Mechanisms of heart development in the Japanese lamprey, Lethenteron japonicum.

Vertebrate hearts have evolved from undivided tubular hearts of chordate ancestors. One of the most intriguing issues in heart evolution is the abrupt appearance of multichambered hearts in the agnathan vertebrates. To explore the developmental mechanisms behind the drastic morphological changes that led to complex vertebrate hearts, we examined the developmental patterning of the agnathan lamprey Lethenteron japonicum. We isolated lamprey orthologs of genes thought to be essential for heart development in chicken and mouse embryos, including genes responsible for differentiation and proliferation of the myocardium (LjTbx20, LjTbx4/5, and LjIsl1/2A), establishment of left-right heart asymmetry (LjPitxA), and partitioning of the heart tube (LjTbx2/3A), and studied their expression patterns during lamprey cardiogenesis. We confirmed the presence of the cardiac progenitors expressing LjIsl1/2A in the pharyngeal and splanchnic mesoderm and the heart tube of the lamprey. The presence of LjIsl1/2A-positive cardiac progenitor cells in cardiogenesis may have permitted an increase of myocardial size in vertebrates. We also observed LjPitxA expression in the left side of lamprey cardiac mesoderm, suggesting that asymmetric expression of Pitx in the heart has been acquired in the vertebrate lineage. Additionally, we observed LjTbx2/3A expression in the nonchambered myocardium, supporting the view that acquisition of Tbx2/3 expression may have allowed primitive tubular hearts to partition, giving rise to multichambered hearts.

Identification of four Engrailed genes in the Japanese lamprey, Lethenteron japonicum.

We have isolated four homologs of Engrailed genes from the Japanese lamprey, Lethenteron japonicum, an agnathan that occupies a critical phylogenic position between cephalochordates and gnathostomes. We named these four genes LjEngrailedA, LjEngrailedB, LjEngrailedC, and LjEngrailedD. LjEngrailedA, LjEngrailedB, and LjEngrailedD share a major expression domain in the presumptive midbrain-hindbrain boundary region of the central nervous system, although their levels and timing of expression differed. On the other hand, LjEngrailedC transcripts were in the pharyngeal ectoderm and the ventral ectoderm of the body wall. In addition, LjEngrailedA was expressed in the ventral side of the epibranchial muscle precursors. LjEngrailedD transcripts were seen in the mesodermal cells of the mandibular arch and later in a group of cells responsible for the formation of the upper lip, lower lip, and velum. Our results provide clues to the evolution of these structures as well as a possible scenario for duplication events of Engrailed genes.

Possible involvement of SINEs in mammalian-specific brain formation.

Retroposons, such as short interspersed elements (SINEs) and long interspersed elements (LINEs), are the major constituents of higher vertebrate genomes. Although there are many examples of retroposons' acquiring function, none has been implicated in the morphological innovations specific to a certain taxonomic group. We previously characterized a SINE family, AmnSINE1, members of which constitute a part of conserved noncoding elements (CNEs) in mammalian genomes. We proposed that this family acquired genomic functionality or was exapted after retropositioning in a mammalian ancestor. Here we identified 53 new AmnSINE1 loci and refined 124 total loci, two of which were further analyzed. Using a mouse enhancer assay, we demonstrate that one SINE locus, AS071, 178 kbp from the gene FGF8 (fibroblast growth factor 8), is an enhancer that recapitulates FGF8 expression in two regions of the developing forebrain, namely the diencephalon and the hypothalamus. Our gain-of-function analysis revealed that FGF8 expression in the diencephalon controls patterning of thalamic nuclei, which act as a relay center of the neocortex, suggesting a role for FGF8 in mammalian-specific forebrain patterning. Furthermore, we demonstrated that the locus, AS021, 392 kbp from the gene SATB2, controls gene expression in the lateral telencephalon, which is thought to be a signaling center during development. These results suggest important roles for SINEs in the development of the mammalian neuronal network, a part of which was initiated with the exaptation of AmnSINE1 in a common mammalian ancestor.

Identification and developmental expression of two Tbx1/10-related genes in the agnathan Lethenteron japonicum.

We have identified two Tbx1/10-related genes, LjTbx1/10A and LjTbx1/10B, from the Japanese river lamprey Lethenteron japonicum. We used in situ hybridization to characterize their expression pattern during embryonic development. LjTbx1/10A and LjTbx1/10B shared common expression in the pharyngeal arches and otic vesicle, although their levels and timing of expression differed markedly. LjTbx1/10A was highly expressed in the mesodermal core of pharyngeal arches and the adjacent endoderm throughout pharyngeal arch development, whereas LjTbx1/10B expression was only transiently upregulated in forming pharyngeal pouches. LjTbx1/10A transcripts first appeared at stage 25 in otic vesicles, whereas LjTbx1/10B transcripts could already be detected in the developing otic placode at stage 20. These results suggest that lamprey LjTbx1/10A and LjTbx1/10B may play distinct roles in the patterning and development of the pharyngeal apparatus. It appears that lamprey Tbx1/10 genes have undergone subfunctionalization independent from gnathostomes, with regard to both regulation and function.