Cyclical Expression of the Notch/Wnt Regulator Nrarp Requires Modulation by Dll3 in Somitogenesis

Developmental Biology 329 (2009) 400–409

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Developmental Biology

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Genomes & Developmental Control Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

William Sewell a,1, Duncan B. Sparrow b,c,1, Allanceson J. Smith a, Dorian M. Gonzalez d, Eric F. Rappaport d, Sally L. Dunwoodie b,c,e, Kenro Kusumi a,d,f,⁎ a School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA b Victor Chang Cardiac Research Institute, Sydney, NSW 2010, Australia c St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, NSW 2052, Australia d The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA e School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, NSW 2052, Australia f Department of Basic Medical Sciences, The University of Arizona College of Medicine–Phoenix in Partnership with Arizona State University, Phoenix, AZ 85004, USA article info abstract

Article history: Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so Received for publication 6 September 2008 far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, Revised 19 January 2009 but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene Accepted 19 February 2009 expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result Available online 3 March 2009 in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using Keywords: fi Nrarp microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identi ed new genes Dll3 enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a Uncx small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib Axin2 and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in Lfng mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this Limch1 gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling Rhpn2 phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase Delta-like 3 with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Notch Wnt Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but Presomitic mesoderm resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null Somitogenesis embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the Hnrnpl role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Segmentation Dll3 but not Lfng for normal function. Oscillatory © 2009 Elsevier Inc. All rights reserved.

Introduction genes Lfng, Hes1, Hes5, and Hes7 and the Wnt pathway genes Axin2, Nkd1, Dact1 and Dkk1 (Aulehla et al., 2003; Bessho et al., 2001; The vertebrate body is shaped from segmental units called somites, Dequéant et al., 2006; Forsberg et al.,1998; Ishikawa et al., 2004; Jouve which are formed in a regular, repeated fashion during embryonic et al., 2000). development. Somites are produced at the anterior end of the The Wnt pathway plays a key role in the presomitic mesoderm. unsegmented presomitic mesoderm (PSM), where oscillatory waves Small, irregular somites have been observed in beta-catenin null of gene expression regulate prepatterning of these segmental units embryos and lengthened presomitic mesoderm observed in beta- (reviewed in Dequéant and Pourquié, 2008; Kageyama et al., 2007; catenin gain-of-function mutants, suggesting that the Wnt pathway Kulesa et al., 2007). To date, many genes have been identiﬁed that regulates somitogenesis by activating target genes such as Dll1 and demonstrate such oscillatory expression, including the Notch pathway positioning boundary determination genes in the anterior presomitic mesoderm (Dunty et al., 2008; Hofmann et al., 2004). Wnt3a mutants disrupt the expression of a number of Notch pathway genes including ⁎ Corresponding author. School of Life Sciences, Arizona State University, PO Box Lfng, whereas the Wnt pathway gene Axin2 has been observed to 874501, Tempe, AZ 85287-4501, USA. Fax: +1 480 965 6899. E-mail address: [email protected] (K. Kusumi). display cycling expression in the Notch pathway Dll1 mutant (Aulehla 1 These authors contributed equally. et al., 2003). These observations and others have been used to support

