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Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development
Gene Expression Patterns 11 (2011) 465–470
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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development Lilian T. Kaufmann a, Mathias S. Gierl a,b, Christof Niehrs a,b,⇑ a b
Division of Molecular Embryology, DKFZ-ZMBH Alliance, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 581, D-69120 Heidelberg, Germany Institut für Molekulare Biologie, Mainz, Germany
a r t i c l e
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Article history: Received 12 June 2011 Received in revised form 28 July 2011 Accepted 29 July 2011 Available online 6 August 2011 Keywords: Gadd45 Expression pattern Mouse embryogenesis Limb buds Neurogenesis Somitogenesis
a b s t r a c t Gadd45 proteins have been implicated in the cellular response to physiological or environmental stress and the accompanying cell cycle arrest, DNA repair, cell survival and senescence or apoptosis. Although their molecular function is well studied, the expression and role of Gadd45 genes during embryonic development in mice is largely unknown. Here we provide a comprehensive comparison of Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development. In situ hybridizations on sectioned and whole mouse embryos show most prominent Gadd45a expression in the tip of the closing neural tube, the cranial and dorsal root ganglia and the somites. Mouse Gadd45b is expressed strongly in the chorion, but only weakly in the embryo proper, including somites and limb buds. Murine Gadd45g expression strongly resembles Xenopus and medaka fish expression in primary neuron precursors and post-mitotic neurons, indicating a conserved role for Gadd45g in vertebrate neurogenesis. Additionally, Gadd45 genes show conserved expression during somitogenesis. In summary, Gadd45 genes are expressed in evolutionary conserved, but also divergent domains, which predominantly encompass areas of cell differentiation, consistent with their established function in growth arrest and DNA demethylation. Ó 2011 Elsevier B.V. All rights reserved.
1. Results and discussion Gadd45 (Growth arrest and DNA damage response) encompasses a small family of stress response genes, encoding small acidic nuclear proteins of the L7Ae/L30e/S12e RNA binding protein superfamily (PFAM entry: PF01248) (Bateman et al., 2004). The three genes, Gadd45a, Gadd45b, Gadd45g, are involved in diverse processes, including cell cycle, apoptosis, DNA repair, and DNA demethylation (Barreto et al., 2007; Hollander and Fornace, 2002; Sheikh et al., 2000; Zhan, 2005). Gadd45a mediates these processes by interacting with many effectors, including cdc2/cyclinB1 (Vairapandi et al., 2002; Zhan et al., 1999), PCNA (Azam et al., 2001; Vairapandi et al., 2000), p21 (Kearsey et al., 1995), nuclear hormone receptors (Yi et al., 2000), histones (Carrier et al., 1999), MEKK4 (Chi et al., 2004; Takekawa and Saito, 1998), XPG (Barreto et al., 2007), AID (Rai et al., 2008) and TAF12 (Schmitz et al., 2009). They play a role in tumor- and autoimmune suppression and UV response (Bulavin et al., 2003; Hollander et al., 1999; Liu et al., 2005). While their function is well characterized in cultured cells and adult mice, much less is known about their role during embryonic
⇑ Corresponding author at: Division of Molecular Embryology, DKFZ-ZMBH Alliance, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 581, D-69120 Heidelberg, Germany. Tel.: +49 6221 42 4690; fax: +49 6221 42 4692. E-mail address: [email protected] (C. Niehrs). 1567-133X/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2011.07.005
development. The phenotypes of the single knock out mice are rather mild. Gadd45a-deficient mice display exencephaly at low penetrance (Hollander et al., 1999). Gadd45b1 and Gadd45b2 in zebrafish are periodically expressed in the anterior presomitic mesoderm and are required for somite segmentation (Kawahara et al., 2005). Gadd45g in medaka fish and Xenopus laevis are expressed in primary neuron precursors and promote differentiation by regulation of cell cycle exit (Candal et al., 2004; de la CalleMustienes et al., 2002). In mouse, the expression of Gadd45 genes during embryonic development has not been analyzed. Here we provide a comprehensive comparison of Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development.
1.1. qPCR expression analysis of Gadd45a, Gadd45b and Gadd45g during early mouse development The temporal expression patterns of the three Gadd45 genes during embryonic development, analyzed by qPCR, were dynamic and differential (Fig. 1). Mouse Gadd45a was expressed at low levels at gastrula stages (E6.5–E7.5) and expression strongly increased towards neurulation, but already reached a plateau at E8.5 (Fig. 1A). Gadd45b was highest expressed in gastrula stages (E6.5–E7.5) and transcripts rapidly decreased during neurulation (Fig. 1B). Gadd45g was not expressed maternally and increased continuously after gastrulation (Fig. 1C). Comparison of
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Fig. 1. qPCR expression analysis of Gadd45 genes in mouse embryos. (A–C) qPCR analysis of mouse Gadd45a (A), Gadd45b (B) and Gadd45g (C) normalised to GAPDH at the indicated embryonic stages. (D) Quantification of relative transcript levels of mouse Gadd45a, Gadd45b and Gadd45g in E10.5 embryos by qPCR. Data represent the mean ± SD of three independent experiments each using one mouse embryo per stage, respectively.
the relative transcript levels of Gadd45a, Gadd45b and Gadd45g at E10.5 by qPCR revealed that among the three Gadd45 genes, Gadd45b showed by far the lowest overall expression (Fig. 1D).
