Figure 1.
Generation and characterization of mice carrying a transgene allowing tissue-specific expression of TIAR.
(A) Schematic representation of the GFP-TIAR transgene and recombination by Cre recombinase. (B) Analysis of transgene expression in mouse tissues by western blot using anti-GFP antibodies. Twenty µg of proteins extracted from the indicated tissues were used for western blot analysis. GFP expression was observed using anti-GFP antibodies and the amount of loaded proteins in the different samples was tested using anti-TIAR antibody (Tg or WT: transgenic or WT tissues respectively).
Figure 2.
Transmission of the transgene.
GFP-TIAR transgene transmission (%) upon mating heterozygous GFP-TIAR males with PGK-Cre or Sycp1-Cre transgenic females. n: number of pups genotyped three weeks after birth using TIAR primers.
Figure 3.
Correlation between transgene copy number and transgene expression.
(A) Left: scheme of partial (TIAR-GFPΔp) or complete (TIAR-GFPΔt) deletion of GFP cassettes upon Cre recombinase activity in vivo. Right: PCR amplification from tail DNA of GFP-TIAR, TIAR-GFPΔp, TIAR-GFPΔt males with primer sets amplifying GFP or endogenous and transgenic TIAR sequences. (B) In vivo expression of TIAR-Flag upon excision of GFP cassettes by PGK-Cre. Semi-quantitative RT-PCR was performed with total RNA from testis of WT, TIAR-GFPΔp and TIAR-GFPΔt mice. Three primer sets were used to detect endogenous and transgenic TIAR (upper lanes), transgenic TIAR (middle lanes) and S16 control mRNAs (lower lanes), respectively. The number of PCR cycles at which a fraction of the PCR reaction was analyzed, is indicated. (C) Western blot analysis of transgenic TIAR expression upon in vivo excision of GFP cassettes by PGK-Cre. Twenty µg of protein extracts of testis from the indicated males were loaded on the gel. The indicated antibodies were used to detect transgenic GFP, transgenic TIAR (FLAG) or endogenous TIAR a and b isoforms (TIAR). Asterisk indicates a non-specific band detected by the anti-Flag antibody. Incubation with anti-tubulin antibody indicates that similar amounts of proteins were loaded on the gel.
Figure 4.
Impaired development of TIAR overexpressing embryos at post-implantation stages.
(A) Embryonic lethality within descendants from crossing GFP-TIAR males with WT or PGK-Cre expressing females. The values correspond to the % of empty decidua or abnormal embryos found in pregnant PGK-Cre or WT females mated with GFP-TIAR transgenic males between 9.5 and 17.5 dpc. n: number of counted decidua. (B-D) Representative pictures of post-implantation development of GFP-TIAR x PGK-Cre embryos collected between 5.5 dpc and 7.5 dpc from pregnant PGK-Cre females mated with heterozygous GFP-TIAR males. Magnification 20× for all embryos but 10× for the normal E7.5 embryo shown in D on the left. (B) Left: pre-streak stage embryo. Note that the distal VE has not started to shift proximally (arrowhead). The anterior movement of distal VE cells converts Proximal-Distal polarity to Anterior-Posterior polarity in the pregastrula embryo; Right: pre-streak stage embryo with abnormal thickening of the VE (arrowhead) and reduced ExE (asterisk). (C) Left: Gastrulation has started in this early-streak stage embryo with the formation of the primitive streak posteriorly; Right: pre-streak stage embryo with distal VE (arrowhead). (D) Left: Gastrulation is completed and the three germ layers are established in this early-neural-plate stage embryo. The primitive streak has extended to reach the distal tip of the embryo where a node has formed (black arrowhead). The amnion is closed and an allantois bud is visible (white arrowhead). Middle: Abnormally small embryo with a reduced ExE (asterisk), thin-looking epiblast layer (white arrowhead) and expanded pro-amniotic cavity. Note distally the abnormal accumulation of the VE (black arrowhead); Right: Abnormally small embryo with a reduced ExE (asterisk) and no visible primitive streak or node. (E) Post-implantation lethality. Embryos from pregnant PGK-Cre females mated with homozygous GFP-TIAR males were collected between 5.5 and 7.5 dpc and the number of empty decidua, delayed/abnormal or normal embryos was counted. n: number of analyzed decidua.
Figure 5.
In utero vs in vitro development of TIAR overexpressing embryos.
(A) Pre-implantation embryos from pregnant WT females mated with homozygous GFP-TIAR males were collected at various pre-implantation stages and observed for GFP expression. GFP expression starts at morula (mo) stage and persists at the early blastocyst stage (bl) where the blastocoele cavity starts to be clearly visible when observed upon bright field. (B) E3.5 embryos were collected from uterus of WT or PGK-Cre females mated with homozygous GFP-TIAR (TIAR) males, stained for TIAR and observed under confocal microscope using TOPRO to visualize the nucleus of each blastomere. The first column shows representative pictures of blastocysts while the second column shows blastomere magnification. IIary antibodies correspond to embryos for which the first step of labelling (anti-TIAR antibody) has been omitted to assess background fluoresence. (C) E3.5 embryos were collected from uterus of WT or PGK-Cre females mated with WT or homozygous GFP-TIAR (TIAR) males and the % of “healthy” blastocysts with no obvious fragmentation was counted. (D) Embryos from WT or PGK-Cre females mated with WT or homozygous GFP-TIA (TIAR) males were collected at E0.5 or E2.5, cultured in M16 medium until WT embryos hatched (for 2-4 days) and the % of “healthy” blastocysts with no obvious fragmentation was counted. n: number of counted embryos.
Figure 6.
Increased sensitivity of PGK-Cre x GFP-TIAR blastocysts to in vitro culture correlated with accumulation of phospho-eIF2α in cytoplasmic granules.
(A,B) Representative pictures of embryos from WT or PGK-Cre x GFP-TIAR (TIAR) crosses grown in vitro in M16 (A) or G2 (B) medium until they reach hatched blastocyst stage. A. in M16 fragmentation and unevensized blastomeres are observed in TIAR compared to healthy looking hatched WT blastocysts. In G2 medium the TIAR embryos are not distinguishable from WT ones (B). (C) WT or PGK-Cre x GFP-TIAR (TIAR) embryos cultured from 8-cell stage to blastocyst stage in G2 or M16 medium were fixed and stained with anti-phospho-eIF2α antibody and TOPRO to label nuclei and observed by confocal microscopy. WT embryos express GFP while GFP has been deleted in Cre-expressing embryos (TIAR), as shown in merge pictures. While artefactual phospho-eIF2α staining due to TOPRO labelling is observed in the nuclei of all types of embryos, phospho-eIF2α accumulates only in the cytoplasm of TIAR embryos grown in M16 medium. Shown are representative images obtained from two independent experiments where 5–20 embryos of each categories were analyzed. The scale bars represent 10 µm.
Figure 7.
Potential associations between proteins encoded by TIAR-associated mRNAs and expressed during pre-implantation stages.
Interconnection revealed using String database (http://string.embl.de/) between 29 proteins encoded by mRNAs specifically bound by TIAR and which are expressed in pre-implantation development.
Table 1.
List of functionally or physically interconnected proteins encoded by mRNAs specifically immunoprecipitated by TIAR in RAW264.7 cells and expressed in pre-implantation development.