0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2009.02.023 W. Sewell et al. / Developmental Biology 329 (2009) 400–409 401 the view that the Wnt pathway is upstream of Notch signaling in the et al., 2000; Dunwoodie et al., 2002; Kusumi et al.,1998; Sparrow et al., PSM. 2006, 2008). During somitogenesis, Notch signaling has been proposed to be Why does loss of Dll3 produce such a severe phenotype? One essential for one or more of the following functions; generation of possibility is that the disruptions in Lfng cycling expression and oscillatory gene expression in PSM cells (Holley et al., 2002; Jouve et activated Notch1 patterns account for the severe phenotype. An al., 2000; Morales et al., 2002), establishment of somite compartment alternate possibility is that as yet unidentified genes are critically polarity (Barrantes et al.,1999; Saga, 2007; Takahashi et al., 2000), and required for somitogenesis, and that these are disrupted in Dll3 communication between neighboring cells to synchronize oscillations mutants. Analysis of Dll3 mutant embryos by microarray studies could (Horikawa et al., 2006; Jiang et al., 2000; Özbudak and Lewis, 2008). identify such factors. Our understanding of somitogenesis continues Recently, pharmacological blockade of the Notch pathway in zebrafish to be advanced by the identification of new genes. This includes genes exhibited somite defects only after long developmental delays, that are highly expressed in the presomitic mesoderm, as well as suggesting Notch signaling is essential for synchronizing oscillations genes that have altered expression in segmental mutants. Functional of neighboring cells in the posterior PSM but not for somite border genomic approaches can be particularly useful to find such genes, formation (Mara et al., 2007; Özbudak and Lewis, 2008). Furthermore, since they can screen the transcriptome, and microarray studies have Feller et al. (2008) suggested a similar role for the Notch pathway in identified genes with oscillatory expression in mouse somitogenesis the caudal PSM in mice as well as demonstrating a requirement for (Dequéant et al., 2006) and in cell culture models of this process Notch signaling in somite compartmentalization and not border (William et al., 2007). Others have used this approach to examine Dll1 formation in the anterior PSM. In mouse, defects in Notch signaling (Machka et al., 2005) and Hes7 (Niwa et al., 2007) mutant embryos. disrupt somite segmentation and oscillatory expression of Notch Previously we reported expression differences in whole 9.5 dpc pathway genes in the PSM (Bessho et al., 2001; Conlon et al., 1995; Notch1 and Dll3 mutant embryos (Loomes et al., 2007). However we Evrard et al., 1998; Feller et al., 2008; Hrab de Angelis et al., 1997; did not identify somitogenesis genes, probably because the PSM is Kusumi et al., 1998, 2004;). In PSM S-1, i.e., somite minus one, the only a minor portion of the entire embryo. Here, we aimed to identify region from which the next somite will form (Pourquié and Tam, genes enriched in the presomitic mesoderm, and we have identified 2001), the transcription factor Mesp2, a direct target of Notch and characterized 3 genes expressed in the caudal PSM. In addition, signaling, appears to regulate segmental border formation through we examined Dll3 mutant PSM and somite level tissues, and identified activation of Epha4 and rostral–caudal compartmentalization through a limited number of genes with disrupted expression. Nrarp (Notch Uncx/Uncx4.1 (reviewed in Saga, 2007). However, the mechanisms by regulated ankyrin repeat protein), which is normally expressed in a which Notch signaling directs expression of downstream genes cycling manner during somitogenesis, emerged from our investigation necessary for paraxial mesoderm segmentation is still not well as a gene that may contribute to the severe Dll3 mutant phenotype. understood. Notch signaling activity can be modified in a number of ways Materials and methods (reviewed in Bray, 2006). Two modifiers of Notch signaling, Lfng and Dll3, are noteworthy given their disruption in humans causes a severe, Generation of mutant mice autosomal recessive vertebral disorder, spondylocostal dysostosis (SCD; Bulman et al., 2000; Sparrow et al., 2006). Disruptions of Lfng Dll3tm1Rbe (referred to as Dll3neo), Lfngtm1Rjo, and Wnt3atm1Amc and Dll3 in the mouse result in somitic and vertebral phenotypes that mutations were crossed into the C57BL/6J background by backcross are morphologically similar to each other and to SCD (reviewed in matings for over 10 generations (Dunwoodie et al., 2002; Kusumi et al., Turnpenny et al., 2007). Lfng is a modifier of Notch signaling. It 1998; Takada et al., 1994). All animals were maintained according to encodes a glycosyltransferase that modifies Notch in the Golgi, and Institutional Animal Care and Use Committee guidelines. Dll3 mutants modulates the ability of Notch to bind to DSL ligands. Loss of Lfng were generated by an intercross mating of Dll3neo/+ mice, Lfng mutants function results in severe rostrocaudal patterning defects (Evrard et were generated by an intercross mating of Lfngtm1Rjo/+ mice, and Wnt3a al., 1998; Shifley et al., 2008; Zhang and Gridley, 1998). In contrast to mutants were produced by an intercross mating of Wnt3atm1Amc/+ Lfng, Dll3 has only recently been identified as a modifier of Notch mice. Genotypes for Dll3neo, Lfngtm1Rjo and Wnt3atm1Amc were deter- activity (Geffers et al., 2007). Dll3 encodes a highly divergent delta- mined by PCR assay as described previously (Dunwoodie et al., 2002; type DSL ligand that, unlike the other DSL ligands, does not appear to Evrard et al., 1998; Kusumi et al., 1998). bind Notch receptors at the cell surface, and instead regulates Notch signaling, perhaps within the Golgi (Dunwoodie et al., 1997; Geffers et Microarray analysis al., 2007; Ladi et al., 2005). Null alleles of Dll3 disrupt transcriptional oscillation of some Notch pathway genes expressed in the PSM, as well Embryos were collected at day 9.5 of gestation, and dissected from as genes involved in determining the rostrocaudal polarity of the decidua in cold M2 medium (Nagy et al., 2003) containing 10% fetal somite (Dunwoodie et al., 2002; Kusumi et al., 1998, 2004). calf serum. All remnants of the allantois were carefully removed from Loss of Notch signaling can lead to failure of Notch pathway gene intact embryos, which were then cut at the boundary of the PSM and expression during somitogenesis, as demonstrated in Dll1 mutations most recently-formed somite. The released tissue (“PSM level” which lead to severely decreased expression of most reported Notch containing tissue from all three germ layers) was placed in cold RNA pathway genes (Barrantes et al., 1999; Kusumi et al., 2004). By lysis solution and frozen at −80 °C for later RNA extraction. The contrast, loss of Dll3 results in decreased levels of gene expression of remainder of the embryo was cut at the level of the otic placode, and only some Notch pathway genes including Hes5 and Hes1; but the the heart and endodermal tissues removed to generate the “somite dynamic expression of the cycling gene Hes7 is unaffected (Dunwoo- level” samples. Total RNA was extracted from 9.5 dpc embryos using a die et al., 2002; Kusumi et al., 2004). Dll3 mutations also lead to the SuperScript RT II Kit (Invitrogen) and subsequently used to synthesize loss of Lfng cycling expression (Kusumi et al., 2004) and disruption of double stranded cDNA. Biotinylated cRNA targets were prepared with cycling patterns of activated Notch1 (Geffers et al., 2007). For a Bioarray HighYield RNA Transcript Labeling Kit (ENZO). Targets were comparison, loss of Lfng expression (Morimoto et al., 2005) or its hybridized to the Affymetrix MOE430A array according to the cyclical component (Shifley et al., 2008) both disrupt cyclical manufacturer's protocol. Arrays were subsequently scanned using activation of Notch1 and Hes7 expression. In both humans and mice, the following parameters: t=0.015, a1 =0.05, a2 =0.065, median DLL3 mutations produce vertebral disruptions as severe as those intensity value=150. CEL files were normalized by the Robust observed in disruptions of LFNG or HES7 (Bessho et al., 2001; Bulman Multichip Average method in order to compare the levels of 402 W. Sewell et al. / Developmental Biology 329 (2009) 400–409 expression between different samples and minimize technical varia- tion accompanying use of the Genespring GX 7.3.2 module (Agilent).

Quantitative PCR

Quantitative PCR was used to conﬁrm the expression of selected candidate genes, using cDNAs assayed by Taqman® Gene Expression Assays (Applied Biosystems). Gapdh was used to normalize Q-PCR results, using assay Mm99999915_g1. Assays for selected genes examined were: Dll3 exons 2–3, Mm00432856_g1; Dll3 exons 5–6, Mm00432853_g1; Nrarp, Mm00482529_s1; Bcat2, Mm00802196_g1; and Hnrnpl, Mm01172981_g1. Cycling conditions used for the Taqman Real-time PCR were: 95 °C 10 min followed by 40 cycles of 95 °C 15 s and 60 °C 1 min. Statistical analysis on expression data was carried out using a t-Test (two sample assuming unequal variance), with a threshold of pb0.05 on a two-tailed test on microarray data and at least a one-tailed test for validation by Q-PCR.

