WDR5 Regulates Left-Right Patterning Via Chromatin-Dependent and -Independent Functions Saurabh S

RESEARCH REPORT WDR5 regulates left-right patterning via chromatin-dependent and -independent functions Saurabh S. Kulkarni and Mustafa K. Khokha*

ABSTRACT Capdevila et al., 2000; Hamada et al., 2002). However, a Congenital heart disease (CHD) is a major cause of infant mortality mechanism connecting cilia, chromatin modifiers, and CHD/Htx and morbidity, yet the genetic causes and mechanisms remain has not been established. Here, we show that WDR5, a histone opaque. In a patient with CHD and heterotaxy, a disorder of left-right modifier, is necessary for both LR patterning and ciliogenesis. (LR) patterning, a de novo mutation was identified in the chromatin LR patterning is established at a conserved ciliated structure, the ’ modifier gene WDR5. WDR5 acts as a scaffolding protein in the H3K4 LR organizer (LRO) [the node in mouse, Kupffer s vesicle in fish methyltransferase complex, but a role in LR patterning is unknown. and gastrocoel roof plate (GRP) in frogs] (Burdine and Schier, 2000; Here, we show that Wdr5 depletion leads to LR patterning defects in Capdevila et al., 2000; Hamada et al., 2002; Blum et al., 2014). Two Xenopus via its role in ciliogenesis. Unexpectedly, we find a dual role types of monociliated cells populate the LRO: cells in the middle are for WDR5 in LR patterning. First, WDR5 is expressed in the nuclei of enriched in motile cilia that create leftward extracellular fluid flow, monociliated cells of the LR organizer (LRO) and regulates foxj1 which is detected by peripheral cells that have a preponderance of expression. LR defects in wdr5 morphants can be partially rescued immotile sensory cilia (Doerks et al., 2002; Basu and Brueckner, with the addition of foxj1. Second, WDR5 localizes to the bases of 2008; Schweickert et al., 2007; Boskovski et al., 2013). Once flow is dand5 cilia. Using a mutant form of WDR5, we demonstrate that WDR5 also sensed at the LRO left margin, (alternatively referred to as Cerl2 coco Xenopus has an H3K4-independent role in LR patterning. Guided by the patient in mouse and in ; a Nodal antagonist) is pitx2 phenotype, we identify multiple roles for WDR5 in LR patterning, downregulated, leading to upregulation in the left lateral plate providing plausible mechanisms for its role in ciliopathies like mesoderm (Burdine and Schier, 2000; Capdevila et al., 2000; heterotaxy and CHD. Hamada et al., 2002; Blum et al., 2014; Vonica and Brivanlou, 2007). The activation of pitx2 in the lateral plate mesoderm is KEY WORDS: Congenital heart disease, Heterotaxy, Xenopus, H3K4 associated with organ lateralization (Lin et al., 1999). methylation, Cilia WDR5 is a core subunit of the human MLL and SET1 histone H3 Lys4 methyltransferase (H3K4MT) complexes that are essential for INTRODUCTION chromatin modification and transcriptional regulation (Wysocka et al., Congenital heart disease (CHD) is the most common developmental 2005; Couture et al., 2006; Dou et al., 2006; Trievel and Shilatifard, defect (Van der Linde et al., 2011). However, our understanding of 2009; Couture and Skiniotis, 2013; Patel et al., 2008b). In particular, the genetic etiologies of CHD remains poor. A recent genetic H3K4MTs function as a complex consisting of one catalytic subunit analysis of CHD patients identified a marked enrichment in genes [SET1A/B (SETD1A/B) or MLL1-4 (KMT2A-D) proteins] and four involved in chromatin modification, specifically genes involved in core regulatory subunits (WDR5, RbBP5, ASH2L and DPY-30) H3K4/H3K27 methylation or H2BK120 ubiquitination (Zaidi et al., (Trievel and Shilatifard, 2009; Couture and Skiniotis, 2013; Takahashi 2013; Homsy et al., 2015). However, how global regulators of et al., 2011). These regulatory subunits form a subcomplex that binds chromatin states could lead to specific phenotypes, such as CHD, the catalytic subunit and dramatically enhances its H3K4MT activity remains unanswered. To address this question, we began our studies (Patel et al., 2009). Depletion of any of the regulatory subunits impairs with WDR5, a recent candidate gene for CHD and a critical member H3K4 methylation (Steward et al., 2006; Dou et al., 2006; Wysocka of the histone (H3K4) methylation pathway (Zaidi et al., 2013; et al., 2005; Cao et al., 2010). WDR5, a highly conserved scaffolding Wysocka et al., 2005). protein, is essential for the association of RbBP5, ASH2L and DPY- The patient with a de novo missense mutation, K7Q, in WDR5 30 with MLL1 via its β-propeller structure (Odho et al., 2010; Trievel had a conotruncal defect with a right aortic arch (normally the aortic and Shlatifard, 2009; Patel et al., 2008b). The central pocket made by arch is on the left), a mild heterotaxy (Htx) phenotype (Zaidi et al., the sevenfold propeller is crucial for binding H3 and MLL (Song and 2013). Htx results from aberrant left-right (LR) patterning of Kingston, 2008; Patel et al., 2008a; Schuetz et al., 2006; Ruthenburg internal organs and can be associated with a severe form of CHD et al., 2006; Couture et al., 2006). Point mutations, for example, in the (Sutherland and Ware, 2009; Amack and Yost, 2010; Hamada et al., arginine-binding cavity of WDR5 (S91K or F133A), can disrupt the 2002). Cilia are well known to be essential for LR patterning (Li H3K4MT complex (Patel et al., 2008b). Interestingly, the patient et al., 2015; Blum et al., 2014; Basu and Brueckner, 2008; mutation (K7Q) lies outside the β-propeller, in the N-terminal tail (first ∼30 amino acids) that is not required for H3K4MT function (Schuetz et al., 2006), making the disease relevance of this mutation uncertain. Pediatric Genomics Discovery Program, Department of Pediatrics and Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. RESULTS AND DISCUSSION *Author for correspondence ([email protected]) Wdr5 depletion alters LR patterning in Xenopus tropicalis S.S.K., 0000-0002-0882-6478; M.K.K., 0000-0002-9846-7076 Previously, in a CHD patient with a right (rather than the normally left) aortic arch, a de novo mutation (K7Q) implicated WDR5 as a

Received 25 September 2017; Accepted 24 October 2018 candidate disease gene (Zaidi et al., 2013). Therefore, we DEVELOPMENT

1 RESEARCH REPORT Development (2018) 145, dev159889. doi:10.1242/dev.159889 investigated whether WDR5 is essential for LR patterning in We investigated multiple potential reasons for cilia defects. First, Xenopus. Depletion of Wdr5 in Xenopus leads to substantial LRO patterning is dependent on proper dorsoventral (DV) pericardial edema that alters the structure of the heart, precluding patterning, so we began by examining DV markers in gastrula accurate determination of its cardiac looping. Therefore, we (stages 10-11) or postgastrula (stage 14) embryos (Fig. S3A,B) examined global markers of LR patterning: pitx2c and dand5 (Khokha et al., 2005; Heasman, 2006; Niehrs et al., 2001). (Fig. S1). Using a wdr5 morpholino oligonucleotide (MO), Wdr5 Interestingly, following Wdr5 depletion, we detected an increase in knockdown led to abnormal patterns of both pitx2c (∼55%) and expression in dkk1, a known WNT protein antagonist (Glinka et al., dand5 (∼40%) (Fig. 1A,B). To examine the specificity and 1998). Previous studies have shown that the transcriptional efficiency of our Wdr5 depletion, we employed multiple tests. First regulation of some WNT regulators and target genes is dependent by western blotting, we previously showed that Wdr5 protein is on WDR5; thus, expansion of dkk1 in our wdr5 morphants supports reduced in morphants, which is rescued by injecting human wild-type these studies (Zhu et al., 2008; Gori et al., 2006). However, we did (WT) WDR5 mRNA (Kulkarni et al., 2018). In addition, we not observe any change in the expression of other key DV patterning demonstrated that the anti-WDR5 antibody was specific using a serial genes, including foxj1, at gastrula stage. Second, although DV dilution of a blocking peptide that reduced the WDR5 signal on the patterning appears generally normal at the gastrula stages, we western blot (Kulkarni et al., 2018). Second, injection of a scrambled considered the possibility that the LRO itself was mispatterned. MO does not alter pitx2c expression compared with that of uninjected Prior to the onset of cilia-mediated flow (stage 16), dand5 controls (Fig. 1C). Third, we can partially rescue alterations in pitx2c expression is symmetric at the lateral margins, which is expression in Wdr5-depleted embryos by co-injecting WT human unchanged in control or Wdr5-depleted embryos (Fig. S3C,F). WDR5 mRNA tagged with 3xGFP (Fig. 1D). Fourth, the depletion of Further, we checked the expression of xnr1, which is also symmetric Wdr5 using a second ATG MO also led to abnormal patterns of at the lateral margins at these stages. It was also similar between pitx2c (Fig. 1E). Together, we conclude that the Wdr5 depletion by controls and morphants, demonstrating that LRO cell fate MO is specific and is essential for global LR patterning. specification was not drastically affected in morphants (Fig. S3C,F). Disruptions in both dand5 and pitx2c point to a defect in the Mispatterning of the LR axis could also arise from defects in LRO, possibly due to cilia-mediated signaling (Hamada et al., 2002; establishing the midline barrier. Therefore, we checked the Doerks et al., 2002; Schweickert et al., 2007). Previous studies have expression of sonic hedgehog (shh) and lefty at the midline and shown that cilia motility on the left but not on the right