Knockout of ERK5 causes multiple defects in placental and embryonic development
© Yan et al; licensee BioMed Central Ltd. 2003
Received: 18 September 2003
Accepted: 16 December 2003
Published: 16 December 2003
ERK5 is a member of the mitogen activated protein kinase family activated by certain mitogenic or stressful stimuli in cells, but whose physiological role is largely unclear.
To help determine the function of ERK5 we have used gene targeting to inactivate this gene in mice. Here we report that ERK5 knockout mice die at approximately E10.5. In situ hybridisation for ERK5, and its upstream activator MKK5, showed strong expression in the head and trunk of the embryo at this stage of development. Between E9.5 and E10.5, multiple developmental problems are seen in the ERK5-/- embryos, including an increase in apoptosis in the cephalic mesenchyme tissue, abnormalities in the hind gut, as well as problems in vascular remodelling, cardiac development and placental defects.
Erk5 is essential for early embryonic development, and is required for normal development of the vascular system and cell survival.
Mitogen activated protein kinase (MAPK) cascades play important roles in many cellular processes including cell proliferation, differentiation, survival and apoptosis. They are also important for many physiological functions in several systems, including in developmental, immune and neuronal systems. At least 12 isoforms of MAPKs exist in mammalian cells, and these can be divided into 4 main groups, the 'classical' MAPKs (ERK1 and ERK2), JNKs (also referred to as SAPK1), p38s (also referred to as SAPK2, SAPK3 and SAPK4) and atypical MAPKs such as ERK3, ERK5 and ERK8. With the exception of ERK3, MAPKs are activated by dual phosphorylation on a Thr-Xaa-Tyr motif by a dual specificity MAPK kinase (MKK). MKKs are in turn activated by a MAPK kinase kinase (MKKK), which are activated in response to appropriate extracellular signals.
ERK5 is an atypical MAPK that can be activated in vivo by a variety of stimuli, including some mitogens such as EGF, and some cellular stress such as oxidative and osmotic shock [1–3]. These stimuli activate a cascade in which the MAPK kinase kinases MEKK3 or MEKK2 activate MKK5, which in turn activates ERK5 [4, 5]. Interest in the ERK5 pathway has been fuelled by reports that the activation of ERK5 by MKK5 can be blocked in vivo by the kinase inhibitors PD184352, PD 98059 and U0126. These inhibitors were developed as inhibitors of the classical MAPK cascade, and have been used extensively to study this cascade in vivo. The discovery that they can also block ERK5 activation, although at higher concentrations than are required to block the activation of ERK1 and ERK2, raised the possibility that ERK5 and ERK1/ERK2 may have some overlapping functions in vivo [6, 7].
The physiological roles of ERK5 are still largely unclear. Overexpression of a constitutively active MKK5 in mice results in cardiac hypertrophy and death of the mice by 8 weeks of age . This is suggestive of a role of ERK5 in the heart, possibly related to cardiac development. ERK5 has also been implicated in the development of smooth muscle, as ERK5 antisense oligonucleotides  or dominant negative ERK5 constructs  have been reported to block the differentiation of smooth muscle cells in cell culture models. At present little is known about the substrates for ERK5 in vivo, however it has been suggested to phosphorylate connexin 43  and the transcription factor MEF2C [12–14]. Mouse knockouts of MEF2C are embryonic lethal, and MEF2C-/- embryos die due to a failure of the developing heart to undergo normal looping at E8.5-9 . Knockout MEKK3 also results in embryonic lethality at E11, MEKK3-/- embryos show problems with myocardium formation, angiogenesis and placental formation . While this could be consistent with a role for ERK5 in linking MEKK3 signalling to MEF2C during cardiac development, it should be noted that MEKK3 can activate other MAPK isoforms, particularly p38α (also referred to as SAPK2A) [17–20]. Knockout of p38α has been reported by several groups, and p38α-/- embryos have also been reported to show problems in cardiac development, angiogenesis and placental formation at E10-11 [21–23].
In order to further examine the role of ERK5 we carried out expression and gene targeting studies in mice. ERK5 knockout was found to be lethal during embryogenesis at E10.5 to E11, and here we report a detailed analysis of these embryos. While this work was in progress, both Regan et al  and Sohn et  also reported ERK5 knockouts, and the effects of these different ERK5 knockouts are considered in the discussion.
