Gene Expression Patterns 13 (2013) 319–327

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Gene Expression Patterns

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Expression of NOL1/NOP2/sun domain (Nsun) RNA methyltransferasefamily genes in early mouse embryogenesis

1567-133X/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.gep.2013.06.003

⇑ Corresponding author at: Physiology and Experimental Medicine, The Hospitalfor Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada. Tel.: +1416 813 5080; fax: +1 416 813 7480.

E-mail addresses: lijun.chi@sickkids.ca (L. Chi), paul.delgadoolguin@sickkids.ca(P. Delgado-Olguín).

Lijun Chi a, Paul Delgado-Olguín a,b,c,⇑a Program in Physiology and Experimental Medicine, The Hospital for Sick Children, Toronto, ON, Canadab Department of Molecular Genetics, University of Toronto, Toronto, ON, Canadac Heart & Stroke Richard Lewar Centre of Excellence, Toronto, ON, Canada

a r t i c l e i n f o

Article history:Received 24 April 2013Received in revised form 18 June 2013Accepted 20 June 2013Available online 29 June 2013

Keywords:NOL1/NOP2/sun domain (Nsun) genesGene expression screenRNA methyltransferaseMouse embryonic development

a b s t r a c t

The NOL1/NOP2/sun domain-containing genes encode the RNA methyltransferases Nsun2, 3, 4, 5, 6 and 7.Methylated RNA pervades the transcriptome, yet the function of RNA methyltransferases is poorly under-stood. Nsun2 and Nsun4 participate in cell proliferation and differentiation, protein biosynthesis and can-cer. In addition, Nsun2 and Nsun7 dysfunction might cause intellectual disability and male sterility,respectively. The functions of Nsun3, Nsun5 and Nsun6 are unknown. Given the widespread distributionof RNA methylation, it is possible that Nsun genes participate in a broader range of relevant biologicalprocesses including the regulation of embryogenesis. Here, we describe the expression pattern of Nsungenes during mouse embryo development. In situ hybridization showed developmentally regulated Nsungene expression. Nsun genes express broadly during gastrulation, but enrich in specific tissues as embryo-genesis proceeds. Nsun transcripts enrich in the developing brain, consistent with proposed functions inneurocognitive development. In addition, Nsun transcripts enrich in the developing ear, eye, olfactory epi-thelium, branchial arches, heart and limb, suggesting possible overlapping functions of NSUN proteins inneural, craniofacial, cardiac, and limb morphogenesis. Furthermore, Nsun2 and Nsun6 enrich in the caudalneural tube and newly formed somites, suggesting possible functions in body axis extension. Theseresults suggest possible overlapping functions of NSUN proteins and RNA methylation in broad aspectsof embryonic development.

� 2013 Elsevier B.V. All rights reserved.

Methylated RNA is extensively distributed throughout codingand noncoding sequences (Dominissini et al., 2012; Meyer et al.,2012; Squires et al., 2012) but the effects of this modification, orthe functions of RNA methyltransferases in cell differentiationand development are poorly understood. This scenario is likely tochange soon, as recent evidence points to an involvement of RNAmethyltransferases in development (Blanco et al., 2011; Tuortoet al., 2012) and disease (Abbasi-Moheb et al., 2012; Khan et al.,2012; Martinez et al., 2012). In addition, advances in our abilityto study RNA modifications globally (Khoddami, 2013), will allowfor identification of the global targets of RNA methyltransferasesin diverse cell types and tissues during development, and in path-ologic processes.

The NOL1/NOP2/sun domain (Nsun) family, which is conservedfrom archaea to eukaryotes, consists of 6 genes: Nsun2 (Misu),Nsun3, Nsun4, Nsun5 (Wbscr20, Wbscr20a), Nsun6 (NOPD1) and

Nsun7. Nsun proteins possess a S-adenosyl methionine binding-do-main and are related to Escherichia coli Sun/Fmu protein, whichmethylates C967 in 16S rRNA (Foster et al., 2003).

