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Integrating technologies for comparing 3D gene expression domains in the developing chick limb.

Fisher ME, Clelland AK, Bain A, Baldock RA, Murphy P, Downie H, Tickle C, Davidson DR, Buckland RA - Dev. Biol. (2008)

Bottom Line: Here we show that OPT data on the developing chick wing from different labs can be reliably integrated into a common database, that OPT is efficient in capturing 3D gene expression domains and that such domains can be meaningfully compared.This reveals previously unappreciated relationships and demonstrates the potential, using modern genomic resources, for building a large scale 3D atlas of gene expression.Such an atlas could be extended to include other types of data, such as fate maps, and the approach is also more generally applicable to embryos, organs and tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Dundee, Dow Street, Dundee, UK. mef103@yahoo.co.uk

ABSTRACT
Chick embryos are good models for vertebrate development due to their accessibility and manipulability. Recent large increases in available genomic data from both whole genome sequencing and EST projects provide opportunities for identifying many new developmentally important chicken genes. Traditional methods of documenting when and where specific genes are expressed in embryos using whole amount and section in-situ hybridisation do not readily allow appreciation of 3-dimensional (3D) patterns of expression, but this can be accomplished by the recently developed microscopy technique, Optical Projection Tomography (OPT). Here we show that OPT data on the developing chick wing from different labs can be reliably integrated into a common database, that OPT is efficient in capturing 3D gene expression domains and that such domains can be meaningfully compared. Novel protocols are used to compare 3D expression domains of 7 genes known to be involved in chick wing development. This reveals previously unappreciated relationships and demonstrates the potential, using modern genomic resources, for building a large scale 3D atlas of gene expression. Such an atlas could be extended to include other types of data, such as fate maps, and the approach is also more generally applicable to embryos, organs and tissues.

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Comparison of WISH/OPT and other methods for detecting a gradient of expression. (A) Dorsal view of wholemount in-situ hybridisation of Wnt5a in a HH22 wing bud. (B) Virtual section of OPT data scanned from A mapped to a reference limb. (C) Section in-situ of Wnt5a from a HH22 wing bud, arrow indicates expression in the AER. (D) A plot of the mean signal intensity in virtual slices of the OPT data set taken along the proximo-distal axis of the limb with 0 representing the most proximal position and 75 the most distal. Colouring under the line represents the domain to which the slices belong, coloured as in panel E. (E) A surface rendering showing the early HH22 reference limb and the three assayed limb domains; proximal (red), medial (orange) and distal (green), arrows indicate the antero-posterior (A-Po) and proximo-distal (Pr-Di) axes of the limb. (F) Comparison of levels of expression in three domains assayed by OPT (purple) and real time RT-PCR (blue), error bars represent standard errors of ± 0.29, ± 1.4 and ± 1.6 for the RT-PCR measurements in the proximal medial and distal regions respectively. OPT values were based on the mean grey level intensity within the domain and standardised against the mean intensity value of the proximal domain to get a relative expression. Domain labels are coloured as in panel E.
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fig1: Comparison of WISH/OPT and other methods for detecting a gradient of expression. (A) Dorsal view of wholemount in-situ hybridisation of Wnt5a in a HH22 wing bud. (B) Virtual section of OPT data scanned from A mapped to a reference limb. (C) Section in-situ of Wnt5a from a HH22 wing bud, arrow indicates expression in the AER. (D) A plot of the mean signal intensity in virtual slices of the OPT data set taken along the proximo-distal axis of the limb with 0 representing the most proximal position and 75 the most distal. Colouring under the line represents the domain to which the slices belong, coloured as in panel E. (E) A surface rendering showing the early HH22 reference limb and the three assayed limb domains; proximal (red), medial (orange) and distal (green), arrows indicate the antero-posterior (A-Po) and proximo-distal (Pr-Di) axes of the limb. (F) Comparison of levels of expression in three domains assayed by OPT (purple) and real time RT-PCR (blue), error bars represent standard errors of ± 0.29, ± 1.4 and ± 1.6 for the RT-PCR measurements in the proximal medial and distal regions respectively. OPT values were based on the mean grey level intensity within the domain and standardised against the mean intensity value of the proximal domain to get a relative expression. Domain labels are coloured as in panel E.

Mentions: We first assayed expression by WISH (method modified after Nieto et al., 1996 see supplementary data) Fig. 1A). The Wnt5a whole mount was scanned using OPT and gene expression data mapped onto a reference limb (Fig. 2D), from which virtual sections were derived (Fig. 1B). These virtual sections were then compared with section in-situs (Fig. 1C) performed as in Moorman et al. (2001). This comparison shows that the virtual section captures the extent and range of the Wnt5a expression pattern as accurately as the section in-situ with the exception of some apical ectodermal ridge (AER) expression (Fig. 1C arrowed). For a further illustration of the effective capture of expression patterns using OPT see supplementary data (Fig. S3–5).