In situ hybridization

Whole mount in situ hybridizations were performed on 8.5, 9.5 and 10.5 dpc embryos as described previously (Harrison et al., 1995; Wilkinson, 1992). DNA templates for RNA probes were generated by RT-PCR from a 9.5 dpc embryo cDNA template. In brief, chimeric PCR templates were generated using 3′ end primers with the T7 RNA polymerase-binding site added. Digoxigenin-UTP labeled (Roche) RNA probes were generated by in vitro transcription (MAXIscript™, Ambion). RNA probes were purified using MC Free filtration units (Millipore). To compare the expression of two genes within the PSM, we bisected the caudal paraxial mesoderm of fixed whole 9.5 and 10.5 dpc embryos prior to in situ hybridization. Hybridized embryos were photographed using a SMZ1000 stereodissecting microscope (Nikon) with a Retiga CCD digital camera (Q-Imaging). To examine cycling gene expression, we bisected the caudal paraxial mesoderm of 9.5 dpc embryos into axial halves, fixing one half in 4% paraformaldehyde while culturing the remaining half for 60, 120 or 180 min. in DMEM/50% FBS prior to fixation (adapted from Forsberg et al., 1998; Kusumi et al., 2004).

Accession numbers

Data sets from Affymetrix microarray analysis of microdissected embryonic tissues (MOE430A) are deposited at the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) with accession number GSE 15178.

Results and discussion

Microarray analysis of PSM and somite tissues

In carrying out microarray analysis of 9.5 dpc embryonic PSM and somite level tissues, we sought to 1.) identify genes that are enriched in PSM and somite tissues, and 2.) identify genes that were up- and down-regulated due to Dll3 mutation. We microdissected tissue from 9.5 dpc embryos to generate somite and PSM level samples (Fig. 1A). Fig. 1. Identification of novel genes expressed in presomitic mesoderm by microarray Since the amount of tissue collected from the PSM of one embryo was analysis of 9.5 dpc embryos. (A) Tissues from presomitic mesoderm and somites from insufficient for microarray analysis using a single round of target ten 9.5 dpc embryos were pooled into each of three biological replicates per tissue. amplification, we pooled dissected samples from ten embryos. We Total RNA extracted from each pool was analyzed using Affymetrix MOE430A arrays sought to avoid double amplification protocols that can lead to with a single round of target preparation. Genes are shown in order of gene-tree clustering. We identified 66 genes that were more than 2-fold increased in expression distortion of gene expression levels. Microarray analysis using in somite level tissue compared to PSM (Supplemental Table S1), with 15 genes with Affymetrix MOE430A arrays was carried out on the biological pool reported expression in paraxial mesoderm (B), 48 genes reported in other tissues, and triplicates, and genes with greater than two-fold differences were 3 genes uncharacterized (Table 1). We identified 87 genes that were more than 2-fold identified (Supplemental Tables S1 and S2). increased expression in PSM compared to somites (Supplemental Table S2), with 48 genes with reported expression in paraxial mesoderm (C), 31 genes reported in other Expression analysis identified 66 genes with greater than two-fold tissues, and 8 genes not previously described (Table 1). Analysis of these 8 genes increased expression in somite when compared to PSM fractions. identified three, A130022J15Rik, Limch1, and Rhpn2 that were highly expressed in the Using the Gene Expression Database (GXD; http://www.informatics. caudal PSM (D–I). W. Sewell et al. / Developmental Biology 329 (2009) 400–409 403 jax.org/expression.shtml) and the published literature, we identified generated and does not report any segmental defects, but the mutant 15 genes that have been reported to be expressed in somites, including was generated to examine thyroid function and segmentation may not Hes5, Meox1, Meox2, Pax1, Uncx/Uncx4.1,andZic1 (Fig. 1Band have been examined in detail (Behrends et al., 2005). Targeted Supplemental Table S1). We found a further 48 genes that were mutations or human mutations of Limch1 and A130022J15Rik have not reported to be expressed in the other embryonic tissues, including the been reported. Another cDNA, 2610528A11Rik was found to be neural tube, surface ectoderm, or endoderm. Expression in the localized to the caudal endoderm (Supplemental Fig. S1). somites was not reported for these 48 genes and remains to be further characterized. Finally, there are also 3 genes whose expression Dll3 mutation disrupts expression of the Notch regulator gene Nrarp has not been reported in embryos. We characterized the expression of these genes by in situ hybridization and observed that Pkdcc (protein To enrich for genes in the paraxial mesoderm that are specifically kinase domain containing, cytoplasmic) was expressed in the disrupted by Dll3 mutation, we compared microdissected tissues from endoderm, Elavl4 (embryonic lethal abnormal vision-like 4) demon- wild-type and Dll3 embryos. We generated biological replicate pools strated a salt-and-pepper expression in the neuroepithelium of the from Dll3+/+ (wild-type) or Dll3neo/neo embryos for a total of twelve midbrain and patches of expression dorsal to the second pharyngeal pools. Microarray analysis using Affymetrix MOE430A arrays was arch (data not shown), and Itih5 (inter-alpha globulin inhibitor H5) carried out on the biological pool triplicates, and genes with expression was not detectable (Table 1 and Supplemental Fig. S1). significant differences of greater than two-fold were selected. Findings were validated by Q-PCR and all expression differences were Identification of Limch1, Rhpn2, and A130022J15Rik as novel genes confirmed as statistically significant. Dll3 itself displayed 3.6 fold expressed in the presomitic mesoderm decreased expression Dll3neo/neo embryos (Fig. 2A). This demonstrates that our microarray approach was capable of detecting relevant We further identified 87 genes with increased expression (≥2 differences in gene expression between wild-type and null mutant fold) in the nascent PSM when compared to somite fractions (Fig. 1C embryos. A complete absence of Dll3 transcripts might be expected in and Supplemental Table S2). As described above, we confirmed that the Dll3 null pools, and the Affymetrix probe set 1449236_at 48 of these genes had been published as being expressed in PSM, comprises oligonucleotides distributed over all of the Dll3 exons, including the somitogenesis genes Dll1, Dll3, Fgf8, Hes7, and Tbx6.31 including exons 1–4 that were not removed in the targeted mutation of these genes were reported to be expressed in the embryo in tissues (Netaffx, www.affymetrix.com; Dunwoodie et al., 2002). We therefore including neural plate or surface ectoderm, but remain to be further used quantitative PCR to examine expression levels of Dll3 exons 2–3 examined in the paraxial mesoderm. This list includes the gene (not targeted) and exons 5–6 (deleted in the mutant; Fig. 2A). As Phlda2, which interestingly is a paralogue of the Fas pathway gene expected, neither were detectable in Dll3neo/neo embryos, indicating Phlda1 that cycles in phase with Wnt pathway genes (Dequéant et that the targeted transcript was likely to have been subject to al., 2006). Finally, there are 8 genes whose expression has not been nonsense-mediated decay. Thus, levels of Dll3 detected by microarray described in embryos (Table 1). We characterized these genes by in in Dll3neo/neo mutants appear to represent baseline noise of the situ hybridization and noted high levels of PSM expression of three of microarray system. them, Limch1 (LIM and calponin homology domains 1), Rhpn2 Surprisingly, few other genes were identified at an initial 2-fold (rhophilin 2), and the cDNA A130022J15Rik (Figs. 1D–I). Gene cut-off level. We next examined a group of genes identified at a Ontogeny predicts that Limch1 encodes an actin binding protein and threshold of 1.5 fold changes (Fig. 2). In addition to Dll3, only Nrarp, that A130022J15Rik encodes a glycosyltransferase, like Lfng (www. which has been previously identified as an inhibitor of the Notch informatics.jax.org). Interestingly, Rhpn2 encodes a rho GTPase signaling pathway, was down-regulated in the Dll3neo/neo PSM binding protein that could play a role in endocytosis (Behrends et fractions. Nrarp will be described in detail below. The only al., 2005). Endocytosis of Notch1 extracellular domain-ligand hetero- upregulated genes in these fractions were Bcat2 (Fig. 2C), which dimers and of Notch1 receptors play a key role in trans- and cis- encodes mitochondrial branched chain aminotransferase 2 that regulation, respectively. A targeted mutation of Rhpn2 has been produces a growth and metabolic defect when knocked out (She et al., 2007) and Hnrnpl (Figs. 2D, E), which encodes heterogeneous

