Biomedical subjects

Carlos E Vanario-Alonso

Publications and source records attributed to Carlos E Vanario-Alonso.

7 recordsLinked to original sources

PubMed2006

Analysis of pattern precision shows that Drosophila segmentation develops substantial independence from gradients of maternal gene products.

We analyze the relation between maternal gradients and segmentation in Drosophila, by quantifying spatial precision in protein patterns. Segmentation is first seen in the striped expression patterns of the pair-rule genes, such as even-skipped (eve). We compare positional precision between Eve and the maternal gradients of Bicoid (Bcd) and Caudal (Cad) proteins, showing that Eve position could be initially specified by the maternal protein concentrations but that these do not have the precision to specify the mature striped pattern of Eve. By using spatial trends, we avoid possible complications in measuring single boundary precision (e.g., gap gene patterns) and can follow how precision changes in time. During nuclear cleavage cycles 13 and 14, we find that Eve becomes increasingly correlated with egg length, whereas Bcd does not. This finding suggests that the change in precision is part of a separation of segmentation from an absolute spatial measure, established by the maternal gradients, to one precise in relative (percent egg length) units.

Animals↗

PubMed2005

Bicoid cooperative DNA binding is critical for embryonic patterning in Drosophila.

Cooperative interactions by DNA-binding proteins have been implicated in cell-fate decisions in a variety of organisms. To date, however, there are few examples in which the importance of such interactions has been explicitly tested in vivo. Here, we tested the importance of cooperative DNA binding by the Bicoid protein in establishing a pattern along the anterior-posterior axis of the early Drosophila embryo. We found that bicoid mutants specifically defective in cooperative DNA binding fail to direct proper development of the head and thorax, leading to embryonic lethality. The mutants did not faithfully stimulate transcription of downstream target genes such as hunchback (hb), giant, and Krüppel. Quantitative analysis of gene expression in vivo indicated that bcd cooperativity mutants were unable to accurately direct the extent to which hb is expressed along the anterior-posterior axis and displayed a reduced ability to generate sharp on/off transitions for hb gene expression. These failures in precise transcriptional control demonstrate the importance of cooperative DNA binding for embryonic patterning in vivo.

Allosteric Regulation↗

PubMed2005

A kinetic mechanism for Drosophila bicoid cooperative binding.

The Bicoid (Bcd) protein is a concentration-dependent transcriptional activator in the embryo of Drosophila melanogaster. Bcd regulates the expression of the maternal and zygotic gene hunchback (hb) that shows a step-like-function expression pattern, in the anterior half of the egg. The regulatory region of hb contains six major binding sites for the Bcd protein, named A1, A2, A3 (strong sites), and X1, X2, X3 (weak sites). Cooperativity between Bcd molecules binding to the hb enhancer element has been characterized as an important mechanism for the step-like shape of hb anterior expression domain. The objective of the present report is to analyse the mechanism of this cooperative binding based on a reaction network model. Using this method we have analysed experimental results from the literature describing how the Bcd protein binds to hb enhancer elements containing the A1 or X1 site alone or these two sites together at wild type distance. This approach allows us to estimate the kinetic constants of protein-protein and protein-DNA interactions. Moreover our results suggest that binding of a Bcd dimer to the hb enhancer element is more stable than binding of a monomer. We propose a cooperative kinetic mechanism for binding of Bcd to the hb enhancer element: First, a monomer binds to the site with a relatively low affinity; after that, another monomer binds to the first one with higher affinity, generating a dimer bound to the site. This yet unreported monomer-monomer cooperative mechanism takes place for occupancy of either one-site or two-site enhancer elements. In addition, we find cooperativity between neighbor sites, as previously reported in the literature.

Animals↗

PubMed2005

A high-throughput method for quantifying gene expression data from early Drosophila embryos.

We describe an automated high-throughput method to measure protein levels in single nuclei in blastoderm embryos of Drosophila melanogaster by means of immunofluorescence. The method consists of a chain of specific algorithms assembled into an image processing pipeline. This pipeline transforms a confocal scan of an embryo stained with fluorescently tagged antibodies into a text file. This text file contains a numerical identifier for each nucleus, the coordinates of its centroid, and the average concentrations of three proteins in that nucleus. The central algorithmic component of the method is the automatic identification of nuclei by edge detection with the use of watersheds as an error-correction step. This method provides high-throughput quantification at cellular resolution.

Algorithms↗

PubMed2004

Dynamic control of positional information in the early Drosophila embryo.

Morphogen gradients contribute to pattern formation by determining positional information in morphogenetic fields. Interpretation of positional information is thought to rely on direct, concentration-threshold-dependent mechanisms for establishing multiple differential domains of target gene expression. In Drosophila, maternal gradients establish the initial position of boundaries for zygotic gap gene expression, which in turn convey positional information to pair-rule and segment-polarity genes, the latter forming a segmental pre-pattern by the onset of gastrulation. Here we report, on the basis of quantitative gene expression data, substantial anterior shifts in the position of gap domains after their initial establishment. Using a data-driven mathematical modelling approach, we show that these shifts are based on a regulatory mechanism that relies on asymmetric gap-gap cross-repression and does not require the diffusion of gap proteins. Our analysis implies that the threshold-dependent interpretation of maternal morphogen concentration is not sufficient to determine shifting gap domain boundary positions, and suggests that establishing and interpreting positional information are not independent processes in the Drosophila blastoderm.

Animals↗

PubMed2004

Dynamical analysis of regulatory interactions in the gap gene system of Drosophila melanogaster.

Genetic studies have revealed that segment determination in Drosophila melanogaster is based on hierarchical regulatory interactions among maternal coordinate and zygotic segmentation genes. The gap gene system constitutes the most upstream zygotic layer of this regulatory hierarchy, responsible for the initial interpretation of positional information encoded by maternal gradients. We present a detailed analysis of regulatory interactions involved in gap gene regulation based on gap gene circuits, which are mathematical gene network models used to infer regulatory interactions from quantitative gene expression data. Our models reproduce gap gene expression at high accuracy and temporal resolution. Regulatory interactions found in gap gene circuits provide consistent and sufficient mechanisms for gap gene expression, which largely agree with mechanisms previously inferred from qualitative studies of mutant gene expression patterns. Our models predict activation of Kr by Cad and clarify several other regulatory interactions. Our analysis suggests a central role for repressive feedback loops between complementary gap genes. We observe that repressive interactions among overlapping gap genes show anteroposterior asymmetry with posterior dominance. Finally, our models suggest a correlation between timing of gap domain boundary formation and regulatory contributions from the terminal maternal system.

Animals↗

PubMed2002

Sharp borders from fuzzy gradients.

Critical boundaries in the early Drosophila embryo are set by morphogenetic gradients. A new quantitative study shows that the placement of one such boundary is more accurate than the gradient thought to set it. Genetic analysis of the accuracy of the process implicates a gene not previously thought to be involved.

Animals↗