Three linked enzyme loci in fishes: implications in the evolution of vertebrate chromosomes.

A three-point linkage group comprised of loci coding for adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phospho-gluconate dehydrogenase (6PGD) is described in fish of the genus Xiphophorus (Poeciliidae). The alleles at loci in this group were shown to assort independently from the alleles at three other loci--isocitrate dehydrogenase 1 and 2, and glyceraldehyde-3-phosphate dehydrogenase 1. Alleles at the latter three loci also assort independently from each other. Data were obtained by observing the segregation of electrophoretically variant alleles in reciprocal backcross hybrids derived from crosses between either X. helleri guentheri or X. h. strigatus and X. maculatus. The linkage component of chi2 was significant (less than 0.01) in all crosses, indicating that the linkage group is conserved in all populations of both species of Xiphophorus examined. While data from X. h. guentheri backcrosses indicate the linkage relationship ADA--6%--G6PDH--24%--6PGD, and ADA--29%--6PGD (30% when corrected for double crossovers), data from backcrosses involving strigatus, while supporting the same gene order, yielded significantly different recombination frequencies. The likelihood of the difference being due to an inversion could not be separated from the possibility of a sex effect on recombination in the present data. The linkage of 6PGD and G6PDH has been shown to exist in species of at least three classes of vertebrates, indicating the possibility of evolutionary conservation of this linkage.

Molecular basis for class V beta-tubulin effects on microtubule assembly and paclitaxel resistance.

Vertebrates produce at least seven distinct beta-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is beta5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of beta5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in beta5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into beta1-tubulin; yet the single mutation of residue Ser-239 in beta5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among beta-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of beta1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with beta1- but not with beta5-tubulin.

The molecular and genetic characterization of the MADS box transcription factor {\it Drosophila\/} MEF2

The myocyte enhancer factor (MEF)-2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates, but the precise position of these regulators within the genetic hierarchy leading to myogenesis is unclear. The MEF2 proteins bind to a conserved A/T-rich DNA sequence present in numerous muscle-specific genes, and they are expressed in the cells of the developing somites and in the embryonic heart at the onset of muscle formation in mammals. The MEF2 genes belong to the MADS box family of transcription factors, which control specific programs of gene expression in species ranging from yeast to humans. Each MEF2 family member contains two highly conserved protein motifs, the MADS domain and the MEF2-specific domain, which together provide the MEF2 factors with their unique DNA binding and dimerization properties. In an effort to further define the function of the MEF2 proteins, and to evaluate the degree of conservation shared among these factors and the phylogenetic pathways that they regulate, we sought to identify MEF2 family members in other species. In Drosophila, a homolog of the vertebrate MEF2 genes was identified and termed D-mef2. The D-MEF2 protein binds to the consensus MEF2 element and can activate transcription through tandem copies of that site. During Drosophila embryogenesis, D-MEF2 is specific to the mesoderm germ layer of the developing embryo and becomes expressed in all muscle cell types within the embryo. The role of D-mef2 in Drosophila embryogenesis was examined by generating a loss-of-function mutation in the D-mef2 gene. In embryos homozygous for this mutant allele, somatic, cardiac, and visceral muscles fail to differentiate, but precursors of these myogenic lineages are normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2 and suggest that this program has been conserved from Drosophila to mammals. ^

Functional studies of the homeobox gene {\it Goosecoid\/} during mouse embryogenesis

A fundamental question in developmental biology is to understand the mechanisms that govern the development of an adult individual from a single cell. Goosecoid (Gsc) is an evolutionarily conserved homeobox gene that has been cloned in vertebrates and in Drosophila. In mice, Gsc is first expressed during gastrulation stages where it marks anterior structures of the embryo, this pattern of expression is conserved among vertebrates. Later, expression is observed during organogenesis of the head, limbs and the trunk. The conserved pattern of expression of Gsc during gastrulation and gain of function experiments in Xenopus suggested a function for Gsc in the development of anterior structures in vertebrates. Also, its expression pattern in mouse suggested a role in morphogenesis of the head, limbs and trunk. To determine the functional requirement of Gsc in mice a loss of function mutation was generated by homologous recombination in embryonic stem cells and mice mutant for Gsc were generated.^ Gsc-null mice survived to birth but died hours after delivery. Phenotypic analysis revealed craniofacial and rib cage abnormalities that correlated with the second phase of Gsc expression in the head and trunk but no anomalies were found that correlated with its pattern of expression during gastrulation or limb development.^ To determine the mode of action of Gsc during craniofacial development aggregation chimeras were generated between Gsc-null and wild-type embryos. Chimeras were generated by the aggregation of cleavage stage embryos, taking advantage of two different Gsc-null alleles generated during gene targeting. Chimeras demonstrated a cell-autonomous function for Gsc during craniofacial development and a requirement for Gsc function in cartilage and mesenchymal tissues.^ Thus, during embryogenesis in mice, Gsc is not an essential component of gastrulation as had been suggested in previous experiments. Gsc is required for craniofacial development where it acts cell autonomously in cartilage and mesenchymal tissues. Gsc is also required for proper development of the rib cage but it is dispensable for limb development in mice. ^