Table 1 nuclear riboprotein L factor that plays a role in mRNA processing Expression of previously uncharacterized genes identified by microarray analysis of (Griffith et al., 2006). We found that Bcat2 is highly expressed in the 9.5 dpc embryonic paraxial mesoderm rostral first pharyngeal arch and also expressed in trunk mesenchyme

Gene Fold Gene description Expression (9.5 dpc) and that Hnrnpl is expressed ubiquitously at 9.5 dpc (Supplemental change Fig. S1). Since mRNA splicing is a key regulatory step for the Genes enriched in somite level tissues dynamically expressed Notch pathway cycling genes, the upregulation Elav14 2.4 Embryonic lethal No expression detected of Hnrnpl may represent a compensatory change in response to abnormal vision-like 4 disruption of the segmentation clock. However, the altered expression Itih5 2.6 Inter-alpha (globulin) No expression detected of both of these genes may be secondary to the disruption in Notch inhibitor H5 Pkdcc 4.8 Protein kinase domain Trunk mesenchyme, ventral signaling. containing, cytoplasmic somites (Supplemental Fig. S1) In tissue collected from somite levels, Hnrnpl was the only gene that displayed increased expression in Dll3 mutants, and given the Genes enriched in PSM level tissues ubiquitous expression that we observed at 9.5 dpc, this is not 2610528A11Rik 7.4 RIKEN 2610528A11 gene Caudal endoderm surprising. We found that Uncx/Uncx4.1 was decreased in expression (Supplemental Fig. S1) A130022J15Rik 2.1 RIKEN A130022J15 gene Caudal PSM (Fig. 1) (Fig. 2E). Uncx is expressed in the caudal compartment of somites, and Arhgap24 3.1 Rho GTPase activating Neural tube we have previously shown that Dll3 mutant embryos display protein 24 disrupted expression in rostral–caudal compartments of somites Gzmk 2.1 Granzyme K No expression detected (Dunwoodie et al., 2002). Uncx expression is also severely down- Hsd17b11 2.4 Hydroxysteroid 17-beta No expression detected dehydrogenase 11 regulated in Dll1 and RBPjk null embryos when Notch1 signaling is Limch1 2.3 LIM and calponin Caudal PSM (Fig. 1) absent (Barrantes et al., 1999; Takahashi et al., 2003). Uncx is required homology domains 1 for the formation of the pedicles and proximal ribs, structures which Mpzl2 2.6 Myelin protein zero-like 2 No expression detected are malformed in Dll3 mutant animals (Kusumi et al., 1998; Mansouri Rhpn2 2.8 Rhophilin 2 Caudal PSM (Fig. 1) et al., 2000). However, the vertebral malformations in Uncx mutants 404 W. Sewell et al. / Developmental Biology 329 (2009) 400–409

