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. Author manuscript; available in PMC: 2013 Nov 1.
Published in final edited form as: Dev Biol. 2012 Aug 25;371(1):77–85. doi: 10.1016/j.ydbio.2012.08.010

Conditional Aurora A deficiency differentially affects early mouse embryo patterning

Yeonsoo Yoon a, Dale O Cowley b,1, Judith Gallant a, Stephen Jones a, Terry Van Dyke b,2, Jaime A Rivera-Pérez a,*
PMCID: PMC3467101  NIHMSID: NIHMS403525  PMID: 22939930

Abstract

Aurora A is a mitotic kinase involved in centrosome maturation, spindle assembly and chromosome segregation during the cell division cycle. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages. Inactivation of Aurora A in the epiblast or visceral endoderm layers of the conceptus leads to apoptosis and inhibition of embryo growth, causing lethality and resorption at approximately E9.5. The effects on embryo patterning, however, depend on the tissue affected by the mutation. Embryos with an epiblast ablation of Aurora A properly establish the anteroposterior axis but fail to progress through gastrulation. In contrast, mutation of Aurora A in the visceral endoderm, leads to posteriorization of the conceptus or failure to elongate the anteroposterior axis. Injection of ES cells into Aurora A epiblast knockout blastocysts reconstitutes embryonic development to E9.5, indicating that the extra-embryonic tissues in these mutant embryos can sustain development to organogenesis stages. Our results reveal new ways to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. Moreover, they show that epiblast-ablated embryos can be used to test the potency of stem cells.

Keywords: Mouse, embryo, patterning, Aurora A, gastrulation, epiblast, visceral endoderm

INTRODUCTION

Cell proliferation and differentiation are tightly regulated processes required for the proper development of multi-cellular organisms (Conlon and Raff, 1999). In mouse embryos, the importance of appropriate cell numbers in embryogenesis has been addressed by experimental manipulations of cell numbers at pre-implantation stages. For example, when one blastomere is removed from a four-cell stage embryo, the resulting conceptus shows a twelve hour delay in gastrulation (Power and Tam, 1993). A similar effect was observed when cell proliferation was inhibited using mitomycin C (Tam, 1988). In the converse experiment, fusion of several morula to produce chimeras leads to reduction in the rate of cell proliferation such that gastrulation can be achieved with similar numbers of cells at 6.5 days of gestation (Lewis and Rossant, 1982).

The mouse embryo at early post-implantation stages is composed of the epiblast, a pseudostratified epithelium that forms the fetus, and several extra-embryonic components that include the visceral endoderm and extra-embryonic ectoderm. At embryonic day 5.5 the embryo contains an average of 120 epiblast and 95 visceral endoderm cells (Snow, 1977). Over the next 24 hours, there is rapid cell proliferation resulting in 660 and 250 epiblast and visceral endoderm cells respectively. In fact, it was calculated that the cell cycle rate in the epiblast could be as short as four and a half hours (Snow, 1977). Because of the fundamental importance of proper cell number in embryonic development we decided to examine the effects of reduced cell numbers at early post-implantation stages using tissue-specific ablation of Aurora A

Aurora A is a serine/threonine kinase required for centrosome maturation, spindle assembly and chromosome segregation during the cell division cycle. Aurora A has been identified as a mitotic regulator and putative oncoprotein that is overexpressed in many human tumors (Katayama et al., 2003). Multiple in vitro studies showed that Aurora A has roles associated with cell proliferation (Berdnik and Knoblich, 2002; Du and Hannon, 2004; Giet et al., 2005; Hirota et al., 2003; Yang et al., 2005). It has also been reported that ablation of AurA in primary embryonic fibroblasts leads to delayed mitotic entry and accumulation of mostly tetraploid cells (Cowley et al., 2009). Mouse embryos devoid of AurA have defects in mitotic spindle assembly and chromosome segregation, which leads to mitotic arrest, cell proliferation failure and embryonic lethality at pre-implantation stages (Cowley et al., 2009; Lu et al., 2008; Sasai et al., 2008). This phenotype prevents further investigation of the function of AurA at post-implantation stages.