Nsun2, encoded by the most studied Nsun family member, is anucleolar protein (Frye and Watt, 2006; Sakita-Suto et al., 2007)that methylates and stabilizes tRNA, and thus participates in pro-tein synthesis (Auxilien et al., 2012; Tuorto et al., 2012). Also in-volved in this process is Nsun4, which is recruited by MTERF4,an essential regulator of translation in mammalian mitochondria,to the mitochondrial ribosome (Cámara et al., 2011; Spåhr et al.,2012; Yakubovskaya et al., 2012). Nsun2 is also involved in cell pro-liferation growth and cancer. It is required for proper spindleassembly, chromosome segregation (Hussain et al., 2009), and forMyc-induced Keratinocyte proliferation and differentiation (Fryeand Watt, 2006). In addition, Nsun2 reduces mRNA decay of thecell cycle regulator p16 by methylating its 30UTR (Zhang et al.,2012), it is up regulated in several tumors, and might be a potentialtarget for cancer treatment (Frye and Watt, 2006). Nsun2 is alsoimportant in some aspects of development. It regulates epidermalstem cell differentiation, and its deficiency in mouse causes weightloss, partial alopecia, partial male sterility (Blanco et al., 2011) anddefective germ cell differentiation (Hussain et al., 2013). Nsun7

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might also function in germ cells, a chemically induced mutation inthe mouse gene is associated with male sterility or subfertility(Harris et al., 2007). Furthermore, NSUN2 has been implicated inneurocognitive conditions. Mutations causing protein miss locali-zation or loss of NSUN2 mRNA are associated with autosomal-recessive intellectual disability (Abbasi-Moheb et al., 2012; Khanet al., 2012), and loss of the ortholog causes short-term memorydeficit in Drosophila (Abbasi-Moheb et al., 2012). Finally, homozy-gous splice mutations causing loss of NSUN2 and tRNA methyla-tion are present in patients with a Dubowitz-like syndrome,which also has a neurocognitive component (Martinez et al.,2012). Although the expression patterns or functions of Nsun3,Nsun5 and Nsun6, have not been explored yet, the widespread ofRNA methylation and the evidence available suggest a possibleinvolvement of NSUN proteins in a broad range of biological

Fig. 1. Nsun2 expression during early embryogenesis. (A and B, E–K, and M and N) Whembryos. (B) Close up to skin. (C and D) Control in situ hybridization with sense probe forto the same as in A. (A, C–E, H, L and M) Lateral view. (F and J) Frontal view. (K) Dorsal viewan = anterior, ane = anterior neuroectoderm, ch = chorion, ee = extra embryonic ectodermda = dorsal aorta, fb = forebrain, fg = facio-acoustic ganglion, fl = forelimb, fnp = frontohb = hindbrain, hl = hindlimb, hp = hepatic primordium, ht = heart, ir = isthmic regionnem = neuro endothelium of the midbrain, nt = neural tube, op = optical plate, ot = oticaland wh = whisker. Reference bars in A–K and M and N = 250 lm, L and O = 100 lm.

processes including cell differentiation, embryonic developmentand disease. As a first approach to uncover the potential functionsof Nsun genes in the regulation of development we analyzedtheir expression pattern in mice at several time points duringembryogenesis.

1. Results and discussion

1.1. Nsun2 expression during early embryogenesis

To validate our probe for Nsun2, we hybridized it with E13.5 andE14 embryos. In agreement with a previous report of a LacZ repor-ter driven by the mouse Nsun2 locus (Blanco et al., 2011), we foundNsun2 in the developing whiskers, and hair follicles at these stages

ole mount in situ hybridization of Nsun2 in E13.5, E14.5, E7.5, E8.5, E9.5 and E10.5Nsun2 in 13.5 and E9.5 embryos. The embryonic region presented in C corresponds. (L and O) In situ hybridization on sections of E9.5 and E10.5 embryos. al = allantois,, ba1 = first branchial arch, ba2 = second branchial arch, cv = cerebral vasculature,

nasal prominence, fs = recently formed somite, hf = hair follicle, (hf) head folds,, mb = midbrain, neh = neuro endothelium of the hindbrain, nl = neural lumen,plate, oe = olfactory epithelium, ps = posterior, so = somite, tg = trigeminal ganglion

Table 2Summary of embryonic structures with enriched Nsun genes expression.