Fig. 2. Identification of genes in the paraxial mesoderm whose expression is disrupted by Dll3 mutation. Tissues from Dll3 mutant and wild-type embryos at 9.5 dpc were microdissected into presomitic mesoderm and somite level tissues. Tissues from ten embryos were pooled into each biological replicate, with three replicates for each genotype, and analyzed on Affymetrix MOE430A arrays. Statistical analysis was carried out using a t-Test (two sample assuming unequal variance) and revealed all differences to be significant on a two-tailed test for initial microarray identification and at least on a one-tailed test for validation by Q-PCR. Two tailed p values are given, except as noted. In the PSM at a 1.5 fold threshold, we only identified 2 genes with decreased expression, Dll3 (A; pb0.035) and Nrarp (B; pb0.001). We observed two genes with increased expression, Bcat2 (C; pb0.021) and Hnrnpl (D; pb0.001). We observed by Q-PCR that Dll3 transcripts were absent in Dll3neo/neo embryos both assayed before (Dll3 exons 2–3; pb10− 8) and after (Dll3 exons 5–6; pb10− 6) the neo-induced mutation, suggesting nonsense-mediated decay of the transcript from the targeted allele. Nrarp displayed decreased expression the Dll3 homozygous mutant PSM (B; one-tailed test pb0.032), and Bcat2 (C; pb0.025) and Hnrnpl (D; pb0.001) displayed increased expression in Dll3 homozygous mutant PSM. In somite tissue with a 1.5 fold threshold, we identified that Uncx displayed decreased expression (E right panel; pb0.006) and Hnrnpl was increased in expression (E left panel; pb0.026). are distinct from those observed in Dll3 and much less severe. et al., 2001). More recently, Nrarp was described to display cycling Therefore, the malformations observed due to Dll3 mutation are not expression based on identification in a microarray-based screen for likely to be mostly accounted for by Uncx-mediated effects. genes with oscillatory expression in mouse PSM (Dequéant et al., 2006); and two expression phases for Nrarp have been described The Notch pathway gene Nrarp displays cycling expression during (Dequéant et al., 2006, Shifley et al., 2008). somitogenesis While Nrarp expression in PSM has been reported at particular stages, we sought to fully characterize the cycling expression of Nrarp Evidence suggests that Nrarp may regulate both the Notch and in the PSM from early to late-somitogenesis at 8.5, 9.5, and 10.5 dpc. Wnt signaling pathways. Nrarp was originally identified in an First we identified that distinct caudal-to-rostral shifts, characteristic expression screen in Xenopus embryos as a member of the Delta– of cycling genes, occurred at each stage (Fig. 3). Rostral bands of Nrarp Notch pathway (Gawantka et al., 1998). In Xenopus, activation of the expression displayed anterograde shifts and rostrocaudal contraction Notch pathway resulted in elevated levels of Nrarp expression, characteristic of cycling genes at all three stages. At 8.5 and 9.5 dpc, demonstrating Nrarp is a target of the Notch pathway (Lamar et al., Nrarp expression in the caudal PSM extended over a large proportion 2001). Once expressed, Nrarp functions as a negative feedback of the PSM, but the most intense areas of expression displayed regulator of Notch signaling by binding the activated form of Notch anterograde shifts. In addition, there was strong expression of Nrarp (Notch-ICD) and promoting a decrease in Notch-ICD levels and a remaining in the primitive streak in 8.5 dpc embryos, similar to other reduction in Notch target gene expression. Nrarp has also been found Notch pathway genes such as Lfng (Barrantes et al., 1999; Dunwoodie to be a positive regulator of the Wnt pathway in zebrafish, by et al., 1997). At 9.5 dpc, there was a residual level of Nrarp expression stabilizing the LEF1 protein (Ishitani et al., 2005). Furthermore, the remaining throughout the PSM. At 10.5 dpc, cycling patterns of Nrarp activation of LEF1 was not found to affect Notch signaling in zebrafish, caudal bands were more clearly evident due to loss of this residual so the actions of Nrarp appear to act independently on the canonical expression in the PSM and the tailbud. Wnt and Notch signaling pathways. The phase of a cycling gene is defined by its spatial expression In the mouse, Nrarp was described as being expressed within the along the caudal-to-rostral axis within the embryonic PSM at a paraxial mesodermal in a rostral band and a broad caudal domain in particular point in time during a segmentation cycle (Pourquié and the PSM; however, in this study Nrarp was not noted as a cycling gene Tam, 2001). Using the phase descriptions used for other cycling genes, (Krebs et al., 2001). The rostral band was localized to the caudal S0 we defined three phases for Nrarp expression (Figs. 3G–I and M–O). In (the newly forming somite). The broad caudal domain of Nrarp in the phase I, caudal expression of Nrarp remained in S1 at 8.5 and 9.5 dpc, PSM was defined by a rostral boundary at the second presumptive but not at 10.5 dpc. Rostral bands of Nrarp expression in S0/S1 somite (S-2). These were spatially verified by comparison with Uncx,a contracted along the rostrocaudal axis between phase I and III. marker for the somite caudal compartment, and Mesp2, a marker for Expression of Nrarp in the PSM was localized to the tailbud and caudal the anterior half of the second presumptive somite in the PSM (Krebs region in phase I, shifted rostrally in phase II, and condensed towards W. Sewell et al. / Developmental Biology 329 (2009) 400–409 405

Fig. 4. Nrarp cycling expression displays a 2 hour periodicity. 9.5 dpc mouse embryos were bisected, with left halves cultured for 15 min and ﬁxed, and right halves cultured for an additional 1 hour (A, n=8), 2 h (B, n=7), or 3 h (C, n=7) prior to ﬁxation. Embryo culture resulted in some shrinkage of tissue; therefore, the halves are aligned with the caudal border of the forming somite S0 (arrowhead). At a 1 hour time difference, anterograde shifts of Nrarp caudal expression in the PSM were observed (A). At a 2 hour time difference, cultured halves displayed similar expression patterns of Nrarp, as shown in (B). At 3 hour time differences, cultured halves again differed in Nrarp caudal expression (C). The observation that cultured halves were in the same phase at 2 h, and out of phase at 1 and 3 h, is consistent with the 2 hour periodicity of cycling genes during mouse somitogenesis.

halves again differed in Nrarp caudal expression (Fig. 4C). The observation that cultured halves were in the same phase at 2 h and out of phase at 1 and 3 h is consistent with the 2 hour periodicity of cycling genes during mouse somitogenesis (Hirata et al., 2002).