1.2. Prominent expression of mouse Gadd45a in ganglia In mouse embryos Gadd45a expression was first detected in the primitive streak and the embryonic mesoderm at E6.5–E7.5
Fig. 2. Expression of Gadd45a during mouse development. (A–I and L–P) Whole mount in situ hybridization of Gadd45a on E7.5–E10.5 mouse embryos. (K,Q-R) Vibratome sections of embryos shown in F and L as indicated. (J), control in situ hybridization with sense probe for Gadd45a of E9.5 embryo. Lateral views (A, C–F, J and L), dorsal views (B, G and N), ventral views (H, I, M, O–P). Key: (a) anterior, (d) dorsal, (da) dorsal aorta, (drg) dorsal root ganglia, (fb) forebrain, (fg) facio-acoustic ganglia, (fl) forelimb bud, (gg) glossopharyngeal ganglia, (hb) hindbrain, (hl) hindlimb bud, (lt) lamina terminalis, (mg) mid gut, (nf) neural fold lip, (np) caudal neuropore, (nt) neural tube, (oe) olfactory epithelium, (ot) otic vesicle, (p) posterior, (ps) primitve streak, (so) somitic mesoderm, (tg) trigeminal ganglia, (tv) telencephalic vesicle, (v) ventral, (vg) vagal ganglia.
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(Fig. 2A). This pattern was weak and replaced by a robust Gadd45a expression in the lips of the closing neural fold (Fig. 2B). The expression in the neuroepithelium strongly resembled the distribution of apoptotic cells (Massa et al., 2009). This might indicate a role of Gadd45a during neural tube closure since Gadd45a homozygous mutants show exencephaly (Hollander et al., 1999). At E9.0 Gadd45a continued to be expressed in the tip of the closing neural tube, except for the midbrain and hindbrain region (Fig. 2C and D). Additional expression started in scattered cells of the placodes of trigeminal- and the dorsal root ganglia (Fig. 2C– E) which became stronger at E9.5 (Fig. 2F–H). At E9.5 expression also appeared in the facio-acoustic, glossopharyngeal and the vagal ganglia and in the olfactory epithelium (Fig. 2F–I). The expression of Gadd45a in the closing neural tube persisted only in the caudal neuropore and the lamina terminalis (Fig. 2I), the regions to undergo neural tube closure last. Furthermore, there was weak expression in the myotomes as seen in vibratome sections (Fig. 2K). From E9.5 onwards a ubiquitous basal expression of Gadd45a was observed, compared to the sense control (Fig. 2J). At E10.5 additional expression occurred in the epithelium of the telencephalic ventricles (Fig. 2L, M and Section 2R). In the limb buds Gadd45a was expressed in the posterior-distal mesenchyme subjacent to the AER with a dorsal and a ventral expression domain separated by an area of lower expression in the centre (Fig. 2O–Q). 1.3. Low level expression of mouse Gadd45b in mesodermal derivatives The transcript of Gadd45b could not be detected in the epiblast of E6.5–E7.5 embryos but strongly in the chorion (Fig. 3A). In E8.5 embryos only weak and diffuse Gadd45b expression was detected
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in the future mid- and hindbrain, anterior heart field, first forming somites and in the posterior remnants of the primitive streak. At E9.0 expression in the dorsal midbrain and in the first epithelial somites became more pronounced (Fig. 3C and D). Somite expression progressed anteriorly such that at E10.5 it was expressed in all trunk somites (Fig. 3L and P). From E9.5 to E10.5 Gadd45b was additionally expressed in the branchial arch arteries and the dorsal aorta (Fig. 3E–J), in the tip of the first branchial arches and the forelimb bud (Fig. 3L, M and O). The strong staining in the forebrain vesicles was due to probe trapping and was unspecific. Within the forelimb bud Gadd45b was expressed in the anterior necrotic zone (ANZ) or ‘‘foyer préaxiale primaire’’ (Milaire, 1971, 1976) and the zone of polarizing activity, strongly resembling the Msx2 expression domain associated with cell death (Ganan et al., 1998; Krabbenhoft and Fallon, 1992). 1.4. Prominent expression of mouse Gadd45g in the nervous system Expression of Gadd45g in E7.5 embryos was detected in the ectoplacental cone and scattered cells of the extraembryonic ectoderm but hardly in the epiblast (Fig. 4A). At E8.5–8.75 extraembryonic expression continued in the allantois, and embryonic expression occured in the presomitic mesoderm and in few neural precursors within the neural fold (Fig. 4B and C). At subsequent stages neural expression dominated and manifested in the dorsal midbrain, the cranial and dorsal root ganglia and the neural tube (Fig. 4E–I). Vibratome sections of E9.5 embryos showed that within the brain Gadd45g was expressed at the forebrain-midbrain boundary, the trigeminal ganglia and the latero-ventral hindbrain
Fig. 3. Expression of Gadd45b during mouse development. (A–J and L–O) Whole mount in situ hybridization of Gadd45b on E7.5–E10.5 mouse embryos. (K) control in situ hybridization with sense probe for Gadd45b of E9.5 embryo. Lateral views (A–C, E, F, and I–L), dorsal views (G, N–O), ventral view (D and H), anterior view (M). The dashed boxes in F and N indicate the regions magnified in I, J and O. Key: (a) anterior, (al) allantois, (ANZ) anterior necrotic zone, (ba) first branchial arch, (br) brain, (ch) chorion, (cs) caudal somites, (da) dorsal aorta, (db) dorsal midbrain, (fl) forelimb bud, (fs) forming somites, (hl) hindlimb bud, (ht) heart field, (p) posterior, (ps) primitive streak, (so) somitic mesoderm, (ZPA) zone of polarizing activity.
