Limits...
Micro-computed tomography-based phenotypic approaches in embryology: procedural artifacts on assessments of embryonic craniofacial growth and development.

Schmidt EJ, Parsons TE, Jamniczky HA, Gitelman J, Trpkov C, Boughner JC, Logan CC, Sensen CW, Hallgrímsson B - BMC Dev. Biol. (2010)

Bottom Line: Subsequent microCT scanning produced negligible changes in size but did appear to reduce or even reverse fixation-induced random shape changes.Mixtures of paraformaldehyde + glutaraldehyde reduced average centroid sizes by 2-3%.Experimental designs will need to address these significant effects, either by employing alternative methods that minimize artifacts in the region of focus or in the interpretation of statistical patterns.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology & Anatomy, The McCaig Bone and Joint Institute, and the Alberta Children's Hospital Institute for Child and Maternal Health, University of Calgary, Calgary, AB, Canada.

ABSTRACT

Background: Growing demand for three dimensional (3D) digital images of embryos for purposes of phenotypic assessment drives implementation of new histological and imaging techniques. Among these micro-computed tomography (microCT) has recently been utilized as an effective and practical method for generating images at resolutions permitting 3D quantitative analysis of gross morphological attributes of developing tissues and organs in embryonic mice. However, histological processing in preparation for microCT scanning induces changes in organ size and shape. Establishing normative expectations for experimentally induced changes in size and shape will be an important feature of 3D microCT-based phenotypic assessments, especially if quantifying differences in the values of those parameters between comparison sets of developing embryos is a primary aim. Toward that end, we assessed the nature and degree of morphological artifacts attending microCT scanning following use of common fixatives, using a two dimensional (2D) landmark geometric morphometric approach to track the accumulation of distortions affecting the embryonic head from the native, uterine state through to fixation and subsequent scanning.

Results: Bouin's fixation reduced average centroid sizes of embryonic mouse crania by approximately 30% and substantially altered the morphometric shape, as measured by the shift in Procrustes distance, from the unfixed state, after the data were normalized for naturally occurring shape variation. Subsequent microCT scanning produced negligible changes in size but did appear to reduce or even reverse fixation-induced random shape changes. Mixtures of paraformaldehyde + glutaraldehyde reduced average centroid sizes by 2-3%. Changes in craniofacial shape progressively increased post-fixation.

Conclusions: The degree to which artifacts are introduced in the generation of random craniofacial shape variation relates to the degree of specimen dehydration during the initial fixation. Fixation methods that better maintain original craniofacial dimensions at reduced levels of dehydration and tissue shrinkage lead to the progressive accumulation of random shape variation during handling and data acquisition. In general, to the degree that embryonic organ size and shape factor into microCT-based phenotypic assessments, procedurally induced artifacts associated with fixation and scanning will influence results. Experimental designs will need to address these significant effects, either by employing alternative methods that minimize artifacts in the region of focus or in the interpretation of statistical patterns.

Show MeSH

Related in: MedlinePlus

A, Ontogenetic series of μCT scans showing the range of shape and size variation from E9.5-12. B, Regression of total shape variation on tail-somite stage (TS) and wireframe deformation showing the corresponding shape trajectory. C, Morphing of a μCT surface render along the same shape trajectory (size constant). D, Comparison of the two groups at three standardized stages. Crf4 mice have a mutation on a C57BL/6J background that produces a complex set of craniofacial changes [15].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2836989&req=5

Figure 1: A, Ontogenetic series of μCT scans showing the range of shape and size variation from E9.5-12. B, Regression of total shape variation on tail-somite stage (TS) and wireframe deformation showing the corresponding shape trajectory. C, Morphing of a μCT surface render along the same shape trajectory (size constant). D, Comparison of the two groups at three standardized stages. Crf4 mice have a mutation on a C57BL/6J background that produces a complex set of craniofacial changes [15].

Mentions: Figure 1 shows an example of μCT-based quantitative analysis of craniofacial shape variation during mouse embryonic development. This analysis employs geometric morphometrics [20,21], which is a body of methods dedicated to the quantitative analysis of shape. Analyses of this kind allow the systematic quantitative assessment of the influence of genetic factors on embryonic growth and morphogenesis, allow for the statistical assessment of differences among genotypes or treatment groups, and also contain methods for quantifying and, if desired, correcting for complex shape transformations such as those that occur during morphogenesis. Figure 1D shows an example of this, where two genotypes were compared and shapes standardized to specific developmental stages by regressing shape on tail-somite stage (Figure 1B). These methods are especially powerful if such data can be obtained from the same individual embryos that are then subsequently processed for molecular assays. This will allow investigators to correlate morphometric shape variation with molecular data, such as the expression of particular genes or density distribution of some immunohistochemical marker using individual embryos. The ability to relate directly quantitative assessment of morphogenesis with assays of molecular determinants offers an important new avenue for querying the mechanistic basis of development and dysmorphology.