side of the found that expression was unaltered between controls and LRO is crucial for proper LR patterning (Vick et al., 2009). Indeed, morphants (Fig. S3D,F). Based on these results, we conclude that left-sided wdr5 knockdown led to a higher percentage of abnormal the LRO in wdr5 morphants is normally patterned overall and expression patterns for both LR markers (dand5 ∼80% and pitx2c sought other explanations for the defects in cilia. ∼70%) compared with knockdown on the right (dand5 ∼12% and pitx2c ∼12%), an effect not seen with scrambled MO injection Wdr5 regulates LR patterning via transcriptional regulation (Fig. 1A,B,D). These results suggest a ciliary role for Wdr5 in LR of foxj1 development. Given the established role for WDR5 as a chromatin modifier, we next examined the mRNA expression of three key cilia-specific genes WDR5 (K7Q) is a loss-of-function allele in the LRO: dnah9, rfx2 and foxj1 (Chung et al., 2012; Yu et al., The WDR5 patient had a de novo K7Q missense mutation; however, 2008; Stubbs et al., 2008; Vick et al., 2009). We did not detect a whether this variant is detrimental to function was unclear. To test difference in dnah9 and rfx2 expression (Fig. S3E,F); however, foxj1 this hypothesis, we attempted to rescue the pitx2c phenotype in expression appeared reduced in the LRO of morphants compared wdr5 morphants. In contrast to the WT protein, the K7Q-WDR5 with controls (Fig. 3A,B, black arrows). Therefore, we tested the variant failed to rescue pitx2c patterning in wdr5 morphants, possibility that Wdr5 regulates LRO cilia via foxj1 transcription using supporting the hypothesis that it is a loss-of-function allele (Fig. 1F) a rescue experiment. We injected foxj1 mRNA into Wdr5-depleted (Zhu et al., 2017). Interestingly, the first 30 amino acids are embryos, which partially rescued the pitx2c defects (Fig. 3C), dispensable for forming the β-propeller structure of WDR5 and supporting our hypothesis. Our results are consistent with multiple assembling a functional H3K4MT complex (Schuetz et al., 2006). different findings: (1) the Foxj1 locus is methylated (Mikkelsen et al., Therefore, we wondered whether this allele might suggest a role 2007), (2) global chromatin immunoprecipitation sequencing independent of chromatin modification. identifies the Foxj1 locus as a target of WDR5, RbBP5 and H3K4m3 (Ang et al., 2011), and (3) Foxj1 is an established regulator Wdr5 is essential for cilia in the LRO of monocilia (Yu et al., 2008; Stubbs et al., 2008). LR patterning is established at the LRO. Because we see defects in dand5 expression, an immediate downstream target of cilia Wdr5 is expressed in the nuclei and at the bases of LRO cilia signaling, we decided to examine cilia morphology in the LRO of Because Wdr5 regulates foxj1 transcription in the LRO, we decided wdr5 morphants. Using immunofluorescence, we observed that to confirm that Wdr5 is localized to the nuclei of the LRO. cilia in morphants were abnormal compared with those in WT Unfortunately, although our anti-WDR5 antibody detects Xenopus embryos (Fig. 2A). Specifically, in wdr5 morphants, the cilia were Wdr5 protein by western blotting, we could not detect Wdr5 in the shorter and fewer in number (Fig. 2B,C). We confirmed our results Xenopus LRO using immunofluorescence. Therefore, we used a with scanning electron microscopy (Fig. S2). In addition, we WDR5-3xGFP construct to examine its localization. We note that measured cilia polarity in the LRO according to established WDR5-3xGFP can partially rescue the wdr5 depletion phenotype methods (Walentek et al., 2013). Normally, the LRO monocilia (Fig. 1D), suggesting that it is properly localized and functioning. are posteriorly polarized, which is essential for establishing We injected WDR5-3xGFP in Xenopus embryos at the one-cell leftward flow. Depletion of Wdr5 mildly affected cilia polarity stage and, as expected, WDR5-3xGFP was localized to the nuclei of (Fig. 2D). These results suggest that Wdr5 is essential for cilia in the LRO cells (Fig. 3D, GFP channel imaged at the level of the the LRO. nucleus). However, to our surprise, we found that WDR5-3xGFP DEVELOPMENT