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Fig. 4. Expression of Gadd45g during mouse development. (A–C and E–I) Whole mount in situ hybridization of Gadd45g on E7.5–E10.5 mouse embryos. (D) Control in situ hybridization with sense probe for Gadd45g of E9.5 embryo. Lateral views (A, C–E and H), dorsal views (B and F), ventral views (G and I). (J, K and M) Vibratome sections of embryos shown in E and H as indicated. (L) Section of embryo with similar staining pattern as shown in H. Key: (a) anterior, (al) residual allantois, (ba) first branchial arch, (db) dorsal midbrain, (drg) dorsal root ganglia, (ec) ectoplacental cone, (ee) extraembryonic ectoderm, (fb) forebrain, (fg) facio-acoustic ganglia, (fl) forelimb bud, (fs) forming somite, (hd) hindbrain, (hl) hindlimb bud, (ht) heart, (mb) midbrain, (nf) neural fold, (nt) neural tube, (oe) olfactory epithelium, (os) optic stalk, (ot) otic vesicle, (p) posterior, (so) somitic mesoderm/myotome, (tg) trigeminal ganglia.
(Fig. 4J). In the neural tube Gadd45g was predominantly expressed in ventral and dorsal peripheral cells (Fig. 4K). This pattern resembled the expression of the Notch effector Hes6 (Vasiliauskas and Stern, 2000). Prominent expression occurred also in the facioacoustic ganglia and the ventral part of the otic cup (Fig. 4K). In E10.5 embryos Gadd45g continued to be expressed in distinct domains within the neural tube, the dorsal root ganglia and the myotome (Fig. 4I, L–M). A summary of the expression domains of Gadd45a, Gadd45b and Gadd45g in mouse embryos can be found in Table 1.
la Calle-Mustienes et al., 2002; Sheng et al., 2010). A common element of Gadd45 genes is their expression in cells undergoing differentiation, such as forming somites and neuronal precursors consistent with their functional implication in cell cycle arrest and DNA demethylation (Barreto et al., 2007; Hollander and Fornace, 2002).
1.5. Conclusion
Female CD-1 outbred mice (Mus musculus) were produced and raised in the research colony of the investigators. Wild type mice were mated overnight and noon of the day of vaginal plug detection was defined as embryonic (E) 0.5 days post coitum. Embryos were dissected in ice cold PBS according to standard protocols (Hogan et al., 1994). Mice were kept according to international standard conditions, and all animal experiments complied with local and international guidelines for the use of experimental animals.
The Gadd45 genes are differentially expressed during embryonic development of mouse. Certain expression domains are exclusive for a given family member, like expression of Gadd45a in closing neural tube, Gadd45b in the chorion, and Gadd45g in the mouse brain, but there are also overlapping expression domains. For example cranial ganglia share prominent expression of Gadd45a and Gadd45g. All three Gadd45 family members are expressed in the somites and Gadd45b is indeed required for somitogenesis in zebrafish (Kawahara et al., 2005). Comparison of Gadd45 gene expression between mouse and other species reveals elements of evolutionary conservation and divergence. Gadd45b expression in somites is observed in mouse and zebrafish (Kawahara et al., 2005). Gadd45g is prominently expressed in neural tissue in frog, mouse, and medaka fish, most likely primary neuronal precursors or post-mitotic neurons, where the frog, chick and fish genes have been implicated in cell cycle arrest and neuronal differentiation (Candal et al., 2004; de
2. Experimental procedures 2.1. Animals
2.2. Quantitative real-time PCR (qPCR) Total RNA was extracted from one mouse embryo per stage and experiment using Trizol (Invitrogen) according to standard protocols. cDNA was synthesised with Superscript II reverse transcriptase and random primers (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time PCR was
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Table 1 Summary of Gadd45a, Gadd45b and Gadd45g tissue expression during mouse development. ‘‘+++’’ gene strongly expressed in the corresponding tissue; ‘‘++’’ moderately expressed; ‘‘ + ’’ weakly expressed. Gadd45a E6.5–E7.5 mesoderm extraembryonic E8.5 neural
Gadd45b
Gadd45g
Chorion +++
Ectoplacental cone ++
