Rolando Rivera-Pomar

From gradients to stripes in Drosophila embryogenesis: filling in the gaps

Pattern formation along the anterior-posterior axis of the Drosophila embryo is organized by asymmetrically distributed maternal transcription factors. They initiate a cascade of spatially restricted and interacting zygotic gene activities that provide a molecular blueprint of the larval body at blastoderm stage. The key players in the pattern forming process have been identified. Recent progress has begun to reveal the mechanisms by which coherent positional information of maternal origin becomes transferred into serially repeated zygotic gene expression domains reflecting the metameric body plan of the larva.

Cooperative DNA-binding by Bicoid provides a mechanism for threshold-dependent gene activation in the Drosophila embryo

The Bicoid morphogen directs pattern formation along the anterior–posterior (A–P) axis of the Drosophila embryo. Bicoid is distributed in a concentration gradient that decreases exponentially from the anterior pole, however, it transcribes target genes such as hunchback in a step‐function‐like pattern; the expression domain is uniform and has a sharply defined posterior boundary. A ‘gradient‐affinity’ model proposed to explain Bicoid action states that (i) cooperative gene activation by Bicoid generates the sharp on/off switch for target gene transcription and (ii) target genes with different affinities for Bicoid are expressed at different positions along the A–P axis. Using an in vivo yeast assay and in vitro methods, we show that Bicoid binds DNA with pairwise cooperativity; Bicoid bound to a strong site helps Bicoid bind to a weak site. These results support the first aspect of the model, providing a mechanism by which Bicoid generates sharp boundaries of gene expression. However, contrary to the second aspect of the model, we find no significant difference between the affinity of Bicoid for the anterior gene hunchback and the posterior gene knirps. We propose, instead, that the arrangement of Bicoids bound to the target gene presents a unique signature to the transcription machinery that, in combination with overall affinity, regulates the extent of gene transcription along the A–P axis.

Functional analysis of seven genes encoding eight translation initiation factor 4E (eIF4E) isoforms in Drosophila

The Drosophila genome-sequencing project has revealed a total of seven genes encoding eight eukaryotic initiation factor 4E (eIF4E) isoforms. Four of them (eIF4E-1,2, eIF4E-3, eIF4E-4 and eIF4E-5) share exon/intron structure in their carboxy-terminal part and form a cluster in the genome. All eIF4E isoforms bind to the cap (m7GpppN) structure. All of them, except eIF4E-6 and eIF4E-8 were able to interact with Drosophila eIF4G or eIF4E-binding protein (4E-BP). eIF4E-1, eIF4E-2, eIF4E-3, eIF4E-4 and eIF4E-7 rescued a yeast eIF4E-deficient mutant in vivo. Only eIF4E-1 mRNAs and, at a significantly lower level, eIF4E3 and eIF4E-8 are expressed in embryos and throughout the life cycle of the fly. The transcripts of the remaining isoforms were detected from the third instar larvae onwards. This indicates the cap-binding activity relies mostly on eIF4E-1 during embryogenesis. This agrees with the proteomic analysis of the eIF4F complex purified from embryos and with the rescue of l(3)67Af, an embryonic lethal mutant for the eIF4E-1,2 gene, by transgenic expression of eIF4E-1. Overexpression of eIF4E-1 in wild-type embryos and eye imaginal discs results in phenotypic defects in a dose-dependent manner.

Sequence interval within the PEST motif of Bicoid is important for translational repression of caudal mRNA in the anterior region of the Drosophila embryo.

The Drosophila body organizer Bicoid (Bcd) is a maternal homeodomain protein. It forms a concentration gradient along the longitudinal axis of the preblastoderm embryo and activates early zygotic segmentation genes in a threshold‐dependent fashion. In addition, Bcd acts as a translational repressor of maternal caudal (cad) mRNA in the anterior region of the embry o. This process involves a distinct Bcd‐binding region (BBR) in the 3′ untranslated region (UTR) of cad mRNA. Using cotransfection assays, we found that Bcd represses translation in a cap‐dependent manner. Bcd‐dependent translational repression involves a portion of the PEST motif of Bcd, a conserved protein motif best known for its function in protein degradation. Rescue experiments with Bcd‐deficient embryos expressing transgene‐derived Bcd mutants indicate that amino acid replacements within the C‐terminal portion of the PEST motif prevent translational repression of cad mRNA but allow for Bcd‐dependent transcriptional activation. Thus, Bcd contains separable protein domains for transcriptional and translational regulation of target genes. Maternally‐derived cad protein in the anterior region of embryos interferes with head morphogenesis, showing that cad mRNA suppression by Bcd is an important control event during early Drosophila embryogenesis.

