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Precise registration of gene expression boundaries by a repressive morphogen in Drosophila.

ABSTRACT: BACKGROUND:Morphogen gradients are thought to create concentration thresholds that differentially position the expression boundaries of multiple target genes. Despite intensive study, it is still unclear how the concentration profiles within gradients are spatially related to the critical patterning thresholds they generate. RESULTS:Here we use a combination of quantitative measurements and ectopic-misexpression experiments to examine the transcriptional-repression activities of the Hunchback (Hb) protein gradient in Drosophila embryos. Our results define five expression boundaries that are set primarily by differences in Hb concentration and two boundaries that are set by combinatorial mechanisms involving Hb and at least one other repressor. CONCLUSIONS:Hb functions as a repressive morphogen, but only within a specific range of concentrations ( approximately 40% to approximately 4.4% of maximum Hb concentration), within which there are at least four distinct concentration thresholds. The lower limit of the range reflects a position where the slope of the gradient becomes too shallow for resolution by specific target genes. Concentrations above the upper limit do not contribute directly to differential-repression mechanisms, but they provide a robust source that permits proper functioning of the gradient in heterozygous embryos that contain only one functional hb gene.

SUBMITTER: Yu D

PROVIDER: S-EPMC2481289 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Precise registration of gene expression boundaries by a repressive morphogen in Drosophila.

Yu Danyang D Small Stephen S

Current biology : CB 20080601 12

<h4>Background</h4>Morphogen gradients are thought to create concentration thresholds that differentially position the expression boundaries of multiple target genes. Despite intensive study, it is still unclear how the concentration profiles within gradients are spatially related to the critical patterning thresholds they generate.<h4>Results</h4>Here we use a combination of quantitative measurements and ectopic-misexpression experiments to examine the transcriptional-repression activities of t ...[more]

PMID: 18571415

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Project description:UnlabelledBackgroundDigital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, the annelid Platynereis dumerilii is particularly well suited for such studies. Recently a reference template with registered gene expression patterns has been generated for the anterior part (episphere) of the Platynereis trochophore larva and used for the detailed study of neuronal development.ResultsHere we introduce and evaluate a method for whole-body gene expression pattern registration for Platynereis trochophore and nectochaete larvae based on whole-mount in situ hybridization, confocal microscopy, and image registration. We achieved high-resolution whole-body scanning using the mounting medium 2,2'-thiodiethanol (TDE), which allows the matching of the refractive index of the sample to that of glass and immersion oil thereby reducing spherical aberration and improving depth penetration. This approach allowed us to scan entire whole-mount larvae stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and the nuclear stain 4'6-diamidino-2-phenylindole (DAPI). Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental stage. We then registered to these templates the expression patterns of cell-type specific genes. In order to evaluate the gene expression pattern registration, we analyzed the absolute deviation of cell-center positions. Both the acetylated-tubulin- and the nuclear-stain-based templates allowed near-cellular-resolution gene expression registration. Nuclear-stain-based templates often performed significantly better than acetylated-tubulin-based templates. We provide detailed guidelines and scripts for the use and further expansion of the Platynereis gene expression atlas.ConclusionsWe established whole-body reference templates for the generation of gene expression atlases for Platynereis trochophore and nectochaete larvae. We anticipate that nuclear-staining-based image registration will be applicable for whole-body alignment of the embryonic and larval stages of other organisms in a similar size range.

Dataset Information

Precise registration of gene expression boundaries by a repressive morphogen in Drosophila.

Publications

Precise registration of gene expression boundaries by a repressive morphogen in Drosophila.

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