Embryonic day Embryonic structure Enriched gene transcript

Nsun2 Nsun3 Nsun4 Nsun5 Nsun6 Nsun7

E7.5 Allantois (al) + + + + +Anterior neuroectoderm (ane) + + + + + +Chorion (ch) + + + + + +Extra embryonic ectoderm (ee) + + + + +

E8.5 Forming somites (fs) + +Head folds (hf) + + + + + +Heart (ht) + + + + + +Neural tube (nt) + + + +Sinus venosus (sv) + + + + +Somites (so) + +Allantois (al) + + + + +

E9.5 Dorsal aorta (da) + + +Facio-acoustic ganglion (fg) + + + +First branchial arch (ba1) + + + + + +Forebrain (fb) + + + +Forelimb (fl) + + + + + +Frontonasal prominence (fnp) + + + + + +Heart (ht) + + +Hindbrain (hb) + + +Midbrain (mb) + + +Neural tube (nt) + + +Optical plate (op) + + + + + +Otical plate (ot) + + + + + +Second branchial arch (ba2) + +Somites (so) + + + +Trigeminal ganglion (tg) + + + +

E10.5 Cerebral vasculature (cv) + +Dorsal aorta (da) + + + +Facio-acoustic ganglion (fg) + + + + + +First branchial arch (ba1) + + + + + +Forebrain (fb) + + + + + +Forelimb (fl) + + + + + +Heart (ht)Hepatic primordium (hp) + + + +Hindbrain (hb) + + + +Hindlimb (hl) + + + + + +Intersomitic vessels (isv) +Isthmic region (ir) + + + +Midbrain (mb) + + +Neural tube (nt) + + + + + +Neuro filaments (nf) + +Olfactory epithelium (oe) + + + + + +Optical plate (op) + + + + + +Otical plate (ot) + + + + + +Second branchial arch (ba2) + + + + + +Somites (so) + +Third branchial arch (ba3) +Trigeminal ganglion (tg) + + + + + +

Table 1Primers used to generate templates for probe labeling.

Target Primer Sequence

Nsun2 Forward 50-ATTTAGGTGACACTATAGAAGNGgaaggtggctatcccgagatcgtaaa-30

Reverse 50-GAAATTAATACGACTCACTATAGGtctaggtatgctggatgcgtcatggt-30

Nsun3 Forward 5’-ATTTAGGTGACACTATAGAAGNGatgctgactcggctgaaagcaaaa-3’Reverse 5’-GAAATTAATACGACTCACTATAGGtcccttactgtactccaggaatcccc-3’

Nsun4 Forward 5’-ATTTAGGTGACACTATAGAAGNGtaatctagctgccaacgacctctcca-3’Reverse 5’-GAAATTAATACGACTCACTATAGGctatggcagcctatgcagtttgca-3’

Nsun5 Forward 5’-ATTTAGGTGACACTATAGAAGNGctccctcaaggggttggtgtattccagca-3’Reverse 5’-GAAATTAATACGACTCACTATAGGcttggcaaggtgcggtctcagcttcttct-3’

Nsun6 Forward 5’-ATTTAGGTGACACTATAGAAGNGttaggctcagttcagcatgtcagaggtctg-3’Reverse 5’-GAAATTAATACGACTCACTATAGGcttggtccagtcatggggatgagcaa-3’

Nsun7 Forward 5’-ATTTAGGTGACACTATAGAAGNGtctttccagcgcttgtcctatgagttgg-3’Reverse 5’-GAAATTAATACGACTCACTATAGGagaggcaaagttgaggccctttgttcct-3’

Sequences for the Sp6 and T7 promoters in forward and reverse primers, respectively, are indicated in uppercase.

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(Fig. 1A and B). This result, together with absent signal from thesense probe (Fig. 1C and D), supports specificity of the antisenseprobe.

At E7.5, Nsun2 was detected constitutively, but enriched extraembryonically in the allantois (al), chorion (ch), and extra embry-onic ectoderm (ee). We also found enriched Nsun2 in anteriorneuroectoderm (ane) at this stage (Fig. 1E), which agrees with laterexpression in the head folds (hf) at E8.5 (Fig. 1F). At this stage,Nsun2 was also detected in the developing heart (ht) and at lowerlevels in the somites (so), but appeared slightly enriched in thecaudal neural tube (nt) and most recently formed somites (fs).Nsun2 was also observed in the allantois (al) (Fig. 1F and G). AtE9.5, Nsun2 was enriched in the frontonasal prominence (fnp),maxillary and mandibular components of the first branchial arch(ba1), second branchial arch (ba2) artery, dorsal aorta (da), trigem-