Integrating technologies for comparing 3D gene expression domains in the developing chick limb.

Fisher ME, Clelland AK, Bain A, Baldock RA, Murphy P, Downie H, Tickle C, Davidson DR, Buckland RA - Dev. Biol. (2008)

Comparison of WISH/OPT and other methods for detecting a gradient of expression. (A) Dorsal view of wholemount in-situ hybridisation of Wnt5a in a HH22 wing bud. (B) Virtual section of OPT data scanned from A mapped to a reference limb. (C) Section in-situ of Wnt5a from a HH22 wing bud, arrow indicates expression in the AER. (D) A plot of the mean signal intensity in virtual slices of the OPT data set taken along the proximo-distal axis of the limb with 0 representing the most proximal position and 75 the most distal. Colouring under the line represents the domain to which the slices belong, coloured as in panel E. (E) A surface rendering showing the early HH22 reference limb and the three assayed limb domains; proximal (red), medial (orange) and distal (green), arrows indicate the antero-posterior (A-Po) and proximo-distal (Pr-Di) axes of the limb. (F) Comparison of levels of expression in three domains assayed by OPT (purple) and real time RT-PCR (blue), error bars represent standard errors of ± 0.29, ± 1.4 and ± 1.6 for the RT-PCR measurements in the proximal medial and distal regions respectively. OPT values were based on the mean grey level intensity within the domain and standardised against the mean intensity value of the proximal domain to get a relative expression. Domain labels are coloured as in panel E.
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Related In: Results  -  Collection

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fig1: Comparison of WISH/OPT and other methods for detecting a gradient of expression. (A) Dorsal view of wholemount in-situ hybridisation of Wnt5a in a HH22 wing bud. (B) Virtual section of OPT data scanned from A mapped to a reference limb. (C) Section in-situ of Wnt5a from a HH22 wing bud, arrow indicates expression in the AER. (D) A plot of the mean signal intensity in virtual slices of the OPT data set taken along the proximo-distal axis of the limb with 0 representing the most proximal position and 75 the most distal. Colouring under the line represents the domain to which the slices belong, coloured as in panel E. (E) A surface rendering showing the early HH22 reference limb and the three assayed limb domains; proximal (red), medial (orange) and distal (green), arrows indicate the antero-posterior (A-Po) and proximo-distal (Pr-Di) axes of the limb. (F) Comparison of levels of expression in three domains assayed by OPT (purple) and real time RT-PCR (blue), error bars represent standard errors of ± 0.29, ± 1.4 and ± 1.6 for the RT-PCR measurements in the proximal medial and distal regions respectively. OPT values were based on the mean grey level intensity within the domain and standardised against the mean intensity value of the proximal domain to get a relative expression. Domain labels are coloured as in panel E.
Mentions: We first assayed expression by WISH (method modified after Nieto et al., 1996 see supplementary data) Fig. 1A). The Wnt5a whole mount was scanned using OPT and gene expression data mapped onto a reference limb (Fig. 2D), from which virtual sections were derived (Fig. 1B). These virtual sections were then compared with section in-situs (Fig. 1C) performed as in Moorman et al. (2001). This comparison shows that the virtual section captures the extent and range of the Wnt5a expression pattern as accurately as the section in-situ with the exception of some apical ectodermal ridge (AER) expression (Fig. 1C arrowed). For a further illustration of the effective capture of expression patterns using OPT see supplementary data (Fig. S3–5).

Bottom Line: Here we show that OPT data on the developing chick wing from different labs can be reliably integrated into a common database, that OPT is efficient in capturing 3D gene expression domains and that such domains can be meaningfully compared.This reveals previously unappreciated relationships and demonstrates the potential, using modern genomic resources, for building a large scale 3D atlas of gene expression.Such an atlas could be extended to include other types of data, such as fate maps, and the approach is also more generally applicable to embryos, organs and tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Dundee, Dow Street, Dundee, UK. mef103@yahoo.co.uk

ABSTRACT
Chick embryos are good models for vertebrate development due to their accessibility and manipulability. Recent large increases in available genomic data from both whole genome sequencing and EST projects provide opportunities for identifying many new developmentally important chicken genes. Traditional methods of documenting when and where specific genes are expressed in embryos using whole amount and section in-situ hybridisation do not readily allow appreciation of 3-dimensional (3D) patterns of expression, but this can be accomplished by the recently developed microscopy technique, Optical Projection Tomography (OPT). Here we show that OPT data on the developing chick wing from different labs can be reliably integrated into a common database, that OPT is efficient in capturing 3D gene expression domains and that such domains can be meaningfully compared. Novel protocols are used to compare 3D expression domains of 7 genes known to be involved in chick wing development. This reveals previously unappreciated relationships and demonstrates the potential, using modern genomic resources, for building a large scale 3D atlas of gene expression. Such an atlas could be extended to include other types of data, such as fate maps, and the approach is also more generally applicable to embryos, organs and tissues.

Show MeSH