Generation of ERK5 knockout mice
Ratios of ERK5 adults and embryos
Expression of ERK5 and MKK5 during embryogenesis
ERK5 is required for normal angiogenesis and placental development
ERK5 is required for normal development of the head and lower trunk regions
In this report, we show that knockout of ERK5 results in embryonic lethality at around E10.25 and show that ERK5-/- embryos have problems with placental development, changes in angiogenesis and problems with the development of the head, (especially the cephalic mesenchyme and neuroepithelium), and lower trunk of the embryo. While this work was in progress, two other groups reported ERK5 knockouts. Regan et al reported that the ERK5 knockout was lethal between E9.5 to E11.5 , while Sohn et al reported lethality between E10.5 and E11.5 . Similar effects on placental development and angiogenesis were found in both reports, and this phenotype is consistent with the effects described here. While both Sohn et al and Regan et al reported that ERK5-/- embryos were growth retarded by E10, neither study reported characterisation of the head and trunk regions of these embryos. It is therefore not possible to say if the defects we report in the cephalic mesenchyme and gut were present in these knockouts. Differences in the targeting strageties between both Sohn et al and Regan et al, and that used here may explain why some differences were seen in the phenotypes observed, as it is not possible to rule out the possibility that truncated fragments of the ERK5 protein were expressed in any one of those knockouts, which may give rise to a dominant negative effect. Interestingly however the most severe phenotype reported was that of Regan et al, and in this study the targeting used here deleted the smallest region of the ERK5 gene of all the knockouts. It should also be stressed that other differences, such as the strain and source of mice and ES cells used, may also explain differences between the phenotypes of the three knockouts.
Interestingly we found two distinct morphologies of ERK5-/- embryos at E10.25, however the reason for this was not clear. Class I embryos were characterised by severe growth retardation compared to wild type embryos, while class II embryos were larger but had severe abnormalities in the development of the head and lower trunk. One possible explanation may relate to the degree of severity of the placental phenotype. Placental defects are a common cause of lethality at this developmental stage in knockout mice [27, 28]. If the severity of these placental defects varied between individual ERK5-/- embryos due to other genetic or environmental factors, then as a result, the problems in placental development may be sufficient to kill some embryos (class I) before E10.25, but other embryos (class II) survive longer, allowing other phenotypes to become more pronounced. A similar effect has also been observed in a knockout of p38α, in which some embryos died at E11.5, presumably due to placental defects, while some embryos survived past this stage . We found that the basic structures of the placenta, including the chorionic plate, labyrinthine trophoblast, labyrinth and spongiotrophoblast layers were able to form in the absence of ERK5. Histological sections did not show significant differences between ERK5-/- and wild type placenta at E9.75 (Fig 9). By E10.25 however the thickness of the labyrinth was reduced in ERK5-/- placentas, and there was less intermixing of the embryonic and maternal blood vessels in the placental labyrinth. This correlated with increased apoptosis in this region in ERK5-/- placentas (Fig 10). This is suggestive of a role for ERK5 in development of the labyrinth and chorioallantoic branching. As the labyrinth is the major site of exchange between the embryonic and maternal blood, the problem seen in the ERK5-/- placentas is likely to be sufficient to cause embryonic lethality. ERK5 is not the only MAP kinase signalling protein whose knockout affects labyrinth development. Knockout of MEKK3, an upstream activator of ERK5 and p38 , resulted in embryonic lethality due in part to a failure of the labyrinth development. Also knockouts of p38 and MEK1  result in problems with the labyrinth, as do knockouts of several receptors known to activate MAPK signalling including LifR , EgfR , PdgfR [32, 33], Met , and the GDP/GTP exchange factor Sos1 .
Consistent with the findings of Regan et al and Sohn et al, we also found that ERK5 knockout resulted in problems in angiogenesis in the embryo. Analysis of ERK5 expression by in situ hybridisation however showed that expression of ERK5 was not restricted to the developing blood vessels, but was instead expressed more widely in the embryos. Both Sohn et al and Regan et al report that expression of various signalling molecules important in angiogenesis were normal in the ERK5 knockout. The reason for the reduction in angiogenesis in the knockout mice is unclear, but may be related to general growth retardation seen in ERK5-/- embryos compared to wild type litermates at E10.25.