Nrarp cycling phases relative to Lfng and the Wnt pathway gene Axin2 at 9.5 and 10.5 dpc

Cycling genes tend to fall into two classes — those which cycle in concert with Notch and FGF pathway genes, and Wnt pathway- associated genes that do not. Comparing expression patterns of cycling genes can help give clues about the dynamics within and between gene pathways. Genes have been described as being “in phase” if similar expression patterns from phase I to III are observed for the two genes. Previous models have proposed that cycling genes within the same pathway will display more similar phases than genes

Fig. 3. Nrarp displays cycling expression in the presomitic mesoderm with distinct patterns at 8.5, 9.5, and 10.5 dpc. Embryos at 8.5 dpc (n=15; A–C; dorsal view), 9.5 dpc (n=50; D–F; lateral view), and 10.5 dpc (n=32; J–L; dorsal view) are shown in order corresponding to phases I–III (diagrammed in G–I for 9.5 dpc, M–O for 10.5 dpc), as deﬁned previously for cycling genes (Pourquié and Tam, 2001). Regions of peak Nrarp expression are indicated (arrowheads). Rostral bands of Nrarp expression display anterograde shifts and contraction at 8.5–10.5 dpc, as characteristic of cycling genes. Nrarp expression in the caudal PSM extends over a larger proportion of the PSM at 8.5 and 9.5 dpc compared to 10.5 dpc. However, within this larger region in the PSM, the area of highest expression is observed to display anterograde shifts. Tailbud expression of Nrarp decreases from phases I to II (G, H) and returns by phase III (I). Nrarp more clearly shows cycling expression at 10.5 dpc (arrowheads) with periodically decreased expression in the tailbud between phases II and III (K, L and N, O).

S-2 in phase III. Thorough description of these phases was a necessary step prior to analysis of effects of Dll3, Lfng, and Wnt3a mutations on Nrarp cycling expression.

Nrarp cycling expression displays a 2 hour periodicity

Mouse embryos were bisected at 9.5 dpc, with left halves cultured for 15 min and then fixed, and right halves cultured for an additional Fig. 5. Nrarp cycling expression is in phase with Lfng and out of phase with Axin2 at 10.5 dpc. Mouse 10.5 dpc embryos were bisected and halves were analyzed by in situ 1h(Fig. 4A, n=8), 2 h (Fig. 4B, n=7), or 3 h (Fig. 4C, n=7) prior to hybridization (A–I). Nrarp expression was compared to Lfng (n=12; A–C) and the Wnt fixation. Culturing led to a slight contraction of embryonic explant pathway cycling gene Axin2 (n=12; D–F); and Lfng and Axin2 were compared to each tissues, therefore halves were aligned with the first morphologically other (n=18; G–I). Areas of peak expression are indicated (arrowheads). The caudal to apparent forming somite. At a 1 hour time difference, anterograde rostral shifts in Nrarp expression corresponded to equivalent phases for Lfng (A–C). Neither the expression of Nrarp nor Lfng corresponded with cycling expression phases shifts of Nrarp caudal expression were observed (Fig. 4A). At a 2 hour of Axin2 (E, F and H, I, respectively). However, the cycling expression of Axin2 at time difference, cultured halves displayed similar expression patterns 10.5 dpc is less notable than at 9.5 dpc (Supplemental Fig. S2). All half embryos are of Nrarp, as shown in (Fig. 4B). At 3 hour time differences, cultured aligned at S0 and oriented with rostral at the top. 406 W. Sewell et al. / Developmental Biology 329 (2009) 400–409

