoic acid receptor antagonists. Early in cardiogenesis, retinoic acid alters the expression of key genes in the lateral plate mesoderm including Nkx2.5 and HAND1, indicating that early patterning of the lateral plate mesoderm is, in part, controlled by retinoic acid. We found that, in Xenopus, the transition of the heart from a sheet of cells to a tube required retinoic acid signaling. The requirement for retinoic acid signaling was determined to take place during a narrow window of time between embryonic stages 14 and 18, well before heart tube closure. At the highest doses used, the lateral fields of myocardium fail to fuse, intermediate doses lead to a fusion of the two sides but failure to form a tube, and embryos exposed to lower concentrations of antagonist form a heart tube that failed to complete all the landmark changes that characterize looping. The myocardial phenotypes observed when exposed to the retinoic acid antagonist resemble the myocardium from earlier stages of cardiogenesis, although precocious expression of cardiac differentiation markers was not seen. The morphology of individual cells within the myocardium appeared immature, closely resembling the shape and size of cells at earlier stages of development. However, the failures in morphogenesis are not merely a slowing of development because, even when allowed to develop through stage 40, the heart tubes did not close when embryos were exposed to high levels of antagonist. Indeed, some aspects of left-right asymmetry also remained even in hearts that never formed a tube. These results demonstrate that components of the retinoic acid signaling pathway are necessary for the progression of cardiac morphogenesis in Xenopus.     

Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns

The neural crest is a uniquely vertebrate cell type present in the most basal vertebrates, but not in cephalochordates. We have studied differences in regulation of the neural crest marker AP-2 across two evolutionary transitions: invertebrate to vertebrate, and agnathan to gnathostome. Isolation and comparison of amphioxus, lamprey and axolotl AP-2 reveals its extensive expansion in the vertebrate dorsal neural tube and pharyngeal arches, implying co-option of AP-2 genes by neural crest cells early in vertebrate evolution. Expression in non-neural ectoderm is a conserved feature in amphioxus and vertebrates, suggesting an ancient role for AP-2 genes in this tissue. There is also common expression in subsets of ventrolateral neurons in the anterior neural tube, consistent with a primitive role in brain development. Comparison of AP-2 expression in axolotl and lamprey suggests an elaboration of cranial neural crest patterning in gnathostomes. However, migration of AP-2-expressing neural crest cells medial to the pharyngeal arch mesoderm appears to be a primitive feature retained in all vertebrates. Because AP-2 has essential roles in cranial neural crest differentiation and proliferation, the co-option of AP-2 by neural crest cells in the vertebrate lineage was a potentially crucial event in vertebrate evolution.     

Lamprey contractile protein genes mark different populations of skeletal muscles during development

Agnathan lampreys retain ancestral characteristics of vertebrates in the morphology of skeletal muscles derived from two mesodermal regions: trunk myotomes and unsegmented head mesoderm. During lamprey development, some populations of myoblasts migrate via pathways that differ from those of gnathostomes. To investigate the evolution of skeletal muscle differentiation in vertebrates, we characterize multiple contractile protein genes expressed in the muscle cells of the Japanese lamprey, Lethenteron japonicum. Lamprey actin gene LjMA2, and myosin heavy chain (MyHC) genes LjMyHC1 and LjMyHC2 are all expressed in the developing skeletal muscle cells of early embryos. However, LjMyHC1 and LjMyHC2 are expressed only in cells originating from myotomes, while LjMA2 is expressed in both myotomal and head musculature. Thus, in lampreys, myotomes and head mesoderm differ in the use of genes encoding contractile protein isoforms. Phylogenetic tree analyses including lamprey MyHCs suggest that the variety of muscle MyHC isoforms in different skeletal muscles may correspond to the morphological complexity of skeletal muscles of different vertebrate species. Another lamprey actin gene LjMA1 is likely to be the first smooth muscle actin gene isolated from non-tetrapods. We conclude that, in vertebrate evolution, the different regulatory systems for striated and smooth muscle-specific genes may have been established before the agnathan/gnathostome divergence.     