Fig. 2. Nsun3 expression during early embryogenesis. (A–G, and I–K) Whole mount in siview. (B and F) Frontal view. (G) Dorsal view. (H and L) In situ hybridization on sections frNsun3 in E9.5 embryo. al = allantois, ane = anterior neuroectoderm, ch = chorion, ee = eda = dorsal aorta, fb = forebrain, fg = facio-acoustic ganglion, fl = forelimb, fnp = frontprimordium, ht = heart, isv = inter somitic vessels, ir = isthmic region, mb = midbrain, nepithelium, so = somite, sv = sinus venosus, and tg = trigeminal ganglion. Reference bars

inal (tg) and facio-acoustic (fg) ganglia, optical (op) and otical (ot)plates, somites (so), forelimb (fl), and in the neuro endothelium ofthe midbrain (nem) (Fig. 1H–L). Nsun2 was also detected in endo-cardium and myocardium of the heart (ht), and in neural tube (nt)at this stage. Consistent with earlier expression in the head folds,Nsun2 was detected in forebrain (fb), midbrain (mb) and hindbrain(hb) (Fig. 1H-L). At E10.5 Nsun2 was enriched in the olfactory epi-thelium (oe) and continued to be expressed in the forebrain (fb),midbrain (mb), hindbrain (hb), first (ba1) and second branchial ar-ches (ba2), trigeminal (tg) and facio-acoustic (fg) ganglia, heart(ht), hepatic primordium (hp), optical and otical plates. Nsun2was also found in cerebral vasculature (cv), the isthmic region(ir), dorsal aorta (da) and neural tube (nt) (Fig. 1M–O), but was lessabundant in somites (so). In addition, Nsun2 was enriched in fore-limb (fl) and hindlimb (hl) (Fig. 1M). Section in situ hybridization

tu hybridization of Nsun3 in E7.5, E8.5, E9.5 and E10.5 embryos. (A, D and I) Lateralom embryos at E9.5 and E10.5. (M) Control in situ hybridization with sense probe forxtra embryonic ectoderm, ba1 = first branchial arch, ba2 = second branchial arch,onasal prominence, (hf) head folds, hb = hindbrain, hl = hindlimb, hp = hepatic

l = neural lumen, nt = neural tube, op = optical plate, ot = otical plate, oe = olfactoryin A–G, and I–K and M = 250 lM, H and L = 100 lm.

Fig. 3. Nsun4 expression during early embryogenesis. (A–G, and I and J) Whole mount in situ hybridization of Nsun4 in E7.5, E8.5, E9.5 and E10.5 embryos. (A, D and I) Lateralview. (B and F) Frontal view. (G) Dorsal view. (H and K) In situ hybridization on sections from embryos at E9.5 and E10.5. (L) Control in situ hybridization with sense probe forNsun4 in E9.5 embryo. al = allantois, ane = anterior neuroectoderm, ch = chorion, ee = extra embryonic ectoderm, ba1 = first branchial arch, ba2 = second branchial arch,da = dorsal aorta, fb = forebrain, fg = facio-acoustic ganglion, fl = forelimb, fnp = frontonasal prominence, (hf) head folds, hb = hindbrain, hl = hindlimb, hp = hepaticprimordium, ht = heart, ir = isthmic region, mb = midbrain, nl = neural lumen, nf = neuro filament, nt = neural tube, op = optical plate, ot = otical plate, oe = olfactoryepithelium, so = somite, sv = sinus venous and tg = trigeminal ganglion. Reference bars in A–G, I, J and L = 250 lm, H and K = 100 lm.

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revealed enrichment in the neuro endothelium of the midbrain(nem) and of the hindbrain (neh) (Fig. 1O). The structures with en-riched Nsun2 gene expression are summarized in Table 2. These re-sults suggest that Nsun2 expression is developmentally regulated.It is broadly expressed during gastrulation at E7.5, and enrichesin specific tissues as development proceeds.

Expression of Nsun2 in the hindbrain agrees with previously re-ported localization in the nucleolus of Purkinje cells in the cerebel-lum (Khan et al., 2012). In addition enrichment in the developingbrain supports proposed altered function in intellectual disability(Abbasi-Moheb et al., 2012; Khan et al., 2012; Martinez et al.,2012). Furthermore, enriched expression in forebrain is consistentwith abnormal telencephalon morphology in double mutant micefor Nsun2 and the DNA methyltransferase Dnmt2. Thesemice,which loose tRNA methylation (5mc) and have deficient proteinsynthesis, are underdeveloped, present incomplete skeleton

ossification and their embryonic fibroblasts and liver cells prolifer-ate and differentiate deficiently (Tuorto et al., 2012), implicatingNsun2 in broad aspects of development.