Here, we utilize a conditional allele of AurA to bypass the early embryonic lethality of AurA mutants and investigate the function of Aurora A in the development of early post-implantation embryos. We induced Cre-mediated tissue-specific null mutations to determine if AurA is required for proliferation of the epiblast and visceral endoderm and to assess the effects of AurA ablation in axial specification and gastrulation.

Our studies indicate that Aurora A is essential for post-implantation embryo growth and survival and suggest that the phenotype of mutant embryos is linked to abnormal growth brought about by a paucity of epiblast or visceral endoderm cells. Our data also indicates that the effects of ablation of Aurora A at post-implantation stages can impact the embryo differently depending on the tissue affected by the mutation. In addition, our results set the stage for novel ways to induce apoptosis and cell ablation in a tissue specific manner in vivo and provide an alternative method to test the potency of a variety of stem cells.

MATERIALS AND METHODS

Embryo staging and analysis

Embryos were staged morphologically as described previously (Downs and Davies, 1993; Rivera-Perez et al., 2010) or were described in terms of dissection time. Noon of the day that a mating plug was observed was considered embryonic day 0.5 of development (E0.5).

Mouse strains and genotyping

AurAfx conditional mice were previously described (Cowley et al., 2009). AurAd2/+ mice were generated by crossing females carrying the AurAfx allele to Sox2Cre transgenic males. Sox2Cre (Hayashi et al., 2002) and ROSA26 reporter (Soriano, 1999) mice were purchased from the Jackson Laboratory (Stock No. 003309 and 004783, respectively). TtrCre transgenics were kindly provided by Dr. Anna-Katerina Hadjantonakis (Kwon and Hadjantonakis, 2009). Embryos were genotyped retrospectively after wholemount hybridization or immunostaining. For each conceptus, the ectoplacental cone was removed using forceps and placed in 20 μl of lysis buffer (50 mM KCl, 10 mM Tris-HCl, 2.5 mM MgCl2, 0.1 mg/ml Gelatin, 0.45% v/v IGEPAL and 0.45% v/v Tween 20, 100 μg/ml Proteinase K). After heat inactivation of the Proteinase K, one or two microliters were used for PCR amplification. Each allele was confirmed using the following primers: AurAfx and AurA+ alleles, forward primer: 5′-CCT GTG AGT TGG AAA GGG ACA TGG CTG-3′, reverse primer: 5′-CCA CCA CGA AGG CAG TGT TCA ATC CTA AA-3′, 2). AurAd2 allele, forward primer: 5′-CAG AGT CTA AGT CGA GAT ATC ACC TGA GGG TTG A-3′, reverse primer: 5′-GAT GGA AAC CCT GAG CAC CTG TG AAC-3′ 3). Sox2Cre and TtrCre alleles, forward primer: 5′-TCC AAT TTA CTG ACC GTA CAC CAA-3′, reverse primer: 5′-CCT GAT CCT GGC AAT TTC GGC TA-3′

Wholemount immunofluorescence analysis

Dissected embryos were fixed for 1 hour in 4% paraformaldehyde in phosphate buffered saline (PBS). After fixation, embryos were washed three times in PBS for 10 minutes, once in PBT (1% BSA and 5% Triton X-100 in PBS) for 10 minutes and incubated in blocking solution (5% normal goat serum in PBT) for 1 hour. Then, embryos were incubated with primary antibodies in blocking solution for 2 hours, washed three times in PBT for 10 minutes, and incubated with secondary antibodies for 1 hour. After secondary antibody incubation, embryos were washed three times in PBT for 10 minutes, once in PBS for 5 minutes, equilibrated in serial Glycerol/PBS solutions, and analyzed in an inverted fluorescence microscope (Leica. DMI4000). Primary antibodies: rabbit anti-Oct4 (Abcam, Cat. No. ab19857) diluted with 1:1000; rabbit anti-cleaved caspase-3 (Cell signaling, Cat. No. 9664) diluted with 1:500. Secondary antibody: Alexa Fluor 594 goat anti-rabbit antibody (Invitrogen Cat. No. A31631) diluted with 1:2000 in PBT.