AmphiNk2-tin,an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart

We isolated a full-length cDNA clone of amphioxus AmphiNk2-tin, an NK2 gene similar in sequence to vertebrate NK2 cardiac genes, suggesting a potentially similar function to Drosophila tinman and to vertebrate NK2 cardiac genes during heart development. During the neurula stage of amphioxus, AmphiNk2-tin is expressed first within the foregut endoderm, then transiently in muscle precursor cells in the somites, and finally in some mesoderm cells of the visceral peritoneum arranged in an approximately midventral row running beneath the midgut and hindgut. The peritoneal cells that express AmphiNk2-tin are evidently precursors of the myocardium of the heart, which subsequently becomes morphologically detectable ventral to the gut. The amphioxus heart is a rostrocaudally extended tube consisting entirely of myocardial cells (at both the larval and adult stages); there are no chambers, valves, endocardium, epicardium, or other differentiated features of vertebrate hearts. Phylogenetic analysis of the AmphiNk2-tin sequence documents its close relationship to vertebrate NK2 class cardiac genes, and ancillary evidence suggests a relationship with the Drosophila NK2 gene tinman. Apparently, an amphioxus-like heart, and the developmental program directing its development, was the foundation upon which the vertebrate heart evolved by progressive modular innovations at the genetic and morphological levels of organization.     

Germ layer patterning in bichir and lamprey; an insight into its evolution in vertebrates

Amphibian holoblastic cleavage in which all blastomeres contribute to any one of the three primary germ layers has been widely thought to be a developmental pattern in the stem lineage of vertebrates, and meroblastic cleavage to have evolved independently in each vertebrate lineage. In extant primitive vertebrates, agnathan lamprey and basal bony fishes also undergo holoblastic cleavage, and their vegetal blastomeres have been generally thought to contribute to embryonic endoderm. However, the present marker analyses in basal ray-finned fish bichir and agnathan lamprey embryos indicated that their mesoderm and endoderm develop in the equatorial marginal zone, and their vegetal cell mass is extraembryonic nutritive yolk cells, having non-cell autonomous meso-endoderm inducing activity. Eomesodermin (eomes), but not VegT, orthologs are expressed maternally in these animals, suggesting that VegT is a maternal factor for endoderm differentiation only in amphibian. The study raises the viewpoint that the lamprey/bichir type holoblastic development would have been ancestral to extant vertebrates and retained in their stem lineage; amphibian-type holoblastic development would have been acquired secondarily, accompanied by the exploitation of new molecular machinery such as maternal VegT.     

Hox cluster organization in the jawless vertebrate Petromyzon marinus

Large-scale gene amplifications may have facilitated the evolution of morphological innovations that accompanied the origin of vertebrates. This hypothesis predicts that the genomes of extant jawless fish, scions of deeply branching vertebrate lineages, should bear a record of these events. Previous work suggests that nonvertebrate chordates have a single Hox cluster, but that gnathostome vertebrates have four or more Hox clusters. Did the duplication events that produced multiple vertebrate Hox clusters occur before or after the divergence of agnathan and gnathostome lineages? Can investigation of lamprey Hox clusters illuminate the origins of the four gnathostome Hox clusters? To approach these questions, we cloned and sequenced 13 Hox cluster genes from cDNA and genomic libraries in the lamprey, Petromyzon marinus. The results suggest that the lamprey has at least four Hox clusters and support the model that gnathostome Hox clusters arose by a two-round-no-cluster-loss mechanism, with tree topology [(AB)(CD)]. A three-round model, however, is not rigorously excluded by the data and, for this model, the tree topologies [(D(C(AB))] and [(C(D(AB))] are most parsimonious. Gene phylogenies suggest that at least one Hox cluster duplication occurred in the lamprey lineage after it diverged from the gnathostome lineage. The results argue against two or more rounds of duplication before the divergence of agnathan and gnathostome vertebrates. If Hox clusters were duplicated in whole-genome duplication events, then these data suggest that, at most, one whole genome duplication occurred before the evolution of vertebrate developmental innovations.     

GATA-6 maintains BMP-4 and Nkx2 expression during cardiomyocyte precursor maturation

GATA-6 is expressed in presumptive cardiac mesoderm before gastrulation, but its role in heart development has been unclear. Here we show that Xenopus and zebrafish embryos, injected with antisense morpholino oligonucleotides designed specifically to knock-down translation of GATA-6 protein, are severely compromised for heart development. Injected embryos express greatly reduced levels of contractile machinery genes and, at the same stage, of regulatory genes such as bone morphogenetic protein-4 (BMP-4) and the Nkx2 family. In contrast, initial BMP and Nkx2 expression is normal, suggesting a maintenance role for GATA-6. Endoderm is critical for heart formation in several vertebrates including Xenopus, and separate perturbation of GATA-6 expression in the deep anterior endoderm and in the overlying heart mesoderm shows that GATA-6 is required in both for cardiogenesis. The GATA-6 requirement in cardiac mesoderm was confirmed in zebrafish, an organism in which endoderm is thought not to be necessary for heart formation. We therefore conclude that proper maturation of cardiac mesoderm requires GATA-6, which functions to maintain BMP-4 and Nkx2 expression.     