1.2. Nsun3 expression during early embryogenesis

At E7.5 Nsun3 was broadly expressed, but enriched extraembryonically in the allantois (al), chorion (ch) and ectoderm(ee). Nsun2 was also enriched in the anterior neuro ectoderm(ane) (Fig 2A). At E8.5, moderate constitutive levels of Nsun2 weredetected. It was detected in somites (so); however, it was enrichedin the head folds (hf), heart (ht) and sinus venosus (sv) (Fig. 2B andC). At E9.5, Nsun3 continued to express constitutively, and was de-tected at low levels in the midbrain (mb), hindbrain (hb), and heart(ht), but was enriched in the frontonasal prominence (fnp), fore-brain (fb), first (ba1) and second (ba2) branchial arches, trigeminal

Fig. 4. Nsun5 expression during early embryogenesis. (A–G, and I and J) Whole mount in situ hybridization of Nsun5 in E7.5, E8.5, E9.5 and E10.5 embryos. (A, D and I) Lateralview. (B and F) Frontal view. (G) Dorsal view. (H and K) In situ hybridization on sections from embryos at E9.5 and E10.5. (L) Control in situ hybridization with sense probe forNsun5 in E9.5 embryo. al = allantois, ane = anterior neuroectoderm, ch = chorion, ee = extra embryonic ectoderm, ba1 = first branchial arch, ba2 = second branchial arch,da = dorsal aorta, fb = forebrain, fg = facio-acoustic ganglion, fl = forelimb, fnp = frontonasal prominence, (hf) head folds, hb = hindbrain, hl = hindlimb, hp = hepaticprimordium, ht = heart, ir = isthmic region, mb = midbrain, nl = neural lumen, nt = neural tube, op = optical plate, ot = otical plate, oe = olfactory epithelium, so = somite,sv = sinus venous and tg = trigeminal ganglion. Reference bars in A–G, I, J and L = 250 lm, H and K = 100 lm.

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(tg) and facio-acoustic (fg) ganglia, dorsal aorta (da), optical (op)and otical (ot) plates, somites (so) and forelimb (fl) (Fig. 2D–H).At E10.5, Nsun3 was detected at low levels in, midbrain (mb), hind-brain (hb), heart (ht), and somites (so), but it was enriched in theolfactory epithelium (oe), forebrain (fb), neural tube (nt) midline,trigeminal (tg) and facio-acoustic (fg) ganglia, the isthmic region(ir), first (ba1), second (ba2) and third (ba3) branchial arches,dorsal aorta (da), hepatic primordium (hp), inter somitic vessels(isv), and in forelimb (fl) and hindlimb (hl) (Fig. 2I–L). The struc-tures with enriched Nsun3 gene expression are summarized inTable 2. Hybridization with a sense probe for Nsun3 with E9.5embryos produced very low background signal (Fig. 2M), support-ing specificity of the antisense probe.

The functions of Nsun3 have not been explored yet. Our resultsraise a possible involvement of Nsun3 in multiple aspects ofembryogenesis.

1.3. Nsun4 expression during early embryogenesis

At E7.5, Nsun4 was enriched extra embryonically in the chorion(ch). It was also enriched in anterior neuro ectoderm (ane), but itwas not detected in anterior visceral endoderm (ave) (Fig. 3A). AtE 8.5, Nsun4 was enriched in head folds (hf), heart (ht), sinus veno-sus (sv), the inner portion of the neural tube (nt), and in allantois(al), but was detected at lower levels in somites (so) (Fig. 3B andC). At E9.5, Nsun4 continued to present homogenous constitutiveexpression, with slight enrichment in the maxillary componentof the first branchial arch (ba1), frontonasal prominence (fnp),forebrain (fb), midbrain (mb), hindbrain (hb), facio-acoustic gan-glion (fg), optic plate (op), otic vesicle (ot) and forelimb (fl)(Fig. 3D–H). At E10.5, constitutive low levels of Nsun4 were still de-tected, however, Nsun4 was enriched in the olfactory epithelium(oe), forebrain (fb), the isthmic region (ir), hindbrain (hb), optical

plate (op), neural tube (nt) midline, first (ba1) and second (ba2)branchial arches, trigeminal (tg) and facio-acoustic ganglia (fg),neuro filaments (nf), hepatic primordium (hp), and in forelimb(fl) and hindlimb (hl) (Fig. 3I–K). The structures with enrichedNsun4 gene expression are summarized in Table 2. Hybridizationwith a sense probe for Nsun4 produced low background signal(Fig. 3L), supporting specificity of the anti sense probe.