Primitive streak ++ Mesoderm + lip of neural fold +++ Brain + Neural precursors ++
mesoderm
Artery precursors + Forming somites +
extraembryonic E9.5 neural
Presomitic mesoderm ++ Allantois ++ Neural tube +++ Cranial ganglia VII–X ++ Dorsal root ganglia + Forebrain–midbrain boundary ++ Dorsal midbrain ++ Hindbrain +++ Presomitic mesoderm +++ Tips of branchial arches + Branchial arch arteries + Telencephalon + Midbrain +++ Hindbrain +++ Cranial ganglia VII–X ++ Dorsal root ganglia +++ Neural tube +++ Olfactory epithelium ++
Tip of closing neural tube (lamina terminalis, caudal neuropore) +++ Cranial ganglia VII–X ++ Dorsal root ganglia ++ Dorsal midbrain +
Mesoderm
Somites +
E10.5 neural
Most caudal somites + Branchial arch arteries + Dorsal aorta + Telencephalon +
Epithelium of telencephalon +
Cranial ganglia VII–X ++ Dorsal root ganglia +++ Olfactory epithelium ++ Caudal neuropore +++ Forelimb posterior-distal mesenchyme ++ Hindlimb posterior-distal mesenchyme ++ Caudal somites +
Mesoderm
carried out on a LightCycler480 using the Universal ProbeLibrary System (Roche Diagnostics) according to the manufacturer’s instructions. Primers were designed for each gene using the Universal ProbeLibrary Assay Design Center (https://www. roche-applied-science.com/) choosing intron spanning assays. The primers and probes used were: Primer Gadd45a-f Gadd45a-r Gadd45b-f Gadd45b-r Gadd45g-f Gadd45g-r GAPDH-f GAPDH-r
Sequence 0
Forelimb ZPA + and ANZ ++ Trunk somites + Presomitic mesoderm ++ Genital ridges +
Gadd45a-50 Gadd45a-30 Gadd45b-50 Gadd45b-30 Gadd45g-50 Gadd45g-30
50 -TGGTGACGAACCCACATTCA-30 50 -TTGAGGGCATAAAGACCAAA-30 50 -TTGCCTCTTGGGTTCGTATC-3 50 -CAAGCGATCTGTCTTGCTCA-30 50 -CCTCCGCACTCTTTTGGATA-30 50 -CAGTCGGCTAAGTCCAGCTC-30
UPL probe 0
5 -GCTGCCAAGCTGCTCAAC-3 50 -TCGTCGTCTTCGTCAGCA-30 50 -CTGCCTCCTGGTCACGAA-30 50 -TTGCCTCTGCTCTCTTCACA-30 50 -GGATAACTTGCTGTTCGTGGA-30 50 -AAGTTCGTGCAGTGCTTTCC-30 50 -AGCTTGTCATCAACGGGAAG-3’ 50 -TTTGATGTTAGTGGGGTCTCG-30
98 10 76 9
The data were analyzed using the LightCycler480 Software release 1.5.0 (Roche Diagnostics). For the temporal expression pattern of Gadd45a, Gadd45b and Gadd45g the mean concentration was calculated using the Analysis type ‘‘Abs Quant/2nd Derivative Max’’ and was normalised to the level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative Gadd45 transcript levels in E10.5 mouse embryos were calculated using the respective primer efficiencies (E) and the raw Cp values: E[Gadd45]^(-Cp[Gadd45])/E[GAPDH]^(-Cp[GAPDH]). 2.3. Whole mount in situ hybridization Gadd45 probe templates were generated by PCR amplification from a cDNA library of E10.5 wild type embryos using the following primers:
Amplicons were cloned into pGEM-T Easy vector (Promega) according to manufacturer’s instruction. Sense and antisense probes were synthesised using digoxygenin (DIG) RNA labelling mix and T7 or Sp6 RNA polymerase (Roche Diagnostics). Mouse embryos were processed for whole mount in situ hybridization essentially following the standard protocols (Wilkinson, 1992). Embryos at E9.5– E10.5 were treated for 20 min with 5 lg/ml proteinase K (Roche), while for E7.5 and E8.5 embryos the treatment was reduced to 8 min. The embryos were photographed using a Nikon SMZ1500 stereomicroscope and 3CCD Colour Video Camera (Sony DXC930P), digitalized using Scion Series 7 software (Scion Corp., Frederick, MD) and processed using Adobe Photoshop 7.0 (Adobe Systems Inc.). 2.4. Vibratome sections For histological examinations tissue sections were obtained from embryos after whole mount in situ hybridization. Embryos were fixed for at least 2 h in 4% PFA, washed three times 30 min in PBS and embedded in 30% gelatine in PBS. After over night fixation of the gelatine block in 4% PFA and at least three repeated 1 h washes in PBS the blocks were cut on a HM 650 V vibratome (Microm) to 34 nm thick sections. Sections were mounted on slides with Mowiol (Calbiochem) and photographed on a Leica DM LB2 microscope using Leica Application Suite software.
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Acknowledgements We thank Nicole Maltry for technical assistance. This work was funded by the DFG and the ERC.