Acquisition of the paired fins: a view from the sequential evolution of the lateral plate mesoderm

The origin of paired fins has long been a focus of both paleontologists and developmental biologists. Fossil records indicate that the first pair of fin‐like structures emerged in the body wall of early vertebrates. However, extant agnathan lampreys and hagfishes lack paired fins, and thus it has been difficult to determine the developmental processes underlying the ancestral acquisition of paired fins in vertebrates. Fortunately, recent advances in our knowledge of the developmental mechanisms of the lateral plate mesoderm among different taxa have provided clues for understanding the evolutionary origin of vertebrate paired appendages.     

Wg signaling in Drosophila heart development as a pioneering model

The heart is one of the first functional embryonic organs occurring during development. The fundamental developmental processes and genes involved in cardiogenesis are conserved between the invertebrates and vertebrates. In the past fifteen years, one of signaling pathways that has been best characterized in heart development in both invertebrates and vertebrates is the Wg/Wnt signaling pathways. Since our discovery of the Wg signaling required for the early heart development in Drosophila, the past fifteen years have witnessed tremendous progress in the understanding of specific Wnt signaling pathways in vertebrate cardiogenesis. This review will summarize the current state of knowledge of Wg signaling transduction in Drosophila heart development, which will benefit our understanding of vertebrate cardiogenesis and human congenital malformations.     

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.     

Wnt signal transduction and the formation of the myocardium

Soon after fertilization, vertebrate embryos grow very rapidly. Thus, early in gestation, a sizeable yet underdeveloped organism requires circulating blood. This need dictates the early appearance of a contractile heart, which is the first functional organ in both the avian and mammalian embryo. The heart arises from paired mesodermal regions within the anterior half of the embryo. As development proceeds, these bilateral precardiac fields merge at the midline to give rise to the primary heart tube. How specific areas of nondifferentiated mesoderm organize into myocardial tissue has been a question that has long intrigued developmental biologists. In recent years, the regulation of Wnt signal transduction has been implicated as an important event that initiates cardiac development. While initial reports in Drosophila and the bird had implicated Wnt proteins as promoters of cardiac tissue formation, subsequent findings that the WNT inhibitors Dkk1 and crescent possess cardiac-inducing activities led to the contrary hypothesis that WNTs actively inhibit cardiogenesis. This seeming contradiction has been resolved, in part, by more recent information indicating that Wnts stimulate multiple signal transduction pathways. In this review, we will examine what is presently known about the importance of regulated Wnt activity for the formation of the heart and the development of the myocardium and discuss this information in context of the emerging complexity of Wnt signal transduction.     

The evolution of chordate neural segmentation

Amphioxus is the closest relative to vertebrates but lacks key vertebrate characters, like rhombomeres, neural crest cells, and the cartilaginous endoskeleton. This reflects major differences in the developmental patterning of neural and mesodermal structures between basal chordates and vertebrates. Here, we analyse the expression pattern of an amphioxus FoxB ortholog and an amphioxus single-minded ortholog to gain insight into the evolution of vertebrate neural segmentation. AmphiFoxB expression shows cryptic segmentation of the cerebral vesicle and hindbrain, suggesting that neuromeric segmentation of the chordate neural tube arose before the origin of the vertebrates. In the forebrain, AmphiFoxB expression combined with AmphiSim and other amphioxus gene expression patterns shows that the cerebral vesicle is divided into several distinct domains: we propose homology between these domains and the subdivided diencephalon and midbrain of vertebrates. In the Hox-expressing region of the amphioxus neural tube that is homologous to the vertebrate hindbrain, AmphiFoxB shows the presence of repeated blocks of cells along the anterior-posterior axis, each aligned with a somite. This and other data lead us to propose a model for the evolution of vertebrate rhombomeric segmentation, in which rhombomere evolution involved the transfer of mechanisms regulating neural segmentation from vertical induction by underlying segmented mesoderm to horizontal induction by graded retinoic acid signalling. A consequence of this would have been that segmentation of vertebrate head mesoderm would no longer have been required, paving the way for the evolution of the unsegmented head mesoderm seen in living vertebrates.