Comprehensive analyses of hox gene expression in Xenopus laevis embryos and adult tissues

From whole genome sequencing of an allotetraploid frog, Xenopus laevis, two homeologous sets (L and S) of four Hox clusters A through D (HoxA.L/S, HoxB.L/S, HoxC.L/S, and HoxD.L/S) and 13 paralogous groups (PGs) with 76 genes were identified, allowing us to carry out the first comprehensive analyses of hox gene expression in vertebrates. Expression of all hox genes during development and in adult tissues was analyzed by RNA‐sequencing. The expression levels of most hox genes were similar between homeologs, but in some pairs, large differences were observed and several of these were confirmed by RT‐PCR and whole mount in situ hybridization experiments. These results indicate that subfunctionalization of hox genes may have occurred since allotetraploidization. Furthermore, comprehensive analysis of hox gene expression during early development did not agree with the hypothesis of temporal collinearity especially in genes belonging to PG2 to PG10 .     

Inhibition of gastrulation in Xenopus embryos by an antibody against a cathepsin L-like protease

An antibody against cathepsin L-like protease (AACLP) was injected into one cell of 2-celled Xenopus embryos. The blastopores of AACLP-injected embryos either did not invaginate or failed to complete invagination. As a result of this failure to complete gastrulation, the body axes could not form normally and tail bud stage embryos were bent dorsally. Embryos injected with a control antibody (CA) developed normally through the tadpole stage. Mesodermal induction was not inhibited in embryos exhibiting this AACLP-induced gastrulation defect, but the mesodermal structure of these embryos was organized incorrectly due to the defective gastrulation during the early stages.     

Inferring the evolutionary history of Drosophila americana and Drosophila novamexicana using a multilocus approach and the influence of chromosomal rearrangements in single gene analyses

The evolutionary history of closely related organisms can prove sometimes difficult to infer. Hybridization and incomplete lineage sorting are the main concerns; however, genome rearrangements can also influence the outcome of analyses based on nuclear sequences. In the present study, DNA sequences from 12 nuclear genes, for which the approximate chromosomal locations are known, have been used to estimate the evolutionary history of two forms of Drosophila americana ( Drosophila   americana americana and Drosophila americana texana ) and Drosophila novamexicana ( virilis group of species). The phylogenetic analysis of the combined data set resulted in a phylogeny showing reciprocal monophyly for D. novamexicana and D. americana . Single gene analyses, however, resulted in incongruent phylogenies influenced by chromosomal rearrangements. Genetic differentiation estimates indicated a significant differentiation between the two species for all genes. Within D. americana , however, there is no evidence for differentiation between the chromosomal forms except at genes located near the X/4 fusion and Xc inversion breakpoint. Thus, the specific status of D. americana and D. novamexicana is confirmed, but there is no overall evidence for genetic differentiation between D. a. americana and D. a. texana , not supporting a subspecific status. Based on levels of allele and nucleotide diversity found in the strains used, it is proposed that D. americana has had a stable, large population during the recent past while D. novamexicana has speciated from a peripheral southwestern population having had an ancestral small effective population size. The influence of chromosomal rearrangements in single gene analyses is also examined.     

DEAF-1 function is essential for the early embryonic development of Drosophila

The Drosophila protein DEAF-1 is a sequence-specific DNA binding protein that was isolated as a putative cofactor of the Hox protein Deformed (Dfd). In this study, we analyze the effects of loss or gain of DEAF-1 function on Drosophila development. Maternal/zygotic mutations of DEAF-1 largely result in early embryonic arrest prior to the expression of zygotic segmentation genes, although a few embryos develop into larvae with segmentation defects of variable severity. Overexpression of DEAF-1 protein in embryos can induce defects in migration/closure of the dorsal epidermis, and overexpression in adult primordia can strongly disrupt the development of eye or wing. The DEAF-1 protein associates with many discrete sites on polytene chromosomes, suggesting that DEAF-1 is a rather general regulator of gene expression.     

