TCreERT2, a transgenic mouse line for temporal control of Cre-mediated recombination in lineages emerging from the primitive streak or tail bud

Abstract

The study of axis extension and somitogenesis has been greatly advanced through the use of genetic tools such as the TCre mouse line. In this line, Cre is controlled by a fragment of the T (Brachyury) promoter that is active in progenitor cells that reside within the primitive streak and tail bud and which give rise to lineages emerging from these tissues as the embryonic axis extends. However, because TCre-mediated recombination occurs early in development, gene inactivation can result in an axis truncation that precludes the study of gene function in later or more posterior tissues. To address this limitation, we have generated an inducible TCre transgenic mouse line, called TCreERT2, that provides temporal control, through tamoxifen administration, in all cells emerging from the primitive streak or tail bud throughout development. TCreERT2 activity is mostly silent in the absence of tamoxifen and, in its presence, results in near complete recombination of emerging mesoderm from E7.5 through E13.5. We demonstrate the utility of the TCreERT2 line for determining rate of posterior axis extension and somite formation, thus providing the first in vivo tool for such measurements. To test the usefulness of TCreERT2 for genetic manipulation, we demonstrate that an early deletion of ß-Catenin via TCreERT2 induction phenocopies the TCre-mediated deletion of ß-Catenin defect, whereas a later induction bypasses this early phenotype and produces a similar defect in more caudal tissues. TCreERT2 provides a useful and novel tool for the control of gene expression of emerging embryonic lineages throughout development.

Figure 1. Tamoxifen adminstration induces TCreERT2-mediated recombination specifically in the primitive streak or tail bud.

Embryos of the indicated age carrying either the TCre (A) or TCreERT2 transgenes (B–M) were assayed for activation of the R26R Cre-reporter by staining for ßGal activity (blue color). TCre; R26R control embryos (A) display widespread recombination at E9.5 compared to TCreERT2;R26R embryos, which, 24 hours after induction, have activated R26R only in tissues recently emerged from the primitive streak at E7.5 and E8.5 (B, C) or tailbud at E 9.5–13.5 (D–H) (white arrows in G and H). An uninduced E9.5 TCreERT2;R26R embryo displays almost no blue cells (I) except for two small clusters (I’ and I”, which are enlargements of the boxed regions in I; arrows point to individual blue cells. A similar region is circled in F). 48 hours after induction, TCreERT2;R26R embryos from E9.5–E12.5 display a recombined region that extends more rostrally (J–M) than in the case of a 24-hour induction period.

Figure 2. TCreERT2 activation results in high levels of recombination.

Sagittal (A–A’) or transverse (B–B”, C–C”) sections of an XGal- stained TCreERT2;R26R E10.5 embryo 24 (A–B”) or 48 hours (C–C”) after tamoxifen induction, illustrates the extent of recombination (blue cells). Lines in A, B and C illustrate the approximate section locations. Blue numbers indicate the percentage of blue mesodermal cells (excluding blood cells), which increase slightly after a 48-hour induction period from about 92% to 95–98% (C’ and C”). Note the near absence of blue cells in the surface ectoderm (se). gt, gut tube; nc, notochord; nt, neural tube; so, somites; ver, ventral ectodermal ridge.

Figure 3. TCreERT2 recombination dynamics allows measurement of the in vivo rate of axis extension and somitogenesis.

A single dose of tamoxifen was injected at 8∶00 AM to dams carrying an E9.0–E9.5 TCreERT2; R26R litter. After the indicated time interval, embryos were harvested and stained for ßGal activity (A–E). The dotted line in all panels marks the border between the anterior PSM and the most caudal somite. After 6 hours, blue cells can just be discerned (A). The intensity of blue increases progressively by 8 hours (B) and 12 hours (C), indicating an increase in the fraction of recombined cells. By 16 hours, the recombined domain has reached the border between the anterior PSM and youngest somite (D). Over the next 8 hours, the four most caudal somites (bracket in E), which together measure approximately 470 µm, are heavily labeled (24-hour point, E) demonstrating that, at this stage, the embryo is extending about one µm/min (470 µm/480 min) and a somite forms about every 2 hours. In the lateral view in E it appears that somites rostral to the bracket are labeled (i.e, there are more than 4 blue somites), but this is an illusion due to viewing a blue labeled neural tube (NT) through translucent, mostly unlabeled, somites. This is demonstrated by a transverse view, shown in panel F, of the embryo in E (white arrowhead E indicates where embryo was sliced).

Figure 4. TCreERT2-mediated deletion of ß-catenin at different axial levels.

All embryos shown, of the indicated genotype and stage, with or without a 48-hour Tam induction, were stained for expression of the markers Uncx4.1 and Msgn1. Both markers were developed with the same color reaction, but they mark mutually exclusive domains: Uncx4.1 is expressed only in stripes in the somites and Msgn1 is expressed only in the PSM. A–E are lateral views and F–J are either dorsal views (F–H) or magnified views of the caudal end of the embryo immediately above (I, J). TCre-mediated deletion of ß-catenin causes caudal truncation and disorganized somites at E8.5 (A, F). These E8.5 defects are phenocopied in the Tam-induced TCreERT2; ß-catenin ^flox/null embryos (C,H) but not in the uninduced control (B,G), which is similar to a normal TCreERT2; ß-catenin ^flox/wt embryo (insert in B). Tam induction can produce similar somitogenesis defects later in E10.5 TCreERT2; ß-catenin ^flox/null embryo (E, J). The yellow arrow in E indicates the axial level where caudal somite defects begin as indicated by Uncx4.1 staining. The red arrow in J points to residual Msgn1 expression. Uninduced E10.5 TCreERT2; ß-catenin ^flox/null control embryos (D, I) are similar to TCreERT2; ß-catenin ^flox/wt embryos (insert in D).