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Stephen Small

Publications and source records attributed to Stephen Small.

10 recordsLinked to original sources
PubMed2006

Developmental mechanisms and cis-regulatory codes.

Complex networks of transcriptional interactions control the processes of animal development. These networks begin with broad positional information that patterns the cells of the early embryo, and end with precise expression profiles that provide the functions of fully differentiated cells. At the heart of these networks are cis-regulatory modules (CRMs), which contain binding sites for regulatory proteins and control the spatial and temporal expression of genes within the network. Recent studies in several model systems have begun to decipher the 'cis-regulatory codes' of CRMs involved in various developmental processes. These studies suggest that CRMs involved in regulating co-expressed genes share sequence characteristics that can be identified by in silico approaches. They also suggest that CRMs involved in specific types of developmental events have common binding site architectures, which can be linked to their specific functions.

Animals↗
PubMed2006

Morphogens: precise outputs from a variable gradient.

The morphogen gradient as a source of embryonic patterning is one of the best accepted concepts in developmental biology. Morphogens can be transcription factors or extracellular signals, but in both cases, they are thought to provide concentration thresholds that position different cell fates within the developing embryo. Several recent papers examine the patterning activities of Drosophila Bicoid, the first known molecular morphogen and reach different conclusions about the patterning power of a single morphogen gradient.

Animals↗
PubMed2005

The role of binding site cluster strength in Bicoid-dependent patterning in Drosophila.

The maternal morphogen Bicoid (Bcd) is distributed in an embryonic gradient that is critical for patterning the anterior-posterior (AP) body plan in Drosophila. Previous work identified several target genes that respond directly to Bcd-dependent activation. Positioning of these targets along the AP axis is thought to be controlled by cis-regulatory modules (CRMs) that contain clusters of Bcd-binding sites of different "strengths." Here we use a combination of Bcd-site cluster analysis and evolutionary conservation to predict Bcd-dependent CRMs. We tested 14 predicted CRMs by in vivo reporter gene assays; 11 show Bcd-dependent activation, which brings the total number of known Bcd target elements to 21. Some CRMs drive expression patterns that are restricted to the most anterior part of the embryo, whereas others extend into middle and posterior regions. However, we do not detect a strong correlation between AP position of target gene expression and the strength of Bcd site clusters alone. Rather, we find that binding sites for other activators, including Hunchback and Caudal correlate with CRM expression in middle and posterior body regions. Also, many Bcd-dependent CRMs contain clusters of sites for the gap protein Kruppel, which may limit the posterior extent of activation by the Bcd gradient. We propose that the key design principle in AP patterning is the differential integration of positive and negative transcriptional information at the level of individual CRMs for each target gene.

Animals↗
PubMed2005

Site-specific transgenesis by Cre-mediated recombination in Drosophila.

Transposons such as P elements are routinely used to stably transfer exogenous DNA (transgenes) into the Drosophila genome. Transgene insertion events, however, are essentially random and are subject to 'position effects' from nearby endogenous regulatory elements. Here we describe a microinjection-based system that uses Cre-mediated recombination to insert transgenes into precise genomic 'landing sites'. The system is simple and efficient, and will permit precise comparisons between multiple transgenic constructs.

Animals↗
PubMed2005

A major role for zygotic hunchback in patterning the Nasonia embryo.

Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. Here, we focus on a comparative analysis of Nasonia hunchback function and expression. We find that a lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, we find that it is strongly expressed maternally, as well as zygotically. Nasonia Hunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. We also find that the maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Finally, we extend the comparisons of Nasonia and Drosophila hunchback mutant phenotypes, and propose that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry.

Animals↗
PubMed2004

Groucho-dependent repression by sloppy-paired 1 differentially positions anterior pair-rule stripes in the Drosophila embryo.