Wholemount in situ hybridization

The wholemount in situ protocol was adapted from Henrique and co-workers (Henrique et al., 1995). Briefly, dissected embryos were fixed overnight in 4% paraformaldehyde in PBS at 4°C. After fixation, embryos were dehydrated in a methanol/PBT (0.1% tween-20 in PBS) series and stored at −20°C in 100% methanol. Embryos were treated with Proteinase K for 8 minutes, hybridized at 70°C for a minimum of 2 hours, and incubated with RNA probes overnight at 70°C. The next day, embryos were washed in MABT (50 mM maleic acid, 75 mM NaCl, 0.1% tween-20), incubated in blocking solution (10% blocking reagent in MAB) for 1 hour, incubated in blocking solution with 10% normal goat serum (NGS-blocking solution) for a minimum of 2 hours, and incubated overnight with alkaline phosphatase-conjugated antibody against digoxigenin (Roche Cat. No. 11 093 274 910) diluted 1: 2000 in NGS-blocking solution at 4°C. After overnight incubation, embryos were washed four times in MABT for 30 minutes, washed twice in NTMT (0.1 M NaCl, 0.1 M Tris-Hcl pH9.5, 50 mM MgCl, 0.1% Tween-20) for 10 minutes and stained with 20 μl/ml NBT/BCIP stock solution (Roche 681451) in NTMT. The color reaction was allowed to proceed for 24 to 48 hours. After that, embryos were washed twice in PBT for 10 minutes, fixed in 4% paraformaldehyde for 20 minutes, and cleared in glycerol/PBT. The following probes were utilized: Hex (291–818 cDNA; 528bp) (Thomas et al., 1998); Brachyury (T) (full length cDNA; 1,784bp) (Herrmann, 1991); Wnt7b (full length cDNA; 3,396bp) (Image Consortium, Image ID 6817404); Fgf8 (full length cDNA; 1,100 bp) (Guo and Li, 2007) and Axin2 (2,420 bp cDNA piece containing part of exon 2, exons 3–9 and a portion of exon 10) (Jho et al., 2002).

Wholemount β-galactosidase assay

Dissected embryos were fixed in fixation solution (0.2% glutaraldehyde, 2% formalin, 5 mM EGTA, 2 mM MgCl2 in 0.1 M phosphate buffer, pH 7.3) for 5–10 minutes, washed three times in rinse solution (0.1% deoxycholate, 0.2% IGEPAL, 2 mM MgCl2 in 0.1 M phosphate buffer, pH 7.3), and stained overnight in staining solution (1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide in rinse solution) at 37°C. The next day, embryos were washed twice in rinse solution for 10 minutes, fixed in 4% Paraformaldehyde in PBS at room temperature for 20 minutes and cleared in glycerol/PBS.

Generation of chimeras

AB1 (McMahon and Bradley, 1990) or KT4 (Tremblay et al., 2000) ES cells were injected into expanded blastocysts collected at E3.5 days of gestation. Ten to fifteen cells were injected per blastocyst. The blastocysts were derived from AurAfx/fx;R26r/r and AurAd2/+;Sox2Cre/Cre or AurAfx/fx and AurAd2/+;Sox2cre/cre crosses. The injected blastocysts were transferred into Swiss Webster (Taconic) pseudopregnant females. Chimeric embryos were dissected at E9.5 or E11.5. Embryos were assayed for β-galactosidase activity to determine the contribution of ES cells in chimeras.

RESULTS

Aurora A is essential for epiblast development

To determine the function of Aurora A in the epiblast of post-implantation embryos, we used a mouse line carrying a floxed allele of AurA (AurAfx) (Cowley et al., 2009). In this allele, exon 2 is flanked by LoxP sites and a null allele (AurAd2) is generated by Cre-mediated excision (Fig. 1A). To obtain epiblast-specific AurA knockout (Epi-KO) embryos, we crossed homozygous AurA floxed (AurAfx/fx) females with males heterozygous for the AurAd2 null allele and heterozygous for the Sox2Cre transgene (AurAd2/+;Sox2Cre/0) (Fig. 1B). In Sox2Cre transgenics, Cre is expressed only in the epiblast at post-implantation stages (Hayashi et al., 2002), leading to the generation of embryos lacking AurA specifically in the epiblast (AurAd2/fx;Sox2Cre/0). This mating strategy also generates control littermates (AurA+/fx;Sox2Cre/0). In embryos with a conditional mutation of AurA in the epiblast, the rest of the conceptus, which includes the visceral endoderm (VE) and extra-embryonic ectoderm, retains a functional allele of AurA (AurAfx).