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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development Lilian T. Kaufmann a, Mathias S. Gierl a,b, Christof Niehrs a,b,⇑ a b
Division of Molecular Embryology, DKFZ-ZMBH Alliance, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 581, D-69120 Heidelberg, Germany Institut für Molekulare Biologie, Mainz, Germany
a r t i c l e
i n f o
Article history: Received 12 June 2011 Received in revised form 28 July 2011 Accepted 29 July 2011 Available online 6 August 2011 Keywords: Gadd45 Expression pattern Mouse embryogenesis Limb buds Neurogenesis Somitogenesis
a b s t r a c t Gadd45 proteins have been implicated in the cellular response to physiological or environmental stress and the accompanying cell cycle arrest, DNA repair, cell survival and senescence or apoptosis. Although their molecular function is well studied, the expression and role of Gadd45 genes during embryonic development in mice is largely unknown. Here we provide a comprehensive comparison of Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development. In situ hybridizations on sectioned and whole mouse embryos show most prominent Gadd45a expression in the tip of the closing neural tube, the cranial and dorsal root ganglia and the somites. Mouse Gadd45b is expressed strongly in the chorion, but only weakly in the embryo proper, including somites and limb buds. Murine Gadd45g expression strongly resembles Xenopus and medaka fish expression in primary neuron precursors and post-mitotic neurons, indicating a conserved role for Gadd45g in vertebrate neurogenesis. Additionally, Gadd45 genes show conserved expression during somitogenesis. In summary, Gadd45 genes are expressed in evolutionary conserved, but also divergent domains, which predominantly encompass areas of cell differentiation, consistent with their established function in growth arrest and DNA demethylation. Ó 2011 Elsevier B.V. All rights reserved.
1. Results and discussion Gadd45 (Growth arrest and DNA damage response) encompasses a small family of stress response genes, encoding small acidic nuclear proteins of the L7Ae/L30e/S12e RNA binding protein superfamily (PFAM entry: PF01248) (Bateman et al., 2004). The three genes, Gadd45a, Gadd45b, Gadd45g, are involved in diverse processes, including cell cycle, apoptosis, DNA repair, and DNA demethylation (Barreto et al., 2007; Hollander and Fornace, 2002; Sheikh et al., 2000; Zhan, 2005). Gadd45a mediates these processes by interacting with many effectors, including cdc2/cyclinB1 (Vairapandi et al., 2002; Zhan et al., 1999), PCNA (Azam et al., 2001; Vairapandi et al., 2000), p21 (Kearsey et al., 1995), nuclear hormone receptors (Yi et al., 2000), histones (Carrier et al., 1999), MEKK4 (Chi et al., 2004; Takekawa and Saito, 1998), XPG (Barreto et al., 2007), AID (Rai et al., 2008) and TAF12 (Schmitz et al., 2009). They play a role in tumor- and autoimmune suppression and UV response (Bulavin et al., 2003; Hollander et al., 1999; Liu et al., 2005). While their function is well characterized in cultured cells and adult mice, much less is known about their role during embryonic
⇑ Corresponding author at: Division of Molecular Embryology, DKFZ-ZMBH Alliance, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 581, D-69120 Heidelberg, Germany. Tel.: +49 6221 42 4690; fax: +49 6221 42 4692. E-mail address: [email protected] (C. Niehrs). 1567-133X/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2011.07.005
development. The phenotypes of the single knock out mice are rather mild. Gadd45a-deficient mice display exencephaly at low penetrance (Hollander et al., 1999). Gadd45b1 and Gadd45b2 in zebrafish are periodically expressed in the anterior presomitic mesoderm and are required for somite segmentation (Kawahara et al., 2005). Gadd45g in medaka fish and Xenopus laevis are expressed in primary neuron precursors and promote differentiation by regulation of cell cycle exit (Candal et al., 2004; de la CalleMustienes et al., 2002). In mouse, the expression of Gadd45 genes during embryonic development has not been analyzed. Here we provide a comprehensive comparison of Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development.
1.1. qPCR expression analysis of Gadd45a, Gadd45b and Gadd45g during early mouse development The temporal expression patterns of the three Gadd45 genes during embryonic development, analyzed by qPCR, were dynamic and differential (Fig. 1). Mouse Gadd45a was expressed at low levels at gastrula stages (E6.5–E7.5) and expression strongly increased towards neurulation, but already reached a plateau at E8.5 (Fig. 1A). Gadd45b was highest expressed in gastrula stages (E6.5–E7.5) and transcripts rapidly decreased during neurulation (Fig. 1B). Gadd45g was not expressed maternally and increased continuously after gastrulation (Fig. 1C). Comparison of
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Fig. 1. qPCR expression analysis of Gadd45 genes in mouse embryos. (A–C) qPCR analysis of mouse Gadd45a (A), Gadd45b (B) and Gadd45g (C) normalised to GAPDH at the indicated embryonic stages. (D) Quantification of relative transcript levels of mouse Gadd45a, Gadd45b and Gadd45g in E10.5 embryos by qPCR. Data represent the mean ± SD of three independent experiments each using one mouse embryo per stage, respectively.
the relative transcript levels of Gadd45a, Gadd45b and Gadd45g at E10.5 by qPCR revealed that among the three Gadd45 genes, Gadd45b showed by far the lowest overall expression (Fig. 1D).