Knockout of ERK5 also affected cardiac development. Using both whole mount in situ hybridisation and immunoblotting of dissected embryos, we show that expression of ERK5, and its upstream activator MKK5, is expressed in the heart at E9.5 to E10.25, although their expression level was low compared to other regions of the embryo (Figs 2,3,4,5). Consistent with this, Sohn et al also reported that ERK5 expression was highest in the heart and trunk of the embryo at E9 to E9.5. Using only RNA in situ hybridisation Regan at al however reported the opposite, with high levels of ERK5 expression localised to the developing heart and little expression in the rest of the embryo. The reasons for this difference between the report of Regan et al, and both our findings and those of Sohn et al is unclear. We found development of the embryonic heart was retarded compared to wild type embryos. Similar to the report of Sohn et al, we observe that basic patterning of the heart can occur in the absence of ERK5. Once formed however, the heart does not develop past it's basic patterning. In particular the thickness of the atrial wall at E9.75 was reduced in ERK5 knockout embryos (Fig 8). Interestingly, the knockout of ERK5 had much less severe effects on heart development compared to the knockout of its potential substrate MEF2C, in which embryos die at E8.5-9 due to failure to undergo normal looping. This suggests that either MEF2C has functions which are independent of its phosphorylation by ERK5 in vivo at this developmental stage, or that other kinases such as p38 can also phosphorylate the same sites on MEF2C as ERK5 in vivo. In this respect it is interesting to note that knockout of p38 resulted in similar problems in cardiac development to the ERK5 knockout. In contrast to this report, and that of Sohn et al, Regan et al reported that the heart did not undergo normal looping at E9.5. The reason for this discrepancy is not clear, but may be due to differences in targeting or the genetic strains of mice used. The knockout of ERK5 has been previously observed to have a similar phenotype to knockouts of receptor tyrosine kinase Tie-2  and its ligand Ang-1 , which may suggest that ERK5 could function downstream of these receptors in the heart. There is however no direct evidence to demonstrate this link and further work would be needed to establish if this were true. In isolated cell lines ERK5 has been reported to be activated by the neuregulin receptors erbB2 and erbB3 , raising the possibility that erk5 may mediate some to the effects of neuregulins in the heart. A further possible reason for the cardiac phenotype is that ERK5 has been reported to inhibit the activity of the VEGF promoter , so that increased VEGF levels in the ERK5-/- embryos may affect cardiac development. A third possibility is that the cardiac defects observed in ERK5-/- embryos may not be directly due to the lack of ERK5 in the heart, and that these phenotypes may be caused wholly or in part by stress induced by the placental defects in the knockouts. It has been shown in other knockout models that cardiac phenotypes can be secondary to other problems in the embryo (for examples see [21, 36]). Further work, including the use of placental rescue or cardiac specific ERK5 knockouts, will be required to fully resolve these issues.
We also observed defects in the development of the cephalic mesenchyme and gut in the ERK5-/- embryos. In ERK5-/- embryos problems were seen in the cephalic mesenchyme from E9.75 onwards. At E9.75 the cephalic mesenchyme appeared less dense with larger spaces between the cells and less contact between the cephalic mesenchyme and the neuroepithelium. However as the embryos developed, this gradually worsened and by E10.25 the cephalic mesenchyme was essentially absent (Fig 11). Several factors suggest that the defects seen in the cephalic mesenchyme are primary phenotypes directly caused by the loss of ERK5 protein in this region. First, in stiu hybridisation showed that ERK5 was expressed in the cephalic mesenchyme from E9.75 (Fig 5). Secondly, these problems could be seen in E9.75 ERK5-/- embryos, while at this stage blood vessel and placental development appeared relatively normal in the knockouts (Fig 6,7,8,9,10), suggesting that the cephalic mesenchyme and gut defects were not secondary to a lack of angiogenesis. Consistent with this, blood vessels were present in the cephalic mesenchyme of E9.75 ERK5-/- embryos, suggesting that the problems with this tissue were not due to a lack of blood supply. The defect in the cephalic mesenchyme appeared to be due to increased apoptosis causing the tissue to be lost, rather than a problem with its initial development. Consistent with the normal initial development of this region, expression patterns of sonic hedgehog and Six3 (L. Yan, unpublished data) at E9.5 were unaffected by the knockout of ERK5. The increase in apoptosis in the ERK5-/- embryos suggests that ERK5 may be involved in regulating cell survival or poliferation. Consistent with this, overexpression of ERK5, or its upstream activator MKK5, has been shown to promote proliferation in some cell types in response to some mitogenic stimuli [1, 37–39].
In summary these results are consistent with a role for ERK5 in angiogenesis and placental development, and show new functions for ERK5 in the survival of the cephalic mesenchyme and regulation of survival and apoptosis. Further work however will be required in order to determine the molecular details of these ERK5 functions.
Antibodies against ERK1/2, p38/SAPK2 and cleaved caspase 3 were from Cell Signalling. The MKK5 antibody was from Stressgen and the CD31 antibody from Pharmhigen. The ERK5 antibody has been described previously .
