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

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.


Examples for stage dependency of important anatomical features. (A–D) Position of palatine shelves (s) in coronal sections (A–C) and bar graph (D). (E–H) Appearance of interventricular foramen in axial sections (E–G), graph of percentiles (5%, 95%) and median of size of interventricular foramen in μm (above) and bar graph (H). (I–L) Rotation of intestine in volume‐rendered 3D models (I–K) and bar graph (L). b, brain; co, colon; e, eye; j, jejunum; la, left atrial appendix; ll, lower limb; lv, left ventricle, ra, right atrial appendix; rv, right ventricle; to, tongue; ul, upper limb; vs, ventricle septum. Scale bars: 500 μm.
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joa12590-fig-0004: Examples for stage dependency of important anatomical features. (A–D) Position of palatine shelves (s) in coronal sections (A–C) and bar graph (D). (E–H) Appearance of interventricular foramen in axial sections (E–G), graph of percentiles (5%, 95%) and median of size of interventricular foramen in μm (above) and bar graph (H). (I–L) Rotation of intestine in volume‐rendered 3D models (I–K) and bar graph (L). b, brain; co, colon; e, eye; j, jejunum; la, left atrial appendix; ll, lower limb; lv, left ventricle, ra, right atrial appendix; rv, right ventricle; to, tongue; ul, upper limb; vs, ventricle septum. Scale bars: 500 μm.

Mentions: Cleft palate. Cleft palate (MP: 0000111) is an important abnormality. It hinders sucking in the neonatal period and consequently may be responsible for perinatal death. The cleft is the result of improper closure of the left and right palatine shelves, which initially develop lateral to the tongue, shifting upwards to fuse in the midline. Almost all DMDD mutants, including those at TS23, show cleft palate. However, examination of control embryos reveals a surprisingly broad variety of positions for the palatine shelves. Reclassifying developmental stage using the forelimb handplate system, it is possible to define a developmental sequence that accounts for this apparent morphological variability. From S21 to S22, palatine plates are positioned laterally to the tongue. From S22+ they start elevating, but in an asymmetric manner. As a result, S22+ embryos can show one shelf above, and one shelf lateral to the tongue (Fig. 4A–C). Finally, from S23− onwards, all embryos have both shelves above their tongues, but only 35% have them yet fully fused along the midline. Having resolved this as a consistent normal developmental sequence, DMDD mutants are only diagnosed as showing cleft palate if they are older than S22+ and still do not have both shelves positioned above the tongue.


A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5)
Examples for stage dependency of important anatomical features. (A–D) Position of palatine shelves (s) in coronal sections (A–C) and bar graph (D). (E–H) Appearance of interventricular foramen in axial sections (E–G), graph of percentiles (5%, 95%) and median of size of interventricular foramen in μm (above) and bar graph (H). (I–L) Rotation of intestine in volume‐rendered 3D models (I–K) and bar graph (L). b, brain; co, colon; e, eye; j, jejunum; la, left atrial appendix; ll, lower limb; lv, left ventricle, ra, right atrial appendix; rv, right ventricle; to, tongue; ul, upper limb; vs, ventricle septum. Scale bars: 500 μm.
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joa12590-fig-0004: Examples for stage dependency of important anatomical features. (A–D) Position of palatine shelves (s) in coronal sections (A–C) and bar graph (D). (E–H) Appearance of interventricular foramen in axial sections (E–G), graph of percentiles (5%, 95%) and median of size of interventricular foramen in μm (above) and bar graph (H). (I–L) Rotation of intestine in volume‐rendered 3D models (I–K) and bar graph (L). b, brain; co, colon; e, eye; j, jejunum; la, left atrial appendix; ll, lower limb; lv, left ventricle, ra, right atrial appendix; rv, right ventricle; to, tongue; ul, upper limb; vs, ventricle septum. Scale bars: 500 μm.
Mentions: Cleft palate. Cleft palate (MP: 0000111) is an important abnormality. It hinders sucking in the neonatal period and consequently may be responsible for perinatal death. The cleft is the result of improper closure of the left and right palatine shelves, which initially develop lateral to the tongue, shifting upwards to fuse in the midline. Almost all DMDD mutants, including those at TS23, show cleft palate. However, examination of control embryos reveals a surprisingly broad variety of positions for the palatine shelves. Reclassifying developmental stage using the forelimb handplate system, it is possible to define a developmental sequence that accounts for this apparent morphological variability. From S21 to S22, palatine plates are positioned laterally to the tongue. From S22+ they start elevating, but in an asymmetric manner. As a result, S22+ embryos can show one shelf above, and one shelf lateral to the tongue (Fig. 4A–C). Finally, from S23− onwards, all embryos have both shelves above their tongues, but only 35% have them yet fully fused along the midline. Having resolved this as a consistent normal developmental sequence, DMDD mutants are only diagnosed as showing cleft palate if they are older than S22+ and still do not have both shelves positioned above the tongue.

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.