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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.

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μCT renderings and cell proliferation data from the same specimens. A, 3D reconstruction of μCT taken after processing but before sectioning shown in anterior and right lateral 3/4 views. B. i, ii Hoescht 33342 staining to visualize cell nuclei (blue) with cells in S-phase visualized using EdU + Alexa Fluor® 488 labeling (green) in frontal sections at the level of the maxillary prominence. B.i at 50× and B.ii and 200×. Small box in B.i. shows the region magnified below. C.ii, μCT rendering and same specimen (C.i) processed wholemount for anti-PHH3 primary antibody to identify M-phase cells shown in right lateral 3/4 view.
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Figure 9: μCT renderings and cell proliferation data from the same specimens. A, 3D reconstruction of μCT taken after processing but before sectioning shown in anterior and right lateral 3/4 views. B. i, ii Hoescht 33342 staining to visualize cell nuclei (blue) with cells in S-phase visualized using EdU + Alexa Fluor® 488 labeling (green) in frontal sections at the level of the maxillary prominence. B.i at 50× and B.ii and 200×. Small box in B.i. shows the region magnified below. C.ii, μCT rendering and same specimen (C.i) processed wholemount for anti-PHH3 primary antibody to identify M-phase cells shown in right lateral 3/4 view.

Mentions: Coupling 3D morphological analyses with histological datasets is viewed as a crucial source of information with much potential to enrich understanding of morphogenetic mechanisms underlying organ growth and development [1-5,7-10,16]. For example, our research group is exploring methods to combine the analysis of regional variation in cell proliferation rates and micro-CT based morphometric data [16]. Such analyses possess the potential to reveal the relationship between cell proliferation data and morphometric variation within samples and can be used to compare this relationship among genotypes or groups that differ in characteristics of interest. We are employing multiple approaches in the attempt to visualize the 3D distribution of proliferating cells within craniofacial primordia (Figure 9). These approaches involve the development of computer-based methods for morphing multiple individuals to a mean shape, superimposing histological and computed microtomography data, averaging multiple individuals for such datasets to construct genotype or other group means, and the development of statistical methods to compare the distribution of immunohistochemical markers or regions of gene expression among groups [27]. Our goal of uniting μCT volumetric data pertaining to embryonic craniofacial size and shape with molecular expression data at histological resolutions will help us to understand how variation in basic morphogenetic processes shape and organize variation at the gross anatomical level. The ability to correlate morphological and molecular data at the individual embryo level will offer a new toolkit to elucidate the relationships between genotypic and phenotypic variation in the contexts of developmental and evolutionary biology as well as in clinical settings.


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)

μCT renderings and cell proliferation data from the same specimens. A, 3D reconstruction of μCT taken after processing but before sectioning shown in anterior and right lateral 3/4 views. B. i, ii Hoescht 33342 staining to visualize cell nuclei (blue) with cells in S-phase visualized using EdU + Alexa Fluor® 488 labeling (green) in frontal sections at the level of the maxillary prominence. B.i at 50× and B.ii and 200×. Small box in B.i. shows the region magnified below. C.ii, μCT rendering and same specimen (C.i) processed wholemount for anti-PHH3 primary antibody to identify M-phase cells shown in right lateral 3/4 view.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: μCT renderings and cell proliferation data from the same specimens. A, 3D reconstruction of μCT taken after processing but before sectioning shown in anterior and right lateral 3/4 views. B. i, ii Hoescht 33342 staining to visualize cell nuclei (blue) with cells in S-phase visualized using EdU + Alexa Fluor® 488 labeling (green) in frontal sections at the level of the maxillary prominence. B.i at 50× and B.ii and 200×. Small box in B.i. shows the region magnified below. C.ii, μCT rendering and same specimen (C.i) processed wholemount for anti-PHH3 primary antibody to identify M-phase cells shown in right lateral 3/4 view.
Mentions: Coupling 3D morphological analyses with histological datasets is viewed as a crucial source of information with much potential to enrich understanding of morphogenetic mechanisms underlying organ growth and development [1-5,7-10,16]. For example, our research group is exploring methods to combine the analysis of regional variation in cell proliferation rates and micro-CT based morphometric data [16]. Such analyses possess the potential to reveal the relationship between cell proliferation data and morphometric variation within samples and can be used to compare this relationship among genotypes or groups that differ in characteristics of interest. We are employing multiple approaches in the attempt to visualize the 3D distribution of proliferating cells within craniofacial primordia (Figure 9). These approaches involve the development of computer-based methods for morphing multiple individuals to a mean shape, superimposing histological and computed microtomography data, averaging multiple individuals for such datasets to construct genotype or other group means, and the development of statistical methods to compare the distribution of immunohistochemical markers or regions of gene expression among groups [27]. Our goal of uniting μCT volumetric data pertaining to embryonic craniofacial size and shape with molecular expression data at histological resolutions will help us to understand how variation in basic morphogenetic processes shape and organize variation at the gross anatomical level. The ability to correlate morphological and molecular data at the individual embryo level will offer a new toolkit to elucidate the relationships between genotypic and phenotypic variation in the contexts of developmental and evolutionary biology as well as in clinical settings.

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