1.2. Prominent expression of mouse Gadd45a in ganglia In mouse embryos Gadd45a expression was first detected in the primitive streak and the embryonic mesoderm at E6.5–E7.5
Fig. 2. Expression of Gadd45a during mouse development. (A–I and L–P) Whole mount in situ hybridization of Gadd45a on E7.5–E10.5 mouse embryos. (K,Q-R) Vibratome sections of embryos shown in F and L as indicated. (J), control in situ hybridization with sense probe for Gadd45a of E9.5 embryo. Lateral views (A, C–F, J and L), dorsal views (B, G and N), ventral views (H, I, M, O–P). Key: (a) anterior, (d) dorsal, (da) dorsal aorta, (drg) dorsal root ganglia, (fb) forebrain, (fg) facio-acoustic ganglia, (fl) forelimb bud, (gg) glossopharyngeal ganglia, (hb) hindbrain, (hl) hindlimb bud, (lt) lamina terminalis, (mg) mid gut, (nf) neural fold lip, (np) caudal neuropore, (nt) neural tube, (oe) olfactory epithelium, (ot) otic vesicle, (p) posterior, (ps) primitve streak, (so) somitic mesoderm, (tg) trigeminal ganglia, (tv) telencephalic vesicle, (v) ventral, (vg) vagal ganglia.
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(Fig. 2A). This pattern was weak and replaced by a robust Gadd45a expression in the lips of the closing neural fold (Fig. 2B). The expression in the neuroepithelium strongly resembled the distribution of apoptotic cells (Massa et al., 2009). This might indicate a role of Gadd45a during neural tube closure since Gadd45a homozygous mutants show exencephaly (Hollander et al., 1999). At E9.0 Gadd45a continued to be expressed in the tip of the closing neural tube, except for the midbrain and hindbrain region (Fig. 2C and D). Additional expression started in scattered cells of the placodes of trigeminal- and the dorsal root ganglia (Fig. 2C– E) which became stronger at E9.5 (Fig. 2F–H). At E9.5 expression also appeared in the facio-acoustic, glossopharyngeal and the vagal ganglia and in the olfactory epithelium (Fig. 2F–I). The expression of Gadd45a in the closing neural tube persisted only in the caudal neuropore and the lamina terminalis (Fig. 2I), the regions to undergo neural tube closure last. Furthermore, there was weak expression in the myotomes as seen in vibratome sections (Fig. 2K). From E9.5 onwards a ubiquitous basal expression of Gadd45a was observed, compared to the sense control (Fig. 2J). At E10.5 additional expression occurred in the epithelium of the telencephalic ventricles (Fig. 2L, M and Section 2R). In the limb buds Gadd45a was expressed in the posterior-distal mesenchyme subjacent to the AER with a dorsal and a ventral expression domain separated by an area of lower expression in the centre (Fig. 2O–Q). 1.3. Low level expression of mouse Gadd45b in mesodermal derivatives The transcript of Gadd45b could not be detected in the epiblast of E6.5–E7.5 embryos but strongly in the chorion (Fig. 3A). In E8.5 embryos only weak and diffuse Gadd45b expression was detected
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in the future mid- and hindbrain, anterior heart field, first forming somites and in the posterior remnants of the primitive streak. At E9.0 expression in the dorsal midbrain and in the first epithelial somites became more pronounced (Fig. 3C and D). Somite expression progressed anteriorly such that at E10.5 it was expressed in all trunk somites (Fig. 3L and P). From E9.5 to E10.5 Gadd45b was additionally expressed in the branchial arch arteries and the dorsal aorta (Fig. 3E–J), in the tip of the first branchial arches and the forelimb bud (Fig. 3L, M and O). The strong staining in the forebrain vesicles was due to probe trapping and was unspecific. Within the forelimb bud Gadd45b was expressed in the anterior necrotic zone (ANZ) or ‘‘foyer préaxiale primaire’’ (Milaire, 1971, 1976) and the zone of polarizing activity, strongly resembling the Msx2 expression domain associated with cell death (Ganan et al., 1998; Krabbenhoft and Fallon, 1992). 1.4. Prominent expression of mouse Gadd45g in the nervous system Expression of Gadd45g in E7.5 embryos was detected in the ectoplacental cone and scattered cells of the extraembryonic ectoderm but hardly in the epiblast (Fig. 4A). At E8.5–8.75 extraembryonic expression continued in the allantois, and embryonic expression occured in the presomitic mesoderm and in few neural precursors within the neural fold (Fig. 4B and C). At subsequent stages neural expression dominated and manifested in the dorsal midbrain, the cranial and dorsal root ganglia and the neural tube (Fig. 4E–I). Vibratome sections of E9.5 embryos showed that within the brain Gadd45g was expressed at the forebrain-midbrain boundary, the trigeminal ganglia and the latero-ventral hindbrain
Fig. 3. Expression of Gadd45b during mouse development. (A–J and L–O) Whole mount in situ hybridization of Gadd45b on E7.5–E10.5 mouse embryos. (K) control in situ hybridization with sense probe for Gadd45b of E9.5 embryo. Lateral views (A–C, E, F, and I–L), dorsal views (G, N–O), ventral view (D and H), anterior view (M). The dashed boxes in F and N indicate the regions magnified in I, J and O. Key: (a) anterior, (al) allantois, (ANZ) anterior necrotic zone, (ba) first branchial arch, (br) brain, (ch) chorion, (cs) caudal somites, (da) dorsal aorta, (db) dorsal midbrain, (fl) forelimb bud, (fs) forming somites, (hl) hindlimb bud, (ht) heart field, (p) posterior, (ps) primitive streak, (so) somitic mesoderm, (ZPA) zone of polarizing activity.