Zebrafish hox paralogue group 2 genes function redundantly as selector genes to pattern the second pharyngeal arch

The pharyngeal arches are one of the defining features of the vertebrates, with the first arch forming the mandibles of the jaw and the second forming jaw support structures. The cartilaginous elements of each arch are formed from separate migratory neural crest cell streams, which derive from the dorsal aspect of the neural tube. The second and more posterior crest streams are characterized by specific Hox gene expression. The zebrafish has a larger overall number of Hox genes than the tetrapod vertebrates, as the result of a duplication event in its lineage. However, in both zebrafish and mouse, there are just two members of Hox paralogue group 2 (PG2): Hoxa2 and Hoxb2. Here, we show that morpholino-mediated "knock-down" of both zebrafish Hox PG2 genes results in major defects in second pharyngeal arch cartilages, involving replacement of ventral elements with a mirror-image duplication of first arch structures, and accompanying changes to pharyngeal musculature. In the mouse, null mutants of Hoxa2 have revealed that this single Hox gene is required for normal second arch patterning. By contrast, loss-of-function of either zebrafish Hox PG2 gene individually has no phenotypic consequence, showing that these two genes function redundantly to confer proper pattern to the second pharyngeal arch. We have also used hoxb1a mis-expression to induce localized ectopic expression of zebrafish Hox PG2 genes in the first arch; using this strategy, we find that ectopic expression of either Hox PG2 gene can confer second arch identity onto first arch structures, suggesting that the zebrafish Hox PG2 genes act as "selector genes."     

Involvement of a urethane-sensitive system in timing the onset of gastrulation in Xenopus laevis embryos

This paper describes success in delaying the onset of gastrulation in Xenopus laevis embryos without damage to their subsequent development by temporarily arresting cleavage with urethane. Exposure of X. laevis embryos to 150 mM urethane before gastrulation resulted in cleavage arrest and its removal led to cleavage resumption. During cleavage arrest, cyclic activities including nuclear replication and the M-phase-promoting factor cycle continued, although their duration was lengthened to nearly 1.8-fold that of the controls. Because of a 30-min time lag from removal of urethane to resumption of cleavage, as well as the retardation of cyclic activities during cleavage arrest, the development of embryos after a 60-min exposure to urethane lagged two cell cycles behind that of control embryos. Here, the two cell cycle delay is equivalent to 50 min at 22-23 degrees C. The start of gastrulation in exposed embryos was accordingly delayed about 50 min, although the delay in mid-blastula transition was as little as 20-25 min. Consistent results were obtained in embryos exposed to urethane for 90 or 120 min and those exposed to procaine or NH4Cl for 60 min. Although these results imply that delay in the start of gastrulation in exposed embryos is ascribed simply to delay in their development raised by cleavage arrest, at the same time they suggest that the onset of gastrulation is timed by systems sensitive to urethane, procaine and NH4Cl in X. laevis embryos.     

Timing system for the start of gastrulation in the Xenopus embryo

This study examined which component of the egg, the nucleus or cytoplasm, is involved in the timing of the start of gastrulation in the Xenopus embryo, and when it starts to measure time. First, nuclei of cells of 256-cell stage embryos were transplanted to enucleated eggs 60 min after activation. These eggs showed first cleavage 20-30 min later than control eggs fertilized at the same time as the activation of recipient eggs, and started gastrulation 25-35 min later than control embryos (depending on the delay in the first cleavage). Second, eggs whose nuclei were temporarily isolated by the extrusion of the portion containing the nucleus out of the fertilization envelope showed first cleavage 60-90 min later than sibling control eggs, because of delayed introduction of the nucleus from the extruded portion. They started gastrulation 60-90 min later than sibling control embryos (depending on the delay in the first cleavage). The portion inside the envelope underwent two to three rounds of oscillation in cell cycle relevant activities before the first cleavage, while the portion outside underwent the same rounds of cleavage as the inside portion. From the present and previous results it is concluded that the putative timing system for the start of gastrulation in the Xenopus embryo, whether it consists of a single or of multiple clocks, starts measuring time at or around the first cleavage, and that the presence of both the nucleus and the cytoplasm in the same cell and occurrence of mitosis and/or cleavage there are indispensable for the timing system to work, although the role of the cytoplasm is superior to that of the nucleus.     