The evolution of paired domains

Pax genes are important developmental control genes. They are involved in nervous system development, organogenesis and oncogenesis. A DNA specific binding domain called the paired domain, which is well conserved during evolution, defines Pax genes. Furthermore, Pax genes are also conserved in terms of their functions. For example, the Pax-6 gene has been showed to be one of the master control genes for eye development both in Drosophila and vertebrates. All of these properties of Pax genes make them an excellent model for studying the evolution of gene function. ^ Molecular evolutionary studies of paired domain are carried out in this study. Five Pax genes from cnidarians, which are the most primitive organisms possessing a nervous system, were isolated and characterized for their DNA binding properties. By combining data obtained from Genbank and this study, the phylogenetic relationship between Pax genes was studied. It was found that Pax genes could be divided into five groups: Pax-1/9, Pax-3 /7, Pax-A, Pax-2/5/ 8/B, and Pax- 4/6. Furthermore, Pax-2/5/8/ B, Pax-A and Pax-4/6 could be clustered into a supergroup I, while Pax-1/9 and Pax-3/7 could be clustered into supergroup II. The phylogeny was also supported by studies on DNA binding properties of paired domains from different groups. A statistical method was applied to infer the critical amino acid residue substitutions between two supergroups and within the supergroup I. It was found that two amino acid residues were mainly responsible for the difference of DNA binding between two supergroups, while only one amino acid was critical for the evolution of novel DNA binding properties of Pax-4/6 group from ancestor. Evolutionary implications of these data are also discussed. ^

The roles of Smads and Pitx2 in cardiac development

The heart is the first organ to form in vertebrates during embryogenesis, and its circulatory function is essential to embryonic survival. Cardiac morphogenesis comprises a complex series of interactions involving cells from several embryonic origins. These cell-cell interactions are regulated temporally and spatially by programs of inductive signaling events, including BMP signaling transduced by Smads and left-right asymmetry signaling mediated by Pitx2. Disruptions of BMP signaling and left-right asymmetry signaling result in abnormal cardiac morphogenesis that causes congenital heart disease in humans. In this study, conventional and conditional gene targeting approaches were employed to dissect the functions of Smad8 and Smad1, intracellular BMP signaling transducers, and Pitx2, a direct target of left-right signaling, in cardiac development. We generated the Smad8mt mutant allele and the Smad8lacZ knock-in allele. Smad8 homozygous mutant mice were viable and fertile without obvious abnormalities. The Smad8lacZ knock-in allele showed that Smad8 was expressed in the myocardium of cardiac outflow tract and atrioventricular cushions. We did not find defects in these Smad8-expressing cardiac regions in Smad8mt/mt and Smad8lacZ/lacZ mutants, indicating that Smad8 is dispensable for cardiac development. Conditional knockout of Smad1 using the Nkx2.5Cre allele in cardiac mesoderm resulted in partial inactivation of Smad1 in the myocardium and complete deletion of Smad1 in the epicardium, and caused ventricular hypoplasia featured with a thinner compact zone, suggesting that Smad1 signaling in the epicardium is required for myocardial morphogenesis in ventricles. Previous data have shown that Pitx2 null mutants exhibit defects in the cardiac outflow tract, a region populated with cells from the cardiac mesoderm and the cardiac neural crest. We found that the cardiac neural crest normally populated into the outflow tract in Pitx2 null mutant. Moreover, specific deletion of Pitx2 in the neural crest resulted in normal heart formation. Deletion of Pitx2 in the cardiac mesoderm caused defective outflow tract, revealing that the function of Pitx2 in the cardiac outflow tract resides in splanchnic and branchial arch mesoderm, and is independent of cardiac neural crest cells. ^

Identification and characterization of mutations in SMC contractile genes involved in thoracic aortic aneurysms and dissections

Aortic aneurysms and dissections are the 15th most common cause of death in the United States. Genetic factors contribute to the pathogenesis of thoracic aortic aneurysms and dissections (TAAD). Currently, six loci and four genes have been identified for familial TAAD. Notably, mutations in smooth muscle cell (SMC) contractile genes, ACTA2 and MYH11, are responsible for 15% of familial TAAD, suggesting that proper SMC contraction is important for normal aorta function. Therefore, we hypothesize that mutations in other genes encoding SMC contractile proteins also cause familial TAAD. ^ To test this hypothesis, we used a candidate gene approach to identify causative mutations in SMC contractile genes for familial TAAD. Sequencing DNA in 80 TAAD patients from unrelated families, we identified putative mutations in eight contractile genes. We chose myosin light chain kinase (MLCK ) S1759P for further study for the following reasons: (1) Serine 1759 is conserved between vertebrates and invertebrates. (2) S1759P is predicted to be functionally deleterious by bioinformatics. (3) Low blood pressure is observed in SMC-selective MLCK-deficient mice. ^ In the presence of Ca2+/Calmodulin (CaM), MLCK containing CaM binding and kinase domains are activated to phosphorylate myosin light chain, thereby initiate SMC contraction. The CaM binding sequence of MLCK forms an α-helix structure required for CaM binding. MLCK Serine 1759 is located within the CaM binding domain. S1759P is predicted to decrease the α-helix composition in the CaM binding domain. Hence, we hypothesize that MLCK mutations cause TAAD through disturbing CaM binding and MLCK activity. ^ We further sequenced MLCK in DNA samples from additional 86 probands with familial TAAD. Two more mutations, MLCK A1754T and R1480Stop, were identified, supporting that MLCK mutations cause familial TAAD. ^ To define whether MLCK mutations disrupted CaM binding and MLCK activity, we performed co-immunoprecipitation and kinase assays. Decreased CaM binding and kinase activity was detected in A1754T and S1759P. Moreover, R1480Stop is predicted to truncate kinase and CaM binding domains. We conclude that MLCK mutations disrupt CaM binding and MLCK activity. ^ Collectively, our study is first to show mutations in genes regulating SMC contraction cause TAAD. This finding further highlights the importance of SMC contraction in maintaining aorta function. ^