dmdd.org.uk@dmdduk contact@dmdd.org.uk

Staging mouse embryos harvested on embryonic day 14 (E14.5)

The DMDD database (dmdd.org.uk)

References

Embryo morphology changes dramatically around E14.5

Staging with inter-digital webbing

Mutants are developmentally delayedConclusion

Precise embryo staging is crucial

WHAT

The DMDD programme is a systematic study of mouse knockout genes that result in embryonic or perinatal death. Our online database of high-resolution embryo images and phenotypes is a unique and expanding resource for both developmental biologists and clinicians.

WHY

Our goal is to further understanding of the genes critical for embryo development and survival. The data has already suggested links between specific genes and abnormal embryonic heart structure, and has huge potential to shed further light on the aetiology of human developmental disorders.

HOW

Embryos are comprehensively examined using the highest-resolution 3D imaging methods. A team of expert anatomists then scores structural abnormalities in tissue organisation and organ structure to identify the likely causes of the failure of the embryo to develop properly.

· DMDD phenotyping is based on the comparison of mutant embryos with wild-type controls.

· Three years of experience has shown that accurate phenotyping can only be achieved after precise developmental staging of each embryo.

· The structure of many organs and tissues changes rapidly during development, and current staging systems for mouse embryos are inadequate for embryos at the end of organogenesis (E14.5).

· We have developed a fast, robust alternative.

This study considered 297 mutant embryos (across 58 different knockout lines) and 215 wild-type controls [1]. All embryos were of the strain C57BL/6N and harvested at E14.5.

Six developmental sub-stages were distinguished, based on the external appearance of the maturing forelimb.

Precise staging revealed that the homozygous mutants were frequently delayed in development.

Mutants must be compared with a stage-matched wild-type to avoid false identification of phenotypes.

Analysis of wild-type control embryos showed that the structure and orientation of many organs can change radically around E14.5, even between these newly defined developmental sub-stages.

Palatal shelvesBetween S21 and S22, the palatal shelves are typically lateral to the tongue. At S22+ they begin to elevate. Around 20% of embryos had asymmetric positioning of the shelves at this time. From S23- onwards, all embryos had both shelves above their tongues.

As a result, comparing a mutant with a littermate that is not stage-matched can lead to false diagnosis.

Perimembraneous Ventricular Septal Defects (PVSDs)PVSDs of varying size were present in 68% of control embryos. The size of the gap in the interventricular septum, and the percentage of embryos with a gap, decreased gradually between S21 and S23.

PVSDs are another phenotype at risk of false diagnosis without precise embryo staging.

Small differences in developmental stage can have profound differences on embryo morphology. Thus, comparison of mutant embryos with wild type littermates is likely to be seriously misleading, unless they are known to be at exactly the same developmental stage.

Embryos can now be staged very precisely, simply by measuring the amount of inter-digital webbing on the developing forelimb.

[1] SH Geyer et al., ‘A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5)’, J. Anat., doi:10.1111/joa.12590.

(t denotes tongue, s denotes palatal shelves).

(t denotes tongue, s denotes palatal shelves).