Figure 1. Growth of the epiblast is inhibited in epiblast-specific AurA knockout embryos.

Figure 1

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A. Schematic representation of AurA wild-type (AurA+), floxed (AurAfx), and null (AurAd2) alleles. Yellow triangles represent LoxP sites, the orange oval represents a FRT site. B. Mating strategy for generating epiblast (red) AurA knockout (Epi-KO) embryos. C & D. DIC (C) and fluorescent (D) images of E6.5 embryos stained with anti-Oct4 antibody, showing epiblast extent. Arrow in C indicates cellular debris. E. E6.25 embryos hybridized with Wnt7b probe. Wnt7b marks the extra-embryonic ectoderm. The epiblast portion (arrow) of Epi-KO embryo is smaller than that of control embryo. Embryos in C–E are shown at the same scale, scale bar, 50 μm.

At E6.5, Epi-KO embryos were smaller than control littermates, the epiblast was reduced in size and cellular debris was visible in the proamniotic cavity (n=14)(Fig. 1C). To determine the extent of the epiblast, we immunostained Epi-KO embryos with an antibody against Oct4 (n=2), a marker of the epiblast (Ovitt and Scholer, 1998). We found that the size of the epiblast in Epi-KO embryos was reduced to approximately one third of the size of control littermates, while the extra-embryonic ectoderm appeared elongated proximo-distally (Fig. 1C, D). These abnormalities were also evident after hybridizing embryos with Wnt7b (n=5) (Fig. 1E), a gene expressed exclusively in the extra-embryonic ectoderm region (Kemp et al., 2007). These results demonstrate that Aurora A is essential for epiblast growth in early post-implantation embryos.

AurA ablation in the epiblast leads to progressive epiblast depletion through apoptosis

AurA Epi-Ko embryos show abnormal accumulation of cellular debris in the proamiotic cavity. To determine if the cellular debris was derived from the epiblast, we marked epiblast cells using the ROSA26 reporter (R26r) mouse line (Soriano, 1999). We crossed AurA floxed mice homozygous for R26r (AurAfx/+;R26r/r) with mice heterozygous for the AurA null allele and homozygous for Sox2Cre (AurAd2/+;Sox2Cre/Cre) to produce Epi-KO embryos carrying the ROSA26 reporter (AurAd2/fx;Sox2Cre/0;R26r/+). At E6.5, β-galactosidase-positive cellular debris was displaced into the proamniotic cavity of Epi-KO embryos (n=6), indicating an epiblast origin (Fig. 2A). Analysis of Epi-KO embryos dissected at E7.5 showed that few epiblast cells remained (Fig. 2B) and by E8.5, the epiblast was almost completely absent, with only extra-embryonic tissues remaining (not shown).

Figure 2. Absence of AurA in the epiblast causes progressive epiblast loss through apoptosis.

Figure 2

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A. E6.5 embryos with the epiblast component labeled with β-galactosidase (blue) derived from ROSA26 reporter. In the Epi-KO embryo, the epiblast portion is reduced and cellular debris are displaced into the proamniotic cavity (arrowhead). B. The epiblast of Epi-KO embryos is almost ablated by E7.5. C and D. DIC (C) and fluorescent (D) images of control and Epi-KO embryos stained with anti-cleaved Caspase-3 antibody. Apoptotic cells in the mutant embryo occupy the proamiotic cavity (arrows). Panels C and D are shown at the same scale. Scale bars, 50 μm.

Previous studies have shown that AurA null blastocysts have increased apoptosis and that knockdown of AurA using RNAi leads to apoptosis in pancreatic cancer cells (Cowley et al., 2009; Hata et al., 2005). To determine if epiblast cells lacking AurA were undergoing apoptosis, we stained E6.5 embryos with an anti-cleaved caspase-3 antibody (Elmore, 2007). Epi-KO embryos displayed strong staining of cleaved caspase-3 in the proamniotic cavity, indicating the presence of apoptotic cells (Fig. 2A, B).

From these results, we conclude that AurA is essential for epiblast survival and that cells lacking AurA undergo apoptosis, leading to embryos devoid of epiblast.