Table 2 9.5, this may be partially accounted for by the greater region of Distribution of patterns of Nrarp, Lfng, and Axin2 gene expression between cycling Nrarp expression along rostrocaudal PSM axis (Fig. 3), but this phases I, II, and III effect is also observed for Nrarp at 10.5 dpc. Therefore, the Gene Embryo Phase I Phase II Phase III p value (deviation assumption that the amount of time that cycling genes spend in age (dpc) from expected) phases I, II, or III may not be equal for all genes. It is possible that Lfng 9.5 38% (18/47) 26% (12/47) 36% (17/47) pb0.517 Nrarp “rushes” through phase III, making it less likely to be b Axin2 9.5 22% (10/45) 33% (15/45) 44% (20/45) p 0.189 represented. Nrarp 9.5 30% (12/40) 60% (24/40) 10% (4/40) pb0.0005 Nrarp 10.5 37% (10/27) 52% (14/27) 11% (3/27) pb0.0319 Nrarp requires Lfng early in somitogenesis but requires Dll3 to maintain Lfng and Axin2 phases were determined as described in Aulehla et al. (2003). Nrarp cycling expression phases were determined as described in Fig. 3. Percentages may not total 100% due to rounding. Given that our microarray analysis of Dll3neo null embryos identified Nrarp as one of a limited number of genes with decreased in different pathways (Aulehla et al., 2003; Dequéant and Pourquié, expression, we examined the spatiotemporal pattern of Nrarp by 2008). When assayed by microarray analysis of caudal PSM tissues, whole mount in situ hybridization. In 9.5 dpc Dll3neo homozygous levels of Nrarp expression were shown to peak in correlation with Lfng embryos, Nrarp expression appeared to be fixed in phase I (n=5; but not with Axin2 (Dequéant et al., 2006). Some genes such as Nkd1 Fig. 6A), and by 10.5 dpc, Nrarp showed decreased expression in the have been shown to display dependency on both Notch and Wnt caudal PSM and in the newly forming somite, S0 (n=3; Fig. 6E). signaling, with Nkd1 transcription requiring Wnt3a and Notch Nrarp cycling expression was observed in Dll3neo heterozygous signaling for oscillatory expression (Ishikawa et al., 2004). embryos (Figs. 6B–D, F–H) and wild-type embryos (data not We used bisected embryos to directly compare Nrarp cycling shown). The static pattern of Nrarp expression in Dll3 null mutants phases I, II, and III with patterns for Lfng and Axin2 in the other half fixed in phase I bares a strong resemblance to the static pattern of (Fig. 5). We observed that the caudal-to-rostral progression and activated Notch1 expression in Dll3 null mutants at 10.5 dpc boundaries of Nrarp within the PSM were more comparable to that of (Geffers et al., 2007). Static patterns of activated Notch1 expression the Notch modulator Lfng at 10.5 dpc (Figs. 5A–C; phases for Nrarp as have also been observed in Lfng null mutants at 11.5 dpc (Morimoto diagrammed in Fig. 2) than at 9.5 dpc (Supplemental Fig. S2A–C). In et al., 2005). In Dll3 mutants, the Notch pathway cycling genes Hes1, comparison, Nrarp patterns were distinct from the caudal-to-rostral Hes5, and Lfng are also frozen in a single phase (Dunwoodie et al., progression of Axin2 at 10.5 dpc (Figs. 5D–F) and 9.5 dpc (Supple- 2002; Kusumi et al., 2004). Interestingly, only Hes7 manages to mental Fig. S2D–F). Previous reports have described Lfng and Axin2 maintain oscillation in Dll3 null embryos, in contrast to findings in expression as being out of phase with each other (Aulehla et al., 2003), embryos constitutively expressing Notch1-ICD (Feller et al., 2008), and we also observed out of phase patterns at 10.5 dpc (Figs. 5G–I). In suggesting that the autoregulatory feedback loop for this bHLH contrast, the phase differences between Lfng and Axin2 were more factor is less dependent on Notch signaling to cycle (Kusumi et al., difficult to detect at 9.5 dpc, suggesting that these genes may grow 2004). increasingly out of phase as somitogenesis progresses (Supplemental To compare the effects of mutations in Lfng with those in Dll3 on Fig. S2G–I, n=9). Nrarp cycling expression, we examined Lfng mutant embryos at To further investigate these observations, we categorized 9.5 9.5 dpc. We observed that Nrarp expression appeared to be cycling in and 10.5 dpc embryos that were probed for Nrarp, Lfng and Axin2 Lfngtm1Rjo null mutants at 9.5 dpc (Figs. 7E–G) and dynamic in the (Table 2). Expression phases I, II, and III have often been caudal PSM at 10.5 dpc (Figs. 7K–M) without the characteristic envisioned as consisting of equal representation of embryos during caudal-to-rostral “wave” seen in wild-type embryos (Figs. 7N–P). cycling, and by inference, relatively equal time periods. We Nrarp expression did not appear to be highly dynamic early in observed for Lfng and Axin2, that departure from equal distribution somitogenesis at 8.5 dpc (Figs. 7A–B). It is interesting that, despite between phases I–III was not significant by X2 analysis (Table 2). In reports that activated Notch1 does not cycle in Lfng null mutants at contrast, Nrarp expression departed significantly from equal 11.5 dpc (Morimoto et al., 2005), Nrarp appears to somehow be “kick- distribution between the three phases. For Nrarp expression at started” to display cycling expression later in somitogenesis. In

Fig. 6. Nrarp displays decreased expression and does not cycle in Dll3 mutants. Nrarp expression was analyzed by in situ hybridization of 9.5 dpc stage Dll3neo/neo mutant (n=5; A), Dll3neo/+ heterozygous (n=7; B), and Dll3+/+ control (n=8; data not shown) embryos. Nrarp expression in the rostral PSM is strongly decreased in Dll3neo/neo 9.5 dpc embryos, and cycling expression was not observed. This observation was conﬁrmed in 10.5 dpc stage Dll3neo/neo mutant (n=3; E), Dll3neo/+ heterozygous (n=4;F–H), and Dll3+/+ control (n=4; data not shown). Both Dll3 heterozygous and wild-type embryos displayed cycling expression at 9.5 and 10.5 dpc (B–D, F–H, and data not shown). W. Sewell et al. / Developmental Biology 329 (2009) 400–409 407

observations; however, this observation was made before Nrarp was known to be a cycling gene and may be describing different normal phases.

Nrarp expression is not observed in Wnt3a mutants

Wnt signaling is required for the expression of many Notch and Wnt pathway genes in the PSM (Aulehla et al., 2003, 2008). Wnt signaling acts to restrict boundary determination genes to the anterior PSM where somite size, morphology and polarity are deﬁned (Dunty et al., 2008). Genes in other pathways such as Cdx2 and Cdx4 do not require Wnt3a for expression within the PSM (Ikeya and Takada, 2001). Given the role of Wnt signaling in regulating PSM formation and maintenance, we examined Nrarp expression in mutants disrupted for