The Drosophila body plan is composed of a linear array of cephalic, thoracic, and abdominal segments along the anterior posterior axis. The number and positions of individual segments are established by a transcriptional network comprised of maternal effect, gap, pair-rule, and segment polarity genes. The sloppy-paired (slp) locus contains two genes (slp1 and slp2) that are expressed in overlapping striped patterns in the presumptive thorax and abdomen. Previous studies suggest that these genes function at the pair-rule and segment polarity levels to establish the spacing and polarity of thoracic and abdominal segments. One of these genes (slp1) is also expressed in a broad anterior domain that appears before the striped patterns. There are severe cephalic defects in slp1 mutants, including the complete loss of the mandibular segment, but the molecular roles played by Slp1 in anterior patterning are not clear. Here, we present evidence that the anterior Slp1 domain acts as a gradient to differentially repress the anteriormost stripes of several different pair-rule genes. This repressive gradient contributes to the precise spatial arrangement of anterior pair-rule stripe borders required for expression of the first engrailed stripe and the formation of the mandibular segment. These results suggest that Slp1 functions as a gap gene-like repressor, in addition to its roles at the pair-rule and segment polarity levels of the hierarchy. The Slp1 protein contains a protein motif (EH1) which mediates binding to the transcriptional corepressor Groucho (Gro). We show that this domain is required for Slp1-mediated repression in vivo.

Animals↗
PubMed2003

A self-organizing system of repressor gradients establishes segmental complexity in Drosophila.

Gradients of regulatory factors are essential for establishing precise patterns of gene expression during development; however, it is not clear how patterning information in multiple gradients is integrated to generate complex body plans. Here we show that opposing gradients of two Drosophila transcriptional repressors, Hunchback (Hb) and Knirps (Kni), position several segments by differentially repressing two distinct regulatory regions (enhancers) of the pair-rule gene even-skipped (eve). Computational and in vivo analyses suggest that enhancer sensitivity to repression is controlled by the number and affinity of repressor-binding sites. Because the kni expression domain is positioned between two gradients of Hb, each enhancer directs expression of a pair of symmetrical stripes, one on each side of the kni domain. Thus, only two enhancers are required for the precise positioning of eight stripe borders (four stripes), or more than half of the whole eve pattern. Our results show that complex developmental expression patterns can be generated by simple repressor gradients. They also support the utility of computational analyses for defining and deciphering regulatory information contained in genomic DNA.

Animals↗
PubMed2002

Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms.

The striped expression pattern of the pair-rule gene even skipped (eve) is established by five stripe-specific enhancers, each of which responds in a unique way to gradients of positional information in the early Drosophila embryo. The enhancer for eve stripe 2 (eve 2) is directly activated by the morphogens Bicoid (Bcd) and Hunchback (Hb). As these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position. The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border. We identify a well-conserved sequence repeat, (GTTT)(4), which is required for repression in a more anterior subregion. This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain. Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior factors. Further genetic analysis suggests that the (GTTT)(4)-mediated mechanism is independent of the Gt-mediated mechanism that sets the anterior stripe border, and suggests that a third mechanism, downregulation of Bcd activity by Torso, prevents activation near the anterior tip. Thus, three distinct mechanisms are required for anterior repression of a single eve enhancer, each in a specific position. Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees, and the eve 1 and eve 3+7 enhancers each contain GTTT repeats similar to the site in the eve 2 enhancer. These results suggest a common mechanism for preventing anterior activation of three different eve enhancers.

Animals↗
PubMed2002

The activity of the Drosophila morphogenetic protein Bicoid is inhibited by a domain located outside its homeodomain.

The Drosophila morphogenetic protein Bicoid (Bcd) is a homeodomain-containing activator that stimulates the expression of target genes during early embryonic development. We demonstrate that a small domain of Bcd located immediately N-terminally of the homeodomain represses its own activity in Drosophila cells. This domain, referred to as a self-inhibitory domain, works as an independent module that does not rely on any other sequences of Bcd and can repress the activity of heterologous activators. We further show that this domain of Bcd does not affect its properties of DNA binding or subcellular distribution. A Bcd derivative with point mutations in the self-inhibitory domain severely affects pattern formation and target gene expression in Drosophila embryos. We also provide evidence to suggest that the action of the self-inhibitory domain requires a Drosophila co-factor(s), other than CtBP or dSAP18. Our results suggest that proper action of Bcd as a transcriptional activator and molecular morphogen during embryonic development is dependent on the downregulation of its own activity through an interaction with a novel co-repressor(s) or complex(es).

Alcohol Oxidoreductases↗