AurA Epi-KO embryos establish the anteroposterior axis but fail to gastrulate

Experiments in which the number of blastomeres was reduced at pre-implantation stages have shown that a threshold number of epiblast cells needs to be reached for gastrulation to proceed (Power and Tam, 1993). Since the size of the epiblast in Epi-KO embryos is reduced to one third of its normal size at the onset of gastrulation (~E6.5), we wondered if specification of the primitive streak and/or gastrulation were affected in Epi-KO embryos.

To determine if the axes of Epi-KO embryos were correctly specified, we examined the position of the anterior visceral endoderm (AVE) and the formation of the primitive streak. At E6.5, the AVE marks the prospective anterior side of the embryo and it is located opposite to the primitive streak which marks the caudal end. To examine the extent of AVE formation and its location in Epi-KO embryos, we conducted wholemount in situ hybridization experiments using Hex, a gene expressed in the AVE (Thomas et al., 1998). Hex is also expressed at the tip of the primitive streak at mid streak stages and later in nascent mesendoderm. Analysis of E6.5 Epi-KO embryos (n=7) revealed that the AVE was properly located on one side of the epiblast, extending from the tip of the epiblast to its junction with the extra-embryonic ectoderm (Fig. 3A). In these embryos, however, we did not observe expression of Hex on the posterior side of the embryo, suggesting a failure in primitive streak formation.

Figure 3. Epiblast-specific AurA knockout embryos establish the anteroposterior axis.

Figure 3

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A. Hex expression in embryos dissected at E6.5 marks the anterior visceral endoderm (red arrowheads) and the tip of the primitive streak (black arrowhead). The Epi-KO embryo shows normal anterior visceral endoderm position but absence of Hex expression in the primitive streak. B–D. Expression of T (B), Fgf8 (C) and Axin2 (D) marks the primitive streak in the posterior region of the embryo (red arrows) at E6.5. T expression is also evident in the extra-embryonic ectoderm (black arrows in B). Pictures are shown at the same scale. Scale bar, 50 μm.

To determine if the primitive streak was specified in Epi-KO embryos, we analyzed the expression of Brachyury (T), Fgf8, and Axin2, three known markers of the primitive streak (Guo and Li, 2007; Jho et al., 2002; Sun et al., 1999; Wilkinson et al., 1990). T also marks the distal portion of the extra-embryonic ectoderm (Perea-Gomez et al., 2004; Rivera-Perez and Magnuson, 2005). In E6.5 Epi-KO embryos, we observed expression of all three genes T (n=4), Fgf8 (n=4) and Axin2 (n=5) in a small area on one side of the proximal epiblast, indicating that the primitive streak was specified (Fig. 3B–D). We also observed expression of T in the distal extra-embryonic ectoderm (Fig. 3B).

These results demonstrate that Epi-KO embryos are able to establish an anteroposterior axis and specify the primitive streak, but the absence of Hex expression in the caudal end of the conceptus suggests that Epi-KO embryos cannot advance to mid-streak stages and fail to gastrulate.

Extra-embryonic AurA Epi-KO tissues support the development of ES cell-derived embryos

The extra-embryonic tissues play a pivotal role in patterning the mouse embryo (see review by Stern and Downs, 2012). In our study, mutation of AurA in the epiblast results in the ablation of the epiblast leaving behind a conceptus composed of extra-embryonic tissues that remain basically intact. To determine if the extra-embryonic tissues of AurA Epi-KO embryos are functionally capable to direct embryonic development, we generated chimeras by injecting ES cells into AurA Epi-KO blastocysts and assessed their development at different stages of development.

To generate chimeras, we injected wild-type AB1 ES cells (McMahon and Bradley, 1990) into Epi-KO blastocysts marked by the R26 reporter (AurAd2/fx;R26r/+;Sox2Cre/0) or ES cells marked by the R26lacZ allele (KT4) (Tremblay et al., 2000) into non-lacZ Epi-KO blastocysts (AurAd2/fx;Sox2Cre/0). In either case, dissection at E9.5 revealed the presence of embryos at early organogenesis stages (n=10) (Fig. 4). ES cell-derived embryos contained blastocyst-derived cells in the gut region (Fig. 4B) and some tended to be smaller than their control littermates. At E11.5, however, we were not able to recover ES-cell derived embryos. Instead, resorption sites were observed that amounted to the expected fifty percent Mendelian ratio (9/20).