Micro-computed tomography-based phenotypic approaches in embryology: procedural artifacts on assessments of embryonic craniofacial growth and development.

Schmidt EJ, Parsons TE, Jamniczky HA, Gitelman J, Trpkov C, Boughner JC, Logan CC, Sensen CW, Hallgrímsson B - BMC Dev. Biol. (2010)

A, Ontogenetic series of μCT scans showing the range of shape and size variation from E9.5-12. B, Regression of total shape variation on tail-somite stage (TS) and wireframe deformation showing the corresponding shape trajectory. C, Morphing of a μCT surface render along the same shape trajectory (size constant). D, Comparison of the two groups at three standardized stages. Crf4 mice have a mutation on a C57BL/6J background that produces a complex set of craniofacial changes [15].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2836989&req=5

Figure 1: A, Ontogenetic series of μCT scans showing the range of shape and size variation from E9.5-12. B, Regression of total shape variation on tail-somite stage (TS) and wireframe deformation showing the corresponding shape trajectory. C, Morphing of a μCT surface render along the same shape trajectory (size constant). D, Comparison of the two groups at three standardized stages. Crf4 mice have a mutation on a C57BL/6J background that produces a complex set of craniofacial changes [15].
Mentions: Figure 1 shows an example of μCT-based quantitative analysis of craniofacial shape variation during mouse embryonic development. This analysis employs geometric morphometrics [20,21], which is a body of methods dedicated to the quantitative analysis of shape. Analyses of this kind allow the systematic quantitative assessment of the influence of genetic factors on embryonic growth and morphogenesis, allow for the statistical assessment of differences among genotypes or treatment groups, and also contain methods for quantifying and, if desired, correcting for complex shape transformations such as those that occur during morphogenesis. Figure 1D shows an example of this, where two genotypes were compared and shapes standardized to specific developmental stages by regressing shape on tail-somite stage (Figure 1B). These methods are especially powerful if such data can be obtained from the same individual embryos that are then subsequently processed for molecular assays. This will allow investigators to correlate morphometric shape variation with molecular data, such as the expression of particular genes or density distribution of some immunohistochemical marker using individual embryos. The ability to relate directly quantitative assessment of morphogenesis with assays of molecular determinants offers an important new avenue for querying the mechanistic basis of development and dysmorphology.

Bottom Line: Subsequent microCT scanning produced negligible changes in size but did appear to reduce or even reverse fixation-induced random shape changes.Mixtures of paraformaldehyde + glutaraldehyde reduced average centroid sizes by 2-3%.Experimental designs will need to address these significant effects, either by employing alternative methods that minimize artifacts in the region of focus or in the interpretation of statistical patterns.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology & Anatomy, The McCaig Bone and Joint Institute, and the Alberta Children's Hospital Institute for Child and Maternal Health, University of Calgary, Calgary, AB, Canada.

ABSTRACT

Background: Growing demand for three dimensional (3D) digital images of embryos for purposes of phenotypic assessment drives implementation of new histological and imaging techniques. Among these micro-computed tomography (microCT) has recently been utilized as an effective and practical method for generating images at resolutions permitting 3D quantitative analysis of gross morphological attributes of developing tissues and organs in embryonic mice. However, histological processing in preparation for microCT scanning induces changes in organ size and shape. Establishing normative expectations for experimentally induced changes in size and shape will be an important feature of 3D microCT-based phenotypic assessments, especially if quantifying differences in the values of those parameters between comparison sets of developing embryos is a primary aim. Toward that end, we assessed the nature and degree of morphological artifacts attending microCT scanning following use of common fixatives, using a two dimensional (2D) landmark geometric morphometric approach to track the accumulation of distortions affecting the embryonic head from the native, uterine state through to fixation and subsequent scanning.

Results: Bouin's fixation reduced average centroid sizes of embryonic mouse crania by approximately 30% and substantially altered the morphometric shape, as measured by the shift in Procrustes distance, from the unfixed state, after the data were normalized for naturally occurring shape variation. Subsequent microCT scanning produced negligible changes in size but did appear to reduce or even reverse fixation-induced random shape changes. Mixtures of paraformaldehyde + glutaraldehyde reduced average centroid sizes by 2-3%. Changes in craniofacial shape progressively increased post-fixation.

Conclusions: The degree to which artifacts are introduced in the generation of random craniofacial shape variation relates to the degree of specimen dehydration during the initial fixation. Fixation methods that better maintain original craniofacial dimensions at reduced levels of dehydration and tissue shrinkage lead to the progressive accumulation of random shape variation during handling and data acquisition. In general, to the degree that embryonic organ size and shape factor into microCT-based phenotypic assessments, procedurally induced artifacts associated with fixation and scanning will influence results. Experimental designs will need to address these significant effects, either by employing alternative methods that minimize artifacts in the region of focus or in the interpretation of statistical patterns.

Show MeSH
Related in: MedlinePlus