Mechanism and Bicoid‐dependent control of hairy stripe 7 expression in the posterior region of the Drosophila embryo

Pair‐rule gene hairy (h) expression in seven evenly spaced stripes, along the longitudinal axis of the Drosophila blastoderm embryo, is mediated by a modular array of separate stripe enhancer elements. The minimal enhancer element, which generates reporter gene expression in place of the most posterior h stripe 7 (h7‐element), contains a dense array of binding sites for factors providing the trans‐acting control of h stripe 7 expression as revealed by genetic analyses. The h7‐element mediates position‐dependent gene expression by sensing region‐specific combinations and concentrations of both the maternal homeodomain transcriptional activators, Caudal and Bicoid, and of transcriptional repressors encoded by locally expressed zygotic gap genes. Caudal and Bicoid, which form complementing concentration gradients along the longitudinal axis of the embryo, function as redundant activators, indicating that the anterior determinant Bicoid is able to activate gene expression in the most posterior region of the embryo. The spatial limits of the h stripe 7 domain are brought about by the local activities of repressors which prevent activation. The results suggest that the gradients of Bicoid and Caudal combine their activities to activate segmentation genes along the entire axis of the embryo.

A cascade of transcriptional control leading to axis determination in Drosophila

embryo develops through a se- tion (7,8), thus complementing the gradient of Bicoidries of rapid syncytial nuclear divisions. The nuclei mi- (Fig. 1a).grate to the egg membrane at the periphery to form The third maternal transcription factor is the zincindividualcells.Thisformationis achievedbythemem- finger protein Hunchback, which becomes asymmetri-brane extending between the nuclei to generate a sin- callydistributedinresponseto

Activation of posterior pair-rule stripe expression in response to maternal caudal and zygotic knirps activities.

Drosophila pair-rule gene expression, in an array of seven evenly spaced stripes along the anterior-posterior axis of the blastoderm embryo, is controlled by distinct cis-acting stripe elements. In the anterior region, such elements mediate transcriptional activation in response to the maternal concentration gradient of the anterior determinant BICOID and repression by spatially distinct activities of zygotic gap genes. In the posterior region, activation of hairy stripe 6 has been shown to depend on the activity of the gap gene knirps, suggesting that posterior stripe expression is exclusively controlled by zygotic regulators. Here we show that the zygotic activation of hairy stripe 6 expression is preceded by activation in response to maternal caudal activity. Thus, transcriptional activation of posterior stripe expression is likely to be controlled by maternal and zygotic factors as has been observed for anterior stripes. The results suggest that activation and the expression level mediated by the hairy stripe 6-element depend on the number of activator binding sites, likely to involve additive rather than synergistic interactions. We found an identical transacting factor requirement for hairy stripe 6 and 7 expression. The arrangement of the corresponding binding sites for the common factors involved in the control of the two stripes share a high degree of similarity, but some of the factors exert opposite regulatory functions within the two enhancer elements.

Functional proteomics of neuropeptidome dynamics during the feeding process of Rhodnius prolixus.

In hematophagous insects, blood intake triggers a prompt response mediated by neuropeptides, which regulates a variety of physiological processes. Here we report a quantitative proteomic analysis of the postfeeding response in the central nervous system of Rhodnius prolixus, a vector of Chagas disease. The concentration of neuropeptides NVP-like, ITG-like, kinin-precursor peptide, and neuropeptide-like precursor 1 (NPLP1) significantly changes in response to blood intake. We also performed a neuropeptidomic analysis of other feeding-related organs, namely salivary glands and gut. We identified NPLP1 in salivary glands and myosuppressin in midgut. This is the first report suggesting a role for NPLP1, involving the peptides processed from this precursor in the hormonal control of the production and/or release of saliva. Our results contribute to the understanding of the postprandial neuroendocrine response in hematophagous and provide important information for physiological and pharmacological studies aimed to the design of next-generation insecticides such as peptidomimetics.

Erratum: Functional analysis of seven genes encoding eight translation initiation factor 4E (eIF4E) isoforms in Drosophila (Mechanisms of Development (2005) 122 (529-543) DOI: 10.1016/j.mod.2004.11.011)

a Max-Planck-Institut für Biophysikalische Chemie, Abt. Molekulare Biologie, Am Fassberg 11, 37077 Göttingen, Germany b Institut für Biochemie und Molekularbiologie, Universität Bern, Bühlstrasse 28, 3012 Bern, Switzerland c Centro de Biologı́a Molecular “Severo Ochoa”, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain d Max-Planck-Institut für Biophysikalische Chemie, Abt. Zelluläre Biochemie, Am Fassberg 11, 37077 Göttingen, Germany e School of Life Sciences, University of Dundee, Wellcome Trust Biocenter, Dundee DD1 5EH, Scotland, UK f Zentrum Anatomie, Abt. Anatomie-Embryologie, Georg-August-Universität Göttingen, Kreuzbergring 36, 37075 Göttingen, Germany

Scanning near-field optical microscopy and microspectroscopy of green fluorescent protein in intact Escherichia coli bacteria

Scanning near-field optical microscopy (SNOM) yields high-resolution topographic and optical information and constitutes an important new technique for visualizing biological systems. By coupling a spectrograph to a near-field microscope, we have been able to perform microspectroscopic measurements with a spatial resolution greatly exceeding that of the conventional optical microscope. Here we present SNOM images of Escherichia coli bacteria expressing a mutant green fluorescent protein (GFP), an important reporter molecule in cell, developmental, and molecular biology. Near-field emission spectra confirm that the fluorescence detected by SNOM arises from bacterially expressed GFP molecules.