Figure 4. AurA Epi-KO embryos support the development of ES cell-derived embryos.

Figure 4

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A. Control and AurA epiblast knockout embryos at E9.5. The AurA Epi-KO conceptus has been reduced to resorbing extra-embryonic components. B. Embryos generated by injection of wild-type ES cells into AurA Epi-KO or control blastocysts marked with R26lacZ. The embryo on the right is a chimera derived from an AurA epiblast knockout embryo and is composed mostly of ES cells-derived tissues. Some blastocyst-derived cells, marked by β-galactosidase activity, are present in the gut region (arrows). Pictures are shown at the same scale. Scale bar, 500 μm.

These results show that the extraembryonic tissues of AurA Epi-KO embryos can support the development of ES cell-derived embryos through gastrulation and early organogenesis stages, but not further development.

Ablation of AurA in visceral endoderm causes apoptosis and embryo lethality

To determine the role of Aurora A in the visceral endoderm (VE), we utilized TtrCre transgenic mice to generate VE-specific AurA knockout (VE-KO) embryos. TtrCre mice express Cre recombinase only in the visceral endoderm at early post-implantation stages (Kwon and Hadjantonakis, 2009; Kwon et al., 2008). To generate VE-KO embryos, we crossed homozygous AurA floxed mice (AurAfx/fx) with transgenic TtrCre mice carrying a null allele of AurA (AurAd2/+;TtrCre/0). VE-KO embryos fail to express AurA in the visceral endoderm, but retain a functional allele of AurA (AurAfx) in the rest of the conceptus.

We recovered VE-KO embryos at E6.5, E7.5 and E9.5 and examined their morphology. At E6.5, VE-KO embryos were smaller than control littermates. In mutant embryos, the visceral endoderm surface was rough and vesicles were apparent (Fig. 5A). At E7.5, the difference in size was more pronounced (Fig. 5B) and by E9.5, the mutant embryos were in the process of resorption (Fig. 5C). These results show that Aurora A is essential in the visceral endoderm for embryo growth and survival.

Figure 5. AurA knockout in the visceral endoderm inhibits embryo growth and causes lethality.

Figure 5

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A. Control and AurA visceral endoderm knockout embryos dissected at E6.25. The VE-KO embryo is smaller than the control embryo. B. Embryos dissected at E7.5, with visceral endoderm cells marked with ROSA26 reporter (blue). Surviving visceral endoderm cells in AurA VE-KO embryos are more prevalent in the visceral endoderm overlying the extra-embryonic ectoderm portion of the conceptus. C. At E9.5, VE-KO embryos (asterisks) are in the process of resorption. Scale bars, 50 μm, 100 μm, and 500 μm in (A), (B), and (C), respectively.

To investigate the fate and integrity of visceral endoderm cells, we labeled AurA-deficient VE cells using the ROSA26 reporter line. β-galactosidase assays revealed the presence of mutant visceral endoderm cells in the extra-embryonic but not in the embryonic region of the conceptus (Fig. 5B). These results suggest that visceral endoderm cells overlying the extraembryonic ectoderm are more tolerant to absence of AurA than visceral endoderm cells overlying the epiblast.

To determine if visceral endoderm cells lacking AurA were undergoing apoptosis, we conducted wholemount immunofluorescence analysis using anti-cleaved caspase-3 antibody. At E6.5, we found extensive apoptosis in the visceral endoderm layer of VE-KO embryos (Fig. 6. A, B). Apoptosis, however, was mostly restricted to the embryonic portion of egg cylinder. These results are consistent with the observation that visceral endoderm cells lacking AurA are more prevalent in the extra-embryonic region of the conceptus (see Fig. 5B) and suggests that embryonic visceral endoderm are eliminated through apoptosis.

Figure 6. AurA visceral endoderm knockout leads to apoptosis and axial defects.

