Evolution of the tbx6/16 subfamily genes in vertebrates: insights from zebrafish.

In any comparative studies striving to understand the similarities and differences of the living organisms at the molecular genetic level, the crucial first step is to establish the homology (orthology and paralogy) of genes between different organisms. Determination of the homology of genes becomes complicated when the genes have undergone a rapid divergence in sequence or when the involved genes are members of a gene family that has experienced a differential gain or loss of its constituents in different taxonomic groups. Organisms with duplicated genomes such as teleost fishes might have been especially prone to these problems because the functional redundancies provided by the duplicate copies of genes would have allowed a rapid divergence or loss of genes during evolution. In this study, we will demonstrate that much of the ambiguities in the determination of the homology between fish and tetrapod genes resulting from the problems like these can be eliminated by complementing the sequence-based phylogenies with nonsequence information, such as the exon-intron structure of a gene or the composition of a gene's genomic neighbors. We will use the Tbx6/16 subfamily genes of zebrafish (tbx6, tbx16, tbx24, and mga genes), which have been well known for the ambiguity of their evolutionary relationships to the Tbx6/16 subfamily genes of tetrapods, as an illustrative example. We will show that, despite the similarity of sequence and expression to the tetrapod Tbx6 genes, zebrafish tbx6 gene is actually a novel T-box gene more closely related to the tetrapod Tbx16 genes, whereas the zebrafish tbx24 gene, hitherto considered to be a novel gene due to the high level of sequence divergence, is actually an ortholog of tetrapod Tbx6 genes. We will also show that, after their initial appearance by the multiplication of a common ancestral gene at the beginning of vertebrate evolution, the Tbx6/16 subfamily of vertebrate T-box genes might have experienced differential losses of member genes in different vertebrate groups and gradual pooling of member gene's functions in surviving members, which might have prevented the revelation of the true identity of member genes by way of the comparison of sequence and function.

Cug2 is essential for normal mitotic control and CNS development in zebrafish.

BACKGROUND: We recently identified a novel oncogene, Cancer-upregulated gene 2 (CUG2), which is essential for kinetochore formation and promotes tumorigenesis in mammalian cells. However, the in vivo function of CUG2 has not been studied in animal models. RESULTS: To study the function of CUG2 in vivo, we isolated a zebrafish homologue that is expressed specifically in the proliferating cells of the central nervous system (CNS). Morpholino-mediated knockdown of cug2 resulted in apoptosis throughout the CNS and the development of neurodegenerative phenotypes. In addition, cug2-deficient embryos contained mitotically arrested cells displaying abnormal spindle formation and chromosome misalignment in the neural plate. CONCLUSIONS: Therefore, our findings suggest that Cug2 is required for normal mitosis during early neurogenesis and has functions in neuronal cell maintenance, thus demonstrating that the cug2 deficient embryos may provide a model system for human neurodegenerative disorders.

The microcephaly gene aspm is involved in brain development in zebrafish.

MCPH is a neurodevelopmental disorder characterized by a global reduction in cerebral cortical volume. Homozygous mutation of the MCPH5 gene, also known as ASPM, is the most common cause of the MCPH phenotype. To elucidate the roles of ASPM during embryonic development, the zebrafish aspm was identified, which is specifically expressed in proliferating cells in the CNS. Morpholino-mediated knock-down of aspm resulted in a significant reduction in head size. Furthermore, aspm-deficient embryos exhibited a mitotic arrest during early development. These findings suggest that the reduction in brain size in MCPH might be caused by lack of aspm function in the mitotic cell cycle and demonstrate that the zebrafish can provide a model system for congenital diseases of the human nervous system.

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages.

During development of the limbs, Hox genes belonging to the paralogous groups 9-13 are expressed in three distinct phases, which play key roles in the segmental patterning of limb skeletons. In teleost fishes, which have a very different organization in their fin skeletons, it is not clear whether a similar patterning mechanism is at work. To determine whether Hox genes are also expressed in several distinct phases during teleost paired fin development, we re-analyzed the expression patterns of hox9-13 genes during development of pectoral fins in zebrafish. We found that, similar to tetrapod Hox genes, expression of hoxa/d genes in zebrafish pectoral fins occurs in three distinct phases, in which the most distal/third phase is correlated with the development of the most distal structure of the fin, the fin blade. Like in tetrapods, hox gene expression in zebrafish pectoral fins during the distal/third phase is dependent upon sonic hedgehog signaling (hoxa and hoxd genes) and the presence of a long-range enhancer (hoxa genes), which indicates that the regulatory mechanisms underlying tri-phasic expression of Hox genes have remained relatively unchanged during evolution. Our results suggest that, although simpler in organization, teleost fins do have a distal structure that might be considered comparable to the autopod region of limbs.

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans.

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.

T-box gene tbx5 is essential for formation of the pectoral limb bud.

The T-box genes Tbx4 and Tbx5 have been shown to have key functions in the specification of the identity of the vertebrate forelimb (Tbx5) and hindlimb (Tbx4). Here we show that in zebrafish, Tbx5 has an additional early function that precedes the formation of the limb bud itself. Functional knockdown of zebrafish tbx5 through the use of an antisense oligonucleotide resulted in a failure to initiate fin bud formation, leading to the complete loss of pectoral fins. The function of the tbx5 gene in the development of zebrafish forelimbs seems to involve the directed migration of individual lateral-plate mesodermal cells into the future limb-bud-producing region. The primary defect seen in the tbx5-knockdown phenotype is similar to the primary defects described in known T-box-gene mutants such as the spadetail mutant of zebrafish and the Brachyury mutant of the mouse, which both similarly exhibit an altered migration of mesodermal cells. A common function for many of the T-box genes might therefore be in mediating the proper migration and/or changes in adhesive properties of early embryonic cells.