Localization and behavior of putative blastopore determinants in the uncleaved Xenopus egg

The present study examines putative blastopore determinants in uncleaved Xenopus eggs. Deletion of marginal and lower portions of Xenopus eggs when between 30 and 50% of the first cell cycle has been completed (0.3-0.5 normalized time (NT)) results in the complete absence of the blastopore, while deletion of the vegetal hemisphere during the same period leads to the formation of a smaller blastopore. Extrusion of only yolk and deep cytoplasm of the vegetal hemisphere during 0.3-0.5 NT does not affect the formation or size of the blastopore. Consistently, transplantation of cortical and subcortical cytoplasm from marginal, but not other, sites of eggs at 0.3-0.5 NT to an animal blastomere from 16-cell stage embryos induces an ectopic blastopore and bottle cell-like cells. This does not occur in the same transplantation from eggs at 0.2 NT. These results suggest that the blastopore determinants become localized to the marginal cortical and/or subcortical cytoplasm during 0.2-0.3 NT. Other results suggest the involvement of a hexyleneglycol-sensitive system in the process of localization of the blastopore determinants to the marginal region during 0.2-0.3 NT. The properties and behavior of the putative blastopore determinants are discussed in relation to those of VegT, which previously has been shown to induce ectopic blastopores.     

Sex differences in vulnerability and maladjustment as a function of parental investment: an evolutionary approach.

Sex differences in aspects of mental health are examined as a function of uneven parental investment in children. Relative vulnerability is a new construct mediating the influence of parental investment on mental health. Couples (129) in three stages of the family life cycle are measured by scales for parental investment, relative vulnerability, anxiety, depression, and ten psychosomatic syndromes. Results show a path of positive correlations from the parent's sex to level of parental investment, to level of relative vulnerability, and to levels of anxiety and depression. Women invest more than men, and hence they are more vulnerable, anxious, and depressed. They reach the summit of their vulnerability while they have three young children. Relative vulnerability was found to have positive effects along with the negative ones and to affect women in different ways than it does men. Results are interpreted in terms of different parental strategies selected by evolution for each sex.     

Hox genes and the regulation of movement in Drosophila

Many animals show regionally specialized patterns of movement along the body axis. In vertebrates, spinal networks regulate locomotion, while the brainstem controls movements of respiration and feeding. Similarly, amongst invertebrates diversification of appendages along the body axis is tied to the performance of characteristically different movements such as those required for feeding, locomotion, and respiration. Such movements require locally specialized networks of nerves and muscles. Here we use the regionally differentiated movements of larval crawling in Drosophila to investigate how the formation of a locally specialized locomotor network is genetically determined. By loss and gain of function experiments we show that particular Hox gene functions are necessary and sufficient to dictate the formation of a neuromuscular network that orchestrates the movements of peristaltic locomotion.     

Novel double promoter approach for identification of transgenic animals: A tool for in vivo analysis of gene function and development of gene-based therapies

Advances in vertebrate genetics have allowed studies of gene function in developing animals through gene knockout and transgenic analyses. These advances have encouraged the development of gene-based therapies through introduction of exogenous genes to enhance and/or replace dysfunctional or missing genes. However, in vertebrates, such analyses often involve tedious screening for transgenic animals, such as PCR-based genotype determinations. Here, we report the use of double-promoter plasmids carrying the transgene of interest and the crystallin-promotor-driven Green fluorescent protein (GFP) in transgenic Xenopus laevis tadpoles. This strategy allows a simple examination for the presence of GFP in the eyes to identify transgenic animals. PCR-based genotyping and functional characterization confirms that all animals expressing GFP in the eyes indeed carry the desired promoter/transgene units. Thus, the use of this and other similar vectors should dramatically improve current transgenesis protocols and reduce the time and cost for identifying transgenic animals.     

Uncovering the structure and function of Pseudomonas aeruginosa periplasmic proteins by an in silico approach

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen highly relevant from a biomedical viewpoint. It is one of the main causes of infection in hospitalized patients and a major cause of mortality of cystic fibrosis patients. This is also due to its ability to develop resistance to antibiotics by various mechanisms. Therefore, it is urgent and desirable to identify novel targets for the development of new antibacterial drugs against Pseudomonas aeruginosa. In this work this problem was tackled by an in silico approach aimed at providing a reliable structural model and functional annotation for the Pseudomonas aeruginosa periplasmic proteins for which these data are not available yet. A total of 83 protein sequences were analyzed, and the corresponding structural models were built, leading to the identification of 32 periplasmic ‘substrate-binding proteins’, 14 enzymes and 4 proteins with different functions, including lipids and metals binding. The most interesting cases were found within the ‘enzymes’ group with the identification of a lipase, which can be regarded as a virulence factor, a protease involved in the assembly of β-barrel membrane proteins and a l,d-transpeptidase, which could contribute to confer resistance to β-lactam antibiotics to the bacterium.

Communicated by Ramaswamy H. Sarma