Figure 6

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A and B. DIC (A) and fluorescent (B) images of E6.5 embryos subjected to cleaved Caspase 3 immunostaining. The AurA VE-KO embryo shows apoptosis in the visceral endoderm overlying the epiblast (arrowhead in B). C and D. Hex expression in embryos dissected at E6.5. The AVE in VE-KO embryos remains at the distal tip (C) or shifts to approximately half of the anterior epiblast (D). E and F. Side view (E) and posterior view (F) of embryos dissected at E6.5 and hybridized with T to mark the primitive streak. The AurA VE-KO embryo shows expansion of the primitive streak to the anterior region of the epiblast (arrow in E). G and H. Side view (G) and posterior view (H) of AurA VE-KO embryos dissected at E6.5 and hybridized with T. In VE-KO embryos, T expression remains associated with one side of the short axis of the conceptus indicating that the anteroposterior axis failed to elongate. All pictures shown at the same scale, scale bar, 50 μm.

From these studies, we conclude that Aurora A is essential for visceral endoderm integrity and survival and that its absence results in apoptosis; however, there are differences in the response to AurA ablation between embryonic and extra-embryonic visceral endoderm cells.

Axial development is affected in AurA VE-KO embryos

In several mutant embryos such as Cripto and Otx2 nulls, the AVE fails to reach the anterior side of the embryo and remains at the tip of the epiblast. These morphogenetic defects lead to expansion of the primitive streak to anterior regions of the epiblast (Ding et al., 1998; Perea-Gomez et al., 2001).

To address whether VE-KO embryos have axialization defects, we conducted wholemount in situ hybridization using Hex and T probes. Analysis of Hex expression revealed defects in the anterior shift of the distal visceral endoderm (DVE) to become the AVE. The position of the AVE varied in VE-KO embryos. In some embryos, it remained at the tip of the embryo (n=2) (Fig. 6C), while in others there was a shift to one side of the epiblast although it failed to reach the epiblast/extraembryonic ectoderm boundary (n=2) (Fig. 6D). Analysis of T expression revealed that T was expanded anteriorly around the epiblast junction with the extraembryonic ectoderm (n=6) (Fig. 6E, F). In other cases, we observed proper localization of T in the posterior side of VE-KO embryos, but in contrast to control embryos, these mutants failed to elongate the anteroposterior axis (Mesnard et al., 2004; Perea-Gomez et al., 2004) (Fig. 6G, H).

Together, these results reveal that ablation of AurA in the visceral endoderm leads to abnormal establishment of the anteroposterior axis of the embryo.

DISCUSSION

Ablation of AurA in mice causes apoptosis and embryonic lethality at pre-implantation stages (Cowley et al., 2009; Lu et al., 2008; Sasai et al., 2008). Our study reveals similar essential roles for Aurora A at early post-implantation stages. Ablation of AurA in the epiblast or in the visceral endoderm of post-implantation embryos leads to apoptosis, inhibition of embryo growth and lethality. The effects of AurA ablation, however, impact embryonic development differently depending on the tissue affected. Mutation of AurA in the epiblast leads to progressive loss of epiblast cells and gastrulation defects and generates a conceptus composed mostly of extra-embryonic tissues by E7.5. Despite the severe reduction of epiblast cells, the axes of the embryo are properly established, suggesting that the defects observed in embryos lacking AurA in the epiblast are due to a paucity of epiblast cells rather than patterning defects. Previous studies have shown that a threshold number of cells is required for gastrulation to proceed (Power and Tam, 1993; Tam, 1988). Our results support these observations, however, the initial specification of the primitive streak, the engine of gastrulation, appears to be less dependent on a cell number threshold.

Lack of AurA in the visceral endoderm leads to defects in the establishment of the anteroposterior axis. Analysis of Hex expression revealed misallocation of the AVE, which is correlated with expansion of the primitive streak to anterior regions of the epiblast. Previous experiments have shown that the AVE regulates the extent of the primitive streak. For example, a double knockout of Cerl1 and Lefty1, two genes expressed in the AVE, leads to the formation of multiple primitive streaks (Perea-Gomez et al., 2002). Also, mutations that affect the shift of the AVE to the anterior side of the epiblast lead to anterior expansion of the primitive streak (Ding et al., 1998; Perea-Gomez et al., 2001). We believe that the defects observed in AurA VE-KO embryos are due to improper positioning of the AVE. How the ablation of AurA in the visceral endoderm leads to misallocation of the AVE is still an open question. One possibility is that a threshold number of visceral endoderm cells is required to allow proper positioning of the AVE. An alternative explanation is that AurA-lacking cells are functionally abnormal and cannot fulfill their normal role in development.