Fig. 7. Nrarp displays cycling expression in the presomitic mesoderm of Lfng mutant embryos. Nrarp expression was analyzed by in situ hybridization at 8.5, 9.5, and 10.5 dpc stages for Lfngtm1Rjo/tm1Rjo mutants (8.5 dpc, n=4; 9.5 dpc, n=5; 10.5 dpc, n=6) and wild-type controls (8.5 dpc, n=5; 9.5 dpc, n=5; 10.5 dpc, n=5).Uncx/Uncx4.1, which is expressed only in the caudal compartment of somites, was also added as a probe to hybridizations of the 9.5 dpc embryos in order highlight disruptions in segmentation in mutant embryos (E–G). Peak areas of Nrarp expression are indicated by arrowheads. In Lfng mutant embryos, Nrarp expression does not appear to be dynamic at 8.5 dpc (A, B) but clearly displays cycling expression at 9.5 dpc (E–G). In Lfng mutant embryos at 10.5 dpc, there was a subtle anterograde shift in peak Nrarp expression and decreased expression in the caudal PSM in phase III (K–M), suggesting dynamic expression. Lfng+/+ 10.5 dpc embryos demonstrate caudal-to-rostral shifts typical of oscillatory genes (N–P). contrast, Nrarp does not cycle in Dll3 mutants, thus suggesting that factors in addition to activated Notch1 cycling are required for somitogenesis. Nrarp has been reported to display stable expression at 8.5 dpc in Lfng mutants with a specific disruption of a regulatory element required for oscillatory expression (Shifley et al., 2008). However, by 10.5 dpc in embryos lacking cycling Lfng, Nrarp cycling recovers (Shifley et al., 2008). Before Nrarp was reported to display cycling expression, the effects of Notch1, Dll1, Lfng and Dll3pu mutations on Nrarp expression had been described for the paraxial mesoderm Fig. 8. Nrarp is not expressed in the PSM of Wnt3a homozygous mutants at 8.5 and tm1Amc/tm1Amc tm1Amc/+ (Krebs et al., 2001). Both Notch1 and Dll1 null mutants result in 9.5 dpc. At 8.5 dpc, Wnt3a (n=6; A), Wnt3a (n=5; B), and Wnt3a+/+ (n=5; C) were analyzed by in situ hybridization with Nrarp probe. Nrarp severe down-regulation of Nrarp in the PSM. Thus, the Dll1 and expression was not observed in mutant embryos, which contained presomitic Dll3 ligands have very different effects on Nrarp expression. mesoderm as confirmed by hybridization with Cdx4 probe (D, E). At 9.5 dpc, Differential functions for delta proteins and splice variants have Wnt3atm1Amc/tm1Amc (n=9; F), Wnt3atm1Amc/+ (n=10; G), and Wnt3a+/+ (n=10; H) also been described in zebrafish (Mara et al., 2007, 2008). Nrarp embryos were analyzed by in situ hybridization with Nrarp probe, which was not expressed in Wnt3atm1Amc/tm1Amc mutants. Panels F–H show approximately equivalent expression in null Lfng embryos was reported to be severely down- tissues that are caudal to somite 3. At 9.5 dpc, Wnt3atm1Amc/tm1Amc mutants had greatly regulated for rostral PSM but not caudal PSM expression. This does reduced amounts of presomitic mesoderm compared to Wnt3a+/+, as demonstrated not agree with the findings of Shifley et al. (2008) and our by in situ hybridization with Cdx4 probe (I, J). 408 W. Sewell et al. / Developmental Biology 329 (2009) 400–409

Wnt signaling (Wnt3atm1Amc mutants; Fig. 8). Wnt3atm1Amc/tm1Amc more careful examination of human and mouse pedicle osteology in embryos lack caudal somites and a tail bud and exhibit axial truncation mutants. As described above, Nrarp mutations in mouse or human (Takada et al., 1994). At 8.5 dpc, Wnt3atm1Amc/tm1Amc embryos have not yet been identified. Given its cycling expression in expressed Cdx4 at comparable levels to +/+ (Figs. 8D, E), suggesting somitogenesis and identification as down-regulated in Dll3 null the presence of paraxial and lateral mesoderm. However, Nrarp mutants, information about the phenotype in Nrarp mutants would expression was severely down-regulated (Fig. 8A), compared to allow us to evaluate this gene in contributing to DLL3-related defects heterozygous and +/+ embryos (Figs. 8B–C). At 9.5 dpc, and as a candidate for human disorders of the spine. Wnt3atm1Amc/tm1Amc embryos exhibited severely malformed and decreased amounts of PSM, confirmed by Cdx4 hybridization (Fig. Acknowledgments 8I), and the remaining tissue also did not show any expression of Nrarp (Fig. 8F), compared to wild-type controls (Figs. 8G–H). Thus, We thank Joshua Gibson, Walter Eckalbar and Michael Chacon for it appears that Nrarp requires Wnt signaling to initiate expression technical assistance. We thank Stephen Pratt for helpful discussions. within the PSM. This work was supported by NIH RO1 AR050687 (KK) and a Hitchings- Elion Fellowship of the Burroughs Wellcome Fund (KK), National Developmental functions of Nrarp Health and Medical Research Council (NHMRC) Project Grants 142006 and 404804 (SLD and DBS), a Westfield-Belconnen Fellowship (DBS), There are two paralogues of Nrarp in the zebrafish. Morpholino- aPfizer Foundation Australia Senior Research Fellowship (SLD) and a based knockdown of Nrarp-a expression in zebrafish has been NHMRC Senior Research Fellowship (SLD). reported to result in slightly smaller animals with altered pigmenta- tion and curly and shortened tail phenotypes (Ishitani et al., 2005). Appendix A. Supplementary data These phenotypes likely result from the destabilization of LEF1 protein by removing the ubiquitylation-blocking function of Nrarp, resulting Supplementary data associated with this article can be found, in in altered neural crest development. The shortened tail phenotype the online version, at doi:10.1016/j.ydbio.2009.02.023. does not appear to be due to major disruptions in somite patterning, but more subtle malformations remain possible (M. Itoh, personal Note added in proof communication). In contrast, Nrarp-b morphants did not display any phenotype. However, knockdown of Nrarp-a and -b enhanced A recently published report characterizes the phenotype of brom expression of her1 indicating they function redundantly as negative bones, a zebrafish mutant carrying a nonsense mutation in hnRNP I, a fi regulators of Notch signaling during somitogenesis in zebra sh polypyrimidine tract binding protein that can form heterodimers (Ishitani et al., 2005). The phenotypes of Nrarp a/b morphants were with Hnrnpl protein (Yang, J., et al., 2009. PLoS Genet. 5, e1000363). not described, therefore, the presence of two Nrarp homologues raises This report suggests that in human, mouse, Xenopus, and zebrafish, fi the possible that overlapping function that might make it dif cult to hnRNP I is a direct inhibitor of Notch signaling, acting by destabilizing detect vertebral phenotypes. the Notch intracellular domain. 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