Normal bias in the direction of fetal rotation depends on blastomere composition during early cleavage in the mouse

Abstract

Interest in establishing the basis of left/right asymmetry during embryogenesis has burgeoned in recent years. Relevant studies in mammals, focused largely on the mouse, have revealed involvement of a variety of genes that are common to the process in other animals. In the mouse, lateral differences in gene expression are first evident late in gastrulation when directional rotation of nodal cilia has been implicated in effecting the normally very strong bias in handedness. Reconstructing cleavage stages with correspondingly positioned blastomeres from appropriate numbers of conceptuses with similar division planes provides a way of testing whether they differ in potency without the confounding effects of reduced cell number. In a study using this strategy, 4-cell stage conceptuses reconstructed from blastomeres produced by equatorial as opposed to meridional second cleavage were found to be compromised in their ability to support normal development. Here, in more refined reconstructions undertaken at both the 4- and 8-cell stage, no significant impairment of development to the 9(th) or 12(th) day of gestation was found for products of equatorial second cleavage or their 8-cell stage progeny. Most surprisingly, however, a significant increase in reversal of the direction of axial rotation was found specifically among fetuses developing from conceptuses reconstructed from 8-cell stage progeny of products of equatorial second cleavage. Hence, manipulations during early cleavage some 6 days before fetal asymmetries are first evident can perturb the normally very strong bias in specification of a facet of left-right asymmetry.

Figure 1. Asymmetries between fetus and rest of conceptus and shape of regular tetrahedral 4-cell stage.

A). Diagram of a rotated fetus in situ with the parietal yolk sac (PYS) reflected back to the base of the chorio-allantoic placenta (CAP). The right side of the fetus faces towards the placenta, as does the umbilical cord (UC). Its tail (FT) is curled back against the right side of the trunk and head (FH), while the vitelline artery and vein (VAV) connect to the visceral yolk sac (VYS) from the left side of the still open abdomen. B) Diagram of a regular tetrahedral 4-cell stage conceptus showing the disposition of the products of meridional (A/B blastomeres) versus equatorial (C and D blastomeres) second cleavage relative to the second polar body (pb).

Figure 2. Reconstruction of 8-cell stage conceptus.

Reconstruction of 8-cell stage conceptus from progeny of the products of E versus M second cleavage. A) One blastomere at the 4-cell stage, typically the product of E second cleavage that is remote from the 2^nd PB, was labelled with DiI. B) This was followed by short-term culture to encourage spread of the label to the sister blastomere and specimens showing spread selected for culture to the 8-cell stage. C) A long hairline slit was then made in the ZP before conceptuses were incubated for up to 35 min. in calcium-free medium plus EGTA before being returned to standard medium. D) Expulsion of fluid from a narrow-tipped pipette inserted through the slit in the ZP was then used to expel the blastomeres whose separation was completed with a pair of fine glass needles (E). F) Finally, the blastomeres isolated from several dissociated conceptuses were sorted into DiI-positive versus negative pools for injection into evacuated ZP. Strongly versus weakly positive blastomeres were separated for some E reconstructions.

Figure 3. Litter of 5 fetuses of which two show clockwise rather than counter-clockwise axial rotation.

A litter of five fetuses from 8-cell stage E reconstructions that have completed axial rotation and shown with their left side oriented uppermost. The tail is curled to the left of the trunk in the upper two and to the right in lower three.