Determination of the fate of mutant visceral endoderm cells in E7.5 AurA VE-KO embryos revealed survival of visceral endoderm cells in the extra-embryonic but not in the embryonic region of mutant embryos. This variability is not due to failure to ablate AurA in the extra-embryonic region since recombination of the ROSA26 reporter indicated activity of Cre recombinase in these cells. The differences in tolerance to AurA ablation may be due to inherent differences between the two groups of visceral endoderm cells, which are morphologically and molecularly distinct (Pfister et al., 2007; Rivera-Perez et al., 2003). Another possibility is that extra-embryonic visceral endoderm cells have lower proliferation rates than embryonic ones. As the apoptotic effects of AurA deletion are tied to cellular proliferation, lower proliferation rates could cause reduced activation of apoptotic signals due to absence of AurA in extra-embryonic visceral endoderm cells and better survival rates in this tissue. These experiments also revealed the presence of β-galactosidase-negative cells in the embryonic visceral endoderm area of the conceptus. These cells may represent lack of recombination of the ROSA26 reporter in visceral endoderm cells in this region of the embryo or non-visceral endoderm cells derived from the epiblast. The first possibility is unlikely since we observed apoptosis in embryonic visceral endoderm cells at E6.5. Moreover, the Ttr-Cre transgene utilized to recombine floxed alleles in the visceral endoderm is expressed in all cells of the visceral endoderm (Kwon and Hadjantonakis, 2009; Kwon et al., 2008). A more plausible explanation is that β-galactosidase-negative cells represent definitive endoderm cells derived from the epiblast. Previous experiments have shown that definitive endoderm cells intercalate with visceral endoderm at primitive streak stages (Kwon et al., 2008). This is also evident in control embryos in our experiments. In the mutant embryos, it is likely that visceral endoderm cells apoptose leaving behind only definitive endoderm cells.

Injection of ES cells into embryos lacking AurA in the epiblast can rescue embryonic development to E9.5 stages. These results reveal that the extra-embryonic tissues of AurA epiblast knockout embryos can support the development of ES cell-derived embryos. Evidence suggest that the extra-embryonic tissues are responsible for patterning the embryo in addition to its nutritional and protective roles (Stern and Downs, 2012). Our chimera results support this view; however, we cannot discard the possibility that residual epiblast cells present at earlier stages of development provide non-cell autonomous signals that direct the differentiation of ES cells into a fully patterned embryo. Interestingly, the chimeras did not survive to late post-implantation stages. Since the embryos die shortly after E10.5, when the placenta becomes functional, we believe that defects in the maternal-fetal interphase such as abnormalities in the allantois, placenta or circulatory system can account for the lethality of the embryos.

In summary, our experiments suggest that at the cellular level Aurora A plays similar roles at pre- and post-implantation stages but that at the organismal level, interfering with its function generates different outcomes depending on the tissue affected. A practical ramification of our study is that mutation of AurA can be used as a way to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. In addition, embryos with an epiblast-specific mutation of AurA can be used in experiments aimed at testing the potency of stem cells. This epiblast complementation technique would be advantageous over tetraploid complementation approaches since they do not require the generation of tetraploid embryos and provide a diploid instead of a multiploid extraembryonic environment.

Highlights.

  • We ablate Aurora A in the epiblast or visceral endoderm of mouse embryos.

  • Aurora A ablation leads to apoptosis, patterning defects and embryo lethality.

  • ES cells can rescue the phenotype of Aurora A deficiency in the epiblast.

  • Embryonic defects vary depending on the tissue affected by the mutation.

  • We provide an alternative to tetraploid complementation experiments.

Acknowledgments

We thank Kat Hadjantonakis for kindly providing TtrCre mice and Kim Tremblay for the generous gift of KT4 ES cells. We also thank Frank Costantini, James Li and Tristan Rodriguez for Axin2, Fgf8 and Hex probes, respectively. We are indebted with Marilyn Keeler for help with ES cell culture. We are grateful to Paul Odgren for critically reading the manuscript. This research was supported by NIH grants GM87130 (JARP), GM94874 (JARP and SNJ) and CA065773 (TvD).

Footnotes

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