NSUN4 and its partner MTERF4 regulate rRNA modification andmitochondrial ribosome biogenesis(Cámara et al., 2011; Spåhret al., 2012; Yakubovskaya et al., 2012), suggesting that NSUN4and MTERF4 function in broad biological processes, however, ourresults suggest that Nsun4 might also have tissue-specific functionsduring embryogenesis.

1.4. Nsun5 expression during early embryogenesis

At E7.5, Nsun5 was broadly expressed in both embryonic andextra embryonic tissues (Fig. 4A). At E8.5 Nsun5 continued to bebroadly expressed, but was enriched in the head folds (hf), heart(ht), sinus venous (sv) and allantois (Fig. 4B and C). At E9.5, Nsun5was expressed constitutively at moderate levels, but was enrichedin the frontonasal prominence (fnp), optical plate (op), neural tube(nt) midline, maxillary and mandibular components of the firstbranchial arch (ba1), trigeminal (tg) and facio-acoustic (ag) ganglia,otic vesicle (ot), dorsal aorta (da) and forelimb (fl) (Fig. 3D–H). AtE10.5 Nsun5 was enriched in the olfactory epithelium (oe), fore-brain (fb), neural tube (nt) midline, isthmic region (ir), neurofilaments (nf), otic vesicle (ot), trigeminal (tg) and facio-acoustic(fg) ganglia, first (ba1) and second (ba2) branchial arches, hepaticprimordium (hp), forelimb (fl), and hindlimb (hl) (Fig. 4I–K). Thestructures with enriched Nsun5 gene expression are summarizedin Table 2. Hybridization of a sense probe for Nsun5 with E9.5

Fig. 5. Nsun6 expression during early embryogenesis. (A–G, and I and J) Whole mount in situ hybridization of Nsun6 in E7.5, E8.5, E9.5 and E10.5 embryos. (A, D and I) Lateralview. (B and F) Frontal view. (G) Dorsal view. (H and K) In situ hybridization on sections from embryos at E9.5 and E10.5. (L) Control in situ hybridization with sense probe forNsun5 in E9.5 embryo. al = allantois, ane = anterior neuroectoderm, ce = cerebellum, ch = chorion, ee = extra embryonic ectoderm, ba1 = first branchial arch, ba2 = secondbranchial arch, da = dorsal aorta, fb = forebrain, fg = facio-acoustic ganglion, fl = forelimb, fnp = frontonasal prominence, fs = recently formed somite, (hf) head folds,hb = hindbrain, hl = hindlimb, ht = heart, mb = midbrain, nl = neural lumen, nt = neural tube, op = optical plate, ot = otical plate, oe = olfactory epithelium, so = somite,sv = sinus venous and tg = trigeminal ganglion. Reference bars in A–G, I, J and L = 250 lm, H and K = 100 lm.

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embryos produced insignificant background (Fig. 4L), supportingspecificity of our antisense probe.

The function of Nsun5 is not known but might be related to dis-ease, as it is located in a genomic region consisting of 18 genes de-leted in patients with Williams–Beuren syndrome (Doll andGrzeschik, 2001), which presents with signs including mental dis-ability, developmental delay, and craniofacial and cardiovascularabnormalities (Martens et al., 2008). The contribution of the de-leted genes to the syndrome is not well established. Enrichedexpression of Nsun5 in the central nervous system, frontonasalprominence and branchial arches feeds speculation on a potentialfunction of Nsun5 in craniofacial development, which is compro-mised in Williams–Beuren syndrome.