Generation of ERK5 knockouts
A genomic clone for ERK5 was obtained by screening a 129SvJ mouse BAC genomic library using a mouse ERK5 EST. Regions of the BAC corresponding to the ERK5 were subcloned by either restriction digestion or random fragmentation and sequenced. A targeting vector was designed based on this sequence to delete exons 4 to 5 of the ERK5 gene. The vector consisted of a first arm of homology (generated by cloning of a Sal I / Eco RI fragment ligated to a PCR product generated using the primers GAATTCAGATCTGTGTAAGG and AAGCTTCTGAAAATGGGAAG) then a neomycin resistance cassette, followed by a second arm of homology (generated by using the primers CATATGAGAAGAGGAAAGCCTGGGA and GCGGCCGCAGCAGGGATCAATATGT) and a thymidine kinase cassette (Fig 1). The targeting vector was linearised using Not I before transfection into mouse ES cells.
Mouse embryonic stem cells were grown and transfected as described previously (), using embryonic fibroblasts from MTK-neo mice as a feeder layer. Colonies resistant to both G418 and ganciclovir were expanded and screened for correct incorporation of the ERK5 targeting vector. A probe external to the targeting vector was generated by PCR using the primers CAAGTAGGGGACCAAGTCAAC and GGCCCAATGGAAAGGCTTCTAT. This probe was used to screen DNA double digested with Hind III and Mfe I from ES cell colonies. Positive cell lines were injected into blastocysts from a C57Bl/6 × BALB/c cross, which were then reimplanted into recipient female mice . Chimeric male offspring were then bred to BALB/c or C57Bl/6 mice as indicated and transmission identified by a combination of coat colour and genotyping by Southern and PCR analysis.
Routine gentoyping of the ERK5 mice was carried out by PCR on tail biopsies. PCR was carried out using the primers AACTAACCAACCCACCTTCCAAGAC and CACTAGTACTCCTACTGGCCCCGTA to identify wild type and AACTAACCAACCCACCTTCCAAGAC and ACCACCAAGCGAAACATCGCATCG to identify targeted alleles.
Isolation of embryos
Male and female mice of known genotype were placed together and time of fertilisation determined by observation of copulation plugs, and noon of that day defined as E0.5. Embryos were dissected from pregnant females at the times indicated, and the yolk sacs separated and used to genotype the embryos by PCR.
Whole mount in situ hybridisation, immunohistochemistry and TUNEL staining
Embryos were harvested and fixed in 4% paraformaldehyde. In situ hybridisation was carried out as described previously . Probes for ERK5 (corresponding to the last 207 amino acid and first 165 bp of the 3' utr) and MKK5 (corresponding to the last 71 amino acid and first 295 bp of the 3' utr) were generated by PCR using the primers ACTAGTACTCCTACTGGC and GCTCAGGTGGCTGCTTAAG or ACTAGTAGGATTCGCCGGTCCTTC and ATCAGTGCTGCTGATAGGGCCTGAC respectively. PCR products were cloned into pBluescript to give antisense sequence when transcribed from the T7 promoter.
Whole mount immunohistochemical analysis of embryos using a CD31 antibody as described . Whole mount terminal deoxynucleotidyl transferase-mediated UTP end labelling (TUNEL) was carried out using the in situ cell death detection kit from Roche.
Embryos placenta were fixed in formaldehyde, then dehydrated in ethanol, cleared in chloroform and then embedded in paraffin as described . Sections were cut and stained using haematoxylin and eosin.
The atrial wall thickness was determined using a modified Cavalieri method . Both the inner and outer areas of the atrial chamber were measured and the average wall thickness was defined as the difference between the average radius of the inner and outer areas of the atrial chamber. Between 6 and 9 sections were analysed per embryo, and 4 wild type and 4 ERK5-/- embryos were analysed.
Tissue was homogenised in 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.27 M sucrose, 1% (v/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol and complete proteinase inhibitor cocktail (Roche). Insoluble material was removed by centrifugation at 13000 g for 5 min at 4°C. Soluble lysate (30 μg) was then run on 4–12% polyacrylamide gels (Novex, Invitrogen) and transferred onto nitrocellulose membranes. Primary antibodies against ERK1/2, p38 and MKK5 were used as described by the supplier, and the ERK5 antibody was used at 0.8 μg/ml. Secondary antibodies conjugated to horseradish peroxidase were from Pierce, and detection was performed using ECL (Amsersham).
We would like to thank Philip Cohen for many helpful discussions and critical reading of the manuscript. We would also like to thank Janet Rossant for the 4311 clone. This research was supported by grants from the UK Medical Research Council, Astra-Zeneca, Boehringer-Ingelheim, GlaxoSmithKline, NovoNordisk and Pfizer.
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