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Fig. 4. Expression of Gadd45g during mouse development. (A–C and E–I) Whole mount in situ hybridization of Gadd45g on E7.5–E10.5 mouse embryos. (D) Control in situ hybridization with sense probe for Gadd45g of E9.5 embryo. Lateral views (A, C–E and H), dorsal views (B and F), ventral views (G and I). (J, K and M) Vibratome sections of embryos shown in E and H as indicated. (L) Section of embryo with similar staining pattern as shown in H. Key: (a) anterior, (al) residual allantois, (ba) first branchial arch, (db) dorsal midbrain, (drg) dorsal root ganglia, (ec) ectoplacental cone, (ee) extraembryonic ectoderm, (fb) forebrain, (fg) facio-acoustic ganglia, (fl) forelimb bud, (fs) forming somite, (hd) hindbrain, (hl) hindlimb bud, (ht) heart, (mb) midbrain, (nf) neural fold, (nt) neural tube, (oe) olfactory epithelium, (os) optic stalk, (ot) otic vesicle, (p) posterior, (so) somitic mesoderm/myotome, (tg) trigeminal ganglia.
(Fig. 4J). In the neural tube Gadd45g was predominantly expressed in ventral and dorsal peripheral cells (Fig. 4K). This pattern resembled the expression of the Notch effector Hes6 (Vasiliauskas and Stern, 2000). Prominent expression occurred also in the facioacoustic ganglia and the ventral part of the otic cup (Fig. 4K). In E10.5 embryos Gadd45g continued to be expressed in distinct domains within the neural tube, the dorsal root ganglia and the myotome (Fig. 4I, L–M). A summary of the expression domains of Gadd45a, Gadd45b and Gadd45g in mouse embryos can be found in Table 1.
la Calle-Mustienes et al., 2002; Sheng et al., 2010). A common element of Gadd45 genes is their expression in cells undergoing differentiation, such as forming somites and neuronal precursors consistent with their functional implication in cell cycle arrest and DNA demethylation (Barreto et al., 2007; Hollander and Fornace, 2002).
1.5. Conclusion
Female CD-1 outbred mice (Mus musculus) were produced and raised in the research colony of the investigators. Wild type mice were mated overnight and noon of the day of vaginal plug detection was defined as embryonic (E) 0.5 days post coitum. Embryos were dissected in ice cold PBS according to standard protocols (Hogan et al., 1994). Mice were kept according to international standard conditions, and all animal experiments complied with local and international guidelines for the use of experimental animals.
The Gadd45 genes are differentially expressed during embryonic development of mouse. Certain expression domains are exclusive for a given family member, like expression of Gadd45a in closing neural tube, Gadd45b in the chorion, and Gadd45g in the mouse brain, but there are also overlapping expression domains. For example cranial ganglia share prominent expression of Gadd45a and Gadd45g. All three Gadd45 family members are expressed in the somites and Gadd45b is indeed required for somitogenesis in zebrafish (Kawahara et al., 2005). Comparison of Gadd45 gene expression between mouse and other species reveals elements of evolutionary conservation and divergence. Gadd45b expression in somites is observed in mouse and zebrafish (Kawahara et al., 2005). Gadd45g is prominently expressed in neural tissue in frog, mouse, and medaka fish, most likely primary neuronal precursors or post-mitotic neurons, where the frog, chick and fish genes have been implicated in cell cycle arrest and neuronal differentiation (Candal et al., 2004; de
2. Experimental procedures 2.1. Animals
2.2. Quantitative real-time PCR (qPCR) Total RNA was extracted from one mouse embryo per stage and experiment using Trizol (Invitrogen) according to standard protocols. cDNA was synthesised with Superscript II reverse transcriptase and random primers (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time PCR was
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Table 1 Summary of Gadd45a, Gadd45b and Gadd45g tissue expression during mouse development. ‘‘+++’’ gene strongly expressed in the corresponding tissue; ‘‘++’’ moderately expressed; ‘‘ + ’’ weakly expressed. Gadd45a E6.5–E7.5 mesoderm extraembryonic E8.5 neural
Gadd45b
Gadd45g
Chorion +++
Ectoplacental cone ++