1.5. Nsun6 expression during early embryogenesis

At E7.5, Nsun6 was broadly expressed in both embryonic andextra embryonic tissues, but slightly enriched in anterior neuroectoderm (ane) (Fig. 5A). At E8.5, Nsun6 was enriched in the headfolds (hf), heart (ht), sinus venosus (sv), the inner portion of theneural tube (nt), and in allantois (al). In addition, similar to Nsun2,Nsun6 appeared enriched in the caudal neural tube (nt) and mostrecently formed somites (fs) (Fig. 5B and C). At E9.5, Nsun6 wasconstitutively expressed at moderate levels, but was enriched inthe frontonasal prominence (fnp), trigeminal ganglion (tg), maxil-lary and mandibular components of the first branchial arch (ba1),optic (op) and otic (ot) plates, somites (so) and forelimb (fl). Nsun6

was less enriched in the heart (ht) (Fig. 5D–H). At E10.5, Nsun6 con-tinued to be expressed ubiquitously at moderate levels, but wasenriched in the olfactory epithelium (oe), forebrain (fb), midbrain(md), hindbrain (hb), neural tube (nt), optical (op) and otical (ot)plates, trigeminal (tg) and facio-acoustic (fg) ganglia, first (ba1)and second (ba2) branchial arches, dorsal aorta (da), forelimband hindlimb (Fig. 5I–K). The structures with enriched Nsun6 geneexpressionare summarized in Table 2. Hybridization with a senseprobe for Nsun6 produced only low background signal (Fig. 5L),supporting specificity of our antisense probe.

The functions of Nsun6 have not been explored yet. Our resultssuggest a possible involvement of Nsun6 in multiple aspects ofembryogenesis. Similar to Nsun2, Nsun6 was detected in the caudalneural tube and most recently formed somites. This feeds specula-tion on a potential involvement of these genes in extension of thebody axis. It would be of interest to determine if Nsun2 and Nsun6respond to, or participate in the molecular regulation of the seg-mentation clock, which controls the periodicity of somite forma-tion (Ozbudak and Pourquie, 2008).

1.6. Nsun7 expression during early embryogenesis

At E7.5, Nsun7 was broadly expressed in embryonic and extraembryonic tissues, but it was slightly enriched in the anterior neu-roectoderm (ane) (Fig. 6A). At E8.5, Nsun7 was expressed ubiqui-tously, but enriched in the head folds (hf), heart (ht), sinusvenosus (sv), the inner portion of the neural tube (nt), and allantois

Fig. 6. Nsun7 expression during early embryogenesis. (A–G, and I and J) Whole mount in situ hybridization of Nsun7 in E7.5, E8.5, E9.5 and E10.5 embryos. (A, D and I) Lateralview. (B and F) Frontal view. (G) Dorsal view. (H and K) In situ hybridization on sections from embryos at E9.5 and E10.5. (L) Control in situ hybridization with sense probe forNsun5 in E9.5 embryo. al = allantois, ane = anterior neuroectoderm, ce = cerebellum, ch = chorion, ee = extra embryonic ectoderm, ba1 = first branchial arch, ba2 = secondbranchial arch, cv = cerebral vasculature, da = dorsal aorta, fb = forebrain, fg = facio-acoustic ganglion, fl = forelimb, fnp = frontonasal prominence, (hf) head folds,hb = hindbrain, hl = hindlimb, ht = heart, mb = midbrain, nl = neural lumen, nt = neural tube, op = optical plate, ot = otical plate, oe = olfactory epithelium, so = somite,sv = sinus venous and tg = trigeminal ganglion. Reference bars in A–G, I, J and L = 250 lm, H and K = 100 lm.

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(al) (Fig. 6B and C). Expression in somites (so) appeared restrictedto the myocele (Fig. 6C). At E9.5, Nsun7 continued to be expressedubiquitously, but was enriched in the forebrain (fb), midbrain(mb), hindbrain (hb), frontonasal prominence (fnp), optical (op)and otical plates (ot), the maxillary and mandibular portions ofthe fist branchial arch (ba1), somites (so) and forelimb (fl)(Fig. 6D–G). At E10.5, Nsun7 continued to be ubiquitously ex-pressed, but enriched in the forebrain (fb), midbrain (mb), hind-brain (hb), olfactory epithelium (oe), trigeminal (tg) and facioacoustic (fg) ganglia, optic (op) and otic (ot) plates, cerebral vascu-lature (cv), first (ba1) and second (ba2) branchial arches, neuraltube (nt), dorsal aorta (da), forelimb (fl), and hindlimb (hl)(Fig. 6I–K). The structures with enriched Nsun7 gene expressionare summarized in Table 2. Hybridization with a sense probe forNsun7 produced only low background signal (Fig. 6L).