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Fig. 1. Wdr5 depletion alters LR patterning in Xenopus tropicalis. (A,B) Percentage of embryos that have abnormal pitx2c (A) or dand5 (B) expression. Embryos were injected at the one-cell stage, or in one cell at the two-cell stage, with wdr5 MO. Absent, signal absent on both sides; bilateral, signal present on both sides; L, left; R, right; Reduced: signal reduced on both sides. (C-G) Percentage of embryos that have abnormal pitx2c expression. Embryos were injected at the one-cell stage with scrambled MO (C); wdr5 MO, wdr5 MO+human WDR5-3xGFP, scrambled MO injected on the right or left side of embryos (D); second wdr5 MO (E); wdr5 MO, wdr5 MO+K7Q-WDR5-3xGFP or K7Q-WDR5-3xGFP (F); or wdr5 MO and wdr5 MO+human 26-334-3xGFP (G). Experiments were repeated three times. ‘n’=number of embryos. ★P<0.05, ★★P<0.005 and ★★★P<0.0005. was also localized to the bases of cilia (Fig. 3E, GFP channel WDR5 has an H3K4-independent role in LR patterning imaged at the apical surface). To see whether this finding was Our unexpected discovery of WDR5 at the bases of monocilia generalized to mammalian monociliated cells, we tested WDR5 suggested a chromatin-independent role, which might also be localization in human retinal pigmented epithelium (hRPE) cells consistent with a deleterious effect of the K7Q allele. To test this using immunofluorescence. Indeed, WDR5 localized to γ-tubulin, a hypothesis, we decided to employ the S91K WDR5 mutant, which component of the basal body in hRPE cells (Fig. 3G), the specificity disrupts binding to MLL and leads to abrogation of H3K4MT activity of which we confirmed using a blocking peptide (Fig. S4). (Patel et al., 2008b). Consistent with a chromatin-independent role, DEVELOPMENT

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Fig. 2. Wdr5 depletion affects cilia morphology in the LRO of X. tropicalis. (A) Cilia in control and wdr5 morphant Xenopus LROs marked by anti-acetylated (Ac.) α-tubulin (green) antibody and F-actin marked by phalloidin (red). (B,C) Number of cilia normalized to the LRO area (B) and length of cilia (C) in uninjected controls and wdr5 morphants. Data are presented as box plot with 95% confidence interval. ‘n’=number of embryos. (D) Number of cilia posteriorly localized in the LRO cells in uninjected controls and wdr5 morphants. Experiments were repeated two times. ★P<0.05 and ★★★P<0.0005. the S91K WDR5 allele partially rescued pitx2c expression, possibly Fig. 4). However, the localizations of these constructs appear more via its localization at the bases of cilia in the LRO (Fig. 3F). diffuse than the full-length constructs, suggesting that an intact β-propeller is required for precise localization of WDR5 to the The β-propeller structure of WDR5 is sufficient for nuclear bases of cilia. On the other hand, the C-terminal constructs (118- but not ciliary localization 334 and 202-334) fail to localize to the bases of cilia or the In ciliogenesis and LR patterning, we found a chromatin-dependent nucleus, producing a diffuse pattern across much of the cell, and a chromatin-independent role for WDR5 that correlates with suggesting that the N-terminal part of the protein is crucial for protein localization. Therefore, we sought to identify any protein localization to the cilia bases. Of note, the construct 118-201 also domains within WDR5 that might define these two different localizes to the bases of cilia, albeit in a more diffuse pattern than localizations. To study protein localization, we made different that of the full-length construct. However, 118-334 does not deletion constructs tagged with 3xGFP and expressed them in the localize to the cilium base, suggesting that the C-terminal end Xenopus LRO (Fig. 4). First, we deleted amino acids 1-26, which might contain a repressive motif for cilia localization. From these contains the patient variant but is dispensable for H3K4MT activity data, we conclude that the N-terminal 26 amino acids (that include (construct 26-334-3xGFP). As expected, the 26-334-3xGFP construct the patient variant) are required for precise cilium localization, partially rescues the pitx2c defect when wdr5 is depleted, indicating although the relationship between ciliary localization and protein that it is functional via the H3K4MT pathway (Fig. 1G) (Schuetz et al., domains appears complex. Interestingly, different parts of WDR5 2006). We also divided WDR5 into five more constructs: 1-117 (first can localize to the cilium base independently of nuclear and second WD repeats), 118-201 (third and fourth WD repeats), 1- localization. 201 (one to four WD repeats), 118-334 (three to seven WD repeats) We also examined the localization of the S91K mutant, which and 202-334 (five to seven WD repeats) (Fig. 4). To begin, we cannot participate in H3K4MT activity, and the patient’s mutation examined nuclear localization. Of the different constructs, only the (K7Q) in the ciliated cells of the LRO (Fig. 4). As predicted, WT-3xGFP and the 26-334-3xGFP localized to the nucleus, localization of the S91K variant to the bases of cilia was similar to suggesting that the whole β-propeller is essential for nuclear that shown by WT-WDR5 (Fig. 4). In addition, the S91K variant localization and confirming that the first 26 N-terminal amino acids also localized to the nucleus, confirming that its effect on H3K4MT are not (