Primitive streak ++ Mesoderm + lip of neural fold +++ Brain + Neural precursors ++
mesoderm
Artery precursors + Forming somites +
extraembryonic E9.5 neural
Presomitic mesoderm ++ Allantois ++ Neural tube +++ Cranial ganglia VII–X ++ Dorsal root ganglia + Forebrain–midbrain boundary ++ Dorsal midbrain ++ Hindbrain +++ Presomitic mesoderm +++ Tips of branchial arches + Branchial arch arteries + Telencephalon + Midbrain +++ Hindbrain +++ Cranial ganglia VII–X ++ Dorsal root ganglia +++ Neural tube +++ Olfactory epithelium ++
Tip of closing neural tube (lamina terminalis, caudal neuropore) +++ Cranial ganglia VII–X ++ Dorsal root ganglia ++ Dorsal midbrain +
Mesoderm
Somites +
E10.5 neural
Most caudal somites + Branchial arch arteries + Dorsal aorta + Telencephalon +
Epithelium of telencephalon +
Cranial ganglia VII–X ++ Dorsal root ganglia +++ Olfactory epithelium ++ Caudal neuropore +++ Forelimb posterior-distal mesenchyme ++ Hindlimb posterior-distal mesenchyme ++ Caudal somites +
Mesoderm
carried out on a LightCycler480 using the Universal ProbeLibrary System (Roche Diagnostics) according to the manufacturer’s instructions. Primers were designed for each gene using the Universal ProbeLibrary Assay Design Center (https://www. roche-applied-science.com/) choosing intron spanning assays. The primers and probes used were: Primer Gadd45a-f Gadd45a-r Gadd45b-f Gadd45b-r Gadd45g-f Gadd45g-r GAPDH-f GAPDH-r
Sequence 0
Forelimb ZPA + and ANZ ++ Trunk somites + Presomitic mesoderm ++ Genital ridges +
Gadd45a-50 Gadd45a-30 Gadd45b-50 Gadd45b-30 Gadd45g-50 Gadd45g-30
50 -TGGTGACGAACCCACATTCA-30 50 -TTGAGGGCATAAAGACCAAA-30 50 -TTGCCTCTTGGGTTCGTATC-3 50 -CAAGCGATCTGTCTTGCTCA-30 50 -CCTCCGCACTCTTTTGGATA-30 50 -CAGTCGGCTAAGTCCAGCTC-30
UPL probe 0
5 -GCTGCCAAGCTGCTCAAC-3 50 -TCGTCGTCTTCGTCAGCA-30 50 -CTGCCTCCTGGTCACGAA-30 50 -TTGCCTCTGCTCTCTTCACA-30 50 -GGATAACTTGCTGTTCGTGGA-30 50 -AAGTTCGTGCAGTGCTTTCC-30 50 -AGCTTGTCATCAACGGGAAG-3’ 50 -TTTGATGTTAGTGGGGTCTCG-30
98 10 76 9
The data were analyzed using the LightCycler480 Software release 1.5.0 (Roche Diagnostics). For the temporal expression pattern of Gadd45a, Gadd45b and Gadd45g the mean concentration was calculated using the Analysis type ‘‘Abs Quant/2nd Derivative Max’’ and was normalised to the level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative Gadd45 transcript levels in E10.5 mouse embryos were calculated using the respective primer efficiencies (E) and the raw Cp values: E[Gadd45]^(-Cp[Gadd45])/E[GAPDH]^(-Cp[GAPDH]). 2.3. Whole mount in situ hybridization Gadd45 probe templates were generated by PCR amplification from a cDNA library of E10.5 wild type embryos using the following primers:
Amplicons were cloned into pGEM-T Easy vector (Promega) according to manufacturer’s instruction. Sense and antisense probes were synthesised using digoxygenin (DIG) RNA labelling mix and T7 or Sp6 RNA polymerase (Roche Diagnostics). Mouse embryos were processed for whole mount in situ hybridization essentially following the standard protocols (Wilkinson, 1992). Embryos at E9.5– E10.5 were treated for 20 min with 5 lg/ml proteinase K (Roche), while for E7.5 and E8.5 embryos the treatment was reduced to 8 min. The embryos were photographed using a Nikon SMZ1500 stereomicroscope and 3CCD Colour Video Camera (Sony DXC930P), digitalized using Scion Series 7 software (Scion Corp., Frederick, MD) and processed using Adobe Photoshop 7.0 (Adobe Systems Inc.). 2.4. Vibratome sections For histological examinations tissue sections were obtained from embryos after whole mount in situ hybridization. Embryos were fixed for at least 2 h in 4% PFA, washed three times 30 min in PBS and embedded in 30% gelatine in PBS. After over night fixation of the gelatine block in 4% PFA and at least three repeated 1 h washes in PBS the blocks were cut on a HM 650 V vibratome (Microm) to 34 nm thick sections. Sections were mounted on slides with Mowiol (Calbiochem) and photographed on a Leica DM LB2 microscope using Leica Application Suite software.
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Acknowledgements We thank Nicole Maltry for technical assistance. This work was funded by the DFG and the ERC.
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