The function of Nsun7 is poorly understood, however, evidencesuggests its requirement for proper germ cell function, gene muta-tion causes male mice subfertility or sterility (Harris et al., 2007).Our results raise the possibility that Nsun7 might have additionaltissue-specific functions during mouse development.

1.7. Conclusion

The expression of Nsun genes is developmentally regulated tis-sue-specifically. Nsun genes express constitutively at moderate

levels during gastrulation and enrich tissue-specifically as embryo-genesis proceeds. Enrichment in several developing organs, mainlyin the central nervous system, craniofacial structures, and limb,suggests redundant functions for Nsun genes and RNA methylationin several aspects of mammalian embryogenesis.

2. Experimental procedures

2.1. Mouse Embryos

CD-1 mice were time-mated; the presence of vaginal plug wasused to designate the embryonic day 0.5. Embryos at E7.5, 8.5,9.5 and 10.5 were used for in situ hybridization. E13.5 and E14.5embryos were used to validate the probe for Nsun2, for which aLacZ reporter was previously reported (Blanco et al., 2011). Em-bryos were dissected in DEPC–PBS, and fixed overnight in 4% para-formaldehyde (PFA) at 4 �C. The Hospital for Sick Children AnimalCare Committee approved all experimental procedures.

2.2. In situ hybridization

To avoid cross hybridization, probes included unique sequencesfor Nsun2, 3, 4, 5, 6 and 7 coding regions. Unique sequences wereidentified in a multiple alignment using the CLC sequence viewer.

L. Chi, P. Delgado-Olguín / Gene Expression Patterns 13 (2013) 319–327 327

PCR products including priming sites for T7 and Sp6 polymerasesin the antisense and sense orientation, respectively, were gener-ated. The primers used are in Table 1. Antisense probes were digox-igenin-labeled using the PCR products as template. Whole-mountin situ hybridization was performed as described (Chi et al.,2011). Briefly, embryos were collected in PBS, fixed with 4% PFA,dehydrated through a methanol series and stored at �20 �C untilthey were processed. Embryos were rehydrated through a metha-nol series, bleached with 6% hydrogen peroxide, treated with10 lg/ml of proteinase K and post fixed with 4% PFA and 0.2% glu-taraldehyde. After washing, embryos were incubated in 50% form-amide, 5� SSC pH 4.5, 50 lg/ml tRNA, 1% SDS and 50 lg/mlHeparin, at 63 �C for 2 h, then the probe was added and incubationproceeded overnight at 63 �C. Embryos were washed 3 times,30 min each, with 50% formamide, 5� SSC pH 4.5 and 1% SDS at70 �C; an then 3 times, 30 min each, with 50% formamide and 5�SSC pH 4.5 at 65 �C. Embryos were then blocked in TBS with 0.1%Tween 20, 2 mM Levamisole and 10% goat serum for 2 h at roomtemperature, and then incubated overnight in blocking solutionwith 1:3000 Anti-DIG antibody (Roche). Embryos were washed atroom temperature multiple times and then overnight at 4 �C inTBS with 0.1% Tween 20 and 2 mM Levamisole. Embryos werewashed in NTMT (100 mM Tris pH 9.5, 50 mM MgCI2, 100 mMNaCl, 1% Tween 20 and 2 mM Levamisole) 3 times, 10 m each, atroom temperature, and the signal was developed with BM purple(Roche). Embryos were dehydrated and rehydrated in an ethanolseries and cleared in 80% glycerol before imaging.

Section in situ hybridization was performed as described(Delgado-Olguín et al., 2012) on 5 lm sections obtained fromparaffin-embedded embryos at E9.5 and E10.5.

2.3. Imaging

Whole mount embryos and sections were imaged usingSMZ1500 and Eclipse Ni-U Nikon microscopes. Images were pro-cessed using NIS-Elements software, Adobe Photoshop Elements9 and CorelDRAW 14.

Acknowledgements

We thank C.C. Hui and Rong Mo (The Hospital for Sick Children)for advice and facilitating reagents, materials and equipment. IanScott and Sivani Paskaradevan (The Hospital for Sick Children) forfacilitating equipment and helping with embryo sectioning. P.D-Ois supported by Operational Funding from the Research Institute,The Hospital for Sick Children.

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