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A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5)

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ABSTRACT

We present a simple and quick system for accurately scoring the developmental progress of mouse embryos harvested on embryonic day 14 (E14.5). Based solely on the external appearance of the maturing forelimb, we provide a convenient way to distinguish six developmental sub‐stages. Using a variety of objective morphometric data obtained from the commonly used C57BL/6N mouse strain, we show that these stages correlate precisely with the growth of the entire embryo and its organs. Applying the new staging system to phenotype analyses of E14.5 embryos of 58 embryonic lethal mutant lines from the DMDD research programme (https://dmdd.org.uk) and its pilot, we show that homozygous mutant embryos are frequently delayed in development. To demonstrate the importance of our staging system for correct phenotype interpretation, we describe stage‐specific changes of the palate, heart and gut, and provide examples in which correct diagnosis of malformations relies on correct staging.

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Stages of E14.5 embryos. (A–F) Volume‐rendered 3D models of the surface showing stages (S) 21–23. (G–L) Appearance of internal structures in coronal sections through the 3D models. (M–R) Volume‐rendered 3D model of hands of embryos classified as S21–S23. All models are set in their original, stage‐dependent size relations. (S) Scheme of development of interdigital space between 3rd and 4th digit from S22− to S23. (T,U) Stages distribution of DMDD control (n = 215) (T) and knock‐out embryos (n = 297) (U). Scale bars: 2 mm.
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joa12590-fig-0002: Stages of E14.5 embryos. (A–F) Volume‐rendered 3D models of the surface showing stages (S) 21–23. (G–L) Appearance of internal structures in coronal sections through the 3D models. (M–R) Volume‐rendered 3D model of hands of embryos classified as S21–S23. All models are set in their original, stage‐dependent size relations. (S) Scheme of development of interdigital space between 3rd and 4th digit from S22− to S23. (T,U) Stages distribution of DMDD control (n = 215) (T) and knock‐out embryos (n = 297) (U). Scale bars: 2 mm.

Mentions: While scoring the organs of the embryos classified as TS22 and TS23, it became apparent that the appearance of the internal structures also varied considerably between the Theiler stages (Fig. 2G–L). We therefore examined which feature proposed by Theiler might provide a means of sub‐dividing TS22 and TS23 into sub‐stages. In the period TS21–TS23, the forelimb gradually and predictably changes its shape, starting as a paddle at TS21 and becoming a hand with separate fingers by TS23 (Fig. 2M–R). We therefore evaluated whether forelimb morphology alone could provide a reproducible, simple and accurate way to assess embryo stage solely on the basis of quick external observation. All 215 wildtype embryos were staged in the order they were produced and imaged, first according to Theiler criteria and then independently using the appearance of the forelimb. Both scorings yielded predominantly the same results, assigning 91% of the embryos to the same stage.


A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5)
Stages of E14.5 embryos. (A–F) Volume‐rendered 3D models of the surface showing stages (S) 21–23. (G–L) Appearance of internal structures in coronal sections through the 3D models. (M–R) Volume‐rendered 3D model of hands of embryos classified as S21–S23. All models are set in their original, stage‐dependent size relations. (S) Scheme of development of interdigital space between 3rd and 4th digit from S22− to S23. (T,U) Stages distribution of DMDD control (n = 215) (T) and knock‐out embryos (n = 297) (U). Scale bars: 2 mm.
© Copyright Policy - creativeCommonsBy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5382591&req=5

joa12590-fig-0002: Stages of E14.5 embryos. (A–F) Volume‐rendered 3D models of the surface showing stages (S) 21–23. (G–L) Appearance of internal structures in coronal sections through the 3D models. (M–R) Volume‐rendered 3D model of hands of embryos classified as S21–S23. All models are set in their original, stage‐dependent size relations. (S) Scheme of development of interdigital space between 3rd and 4th digit from S22− to S23. (T,U) Stages distribution of DMDD control (n = 215) (T) and knock‐out embryos (n = 297) (U). Scale bars: 2 mm.
Mentions: While scoring the organs of the embryos classified as TS22 and TS23, it became apparent that the appearance of the internal structures also varied considerably between the Theiler stages (Fig. 2G–L). We therefore examined which feature proposed by Theiler might provide a means of sub‐dividing TS22 and TS23 into sub‐stages. In the period TS21–TS23, the forelimb gradually and predictably changes its shape, starting as a paddle at TS21 and becoming a hand with separate fingers by TS23 (Fig. 2M–R). We therefore evaluated whether forelimb morphology alone could provide a reproducible, simple and accurate way to assess embryo stage solely on the basis of quick external observation. All 215 wildtype embryos were staged in the order they were produced and imaged, first according to Theiler criteria and then independently using the appearance of the forelimb. Both scorings yielded predominantly the same results, assigning 91% of the embryos to the same stage.

View Article: PubMed Central - PubMed

ABSTRACT

We present a simple and quick system for accurately scoring the developmental progress of mouse embryos harvested on embryonic day 14 (E14.5). Based solely on the external appearance of the maturing forelimb, we provide a convenient way to distinguish six developmental sub‐stages. Using a variety of objective morphometric data obtained from the commonly used C57BL/6N mouse strain, we show that these stages correlate precisely with the growth of the entire embryo and its organs. Applying the new staging system to phenotype analyses of E14.5 embryos of 58 embryonic lethal mutant lines from the DMDD research programme (https://dmdd.org.uk) and its pilot, we show that homozygous mutant embryos are frequently delayed in development. To demonstrate the importance of our staging system for correct phenotype interpretation, we describe stage‐specific changes of the palate, heart and gut, and provide examples in which correct diagnosis of malformations relies on correct staging.

No MeSH data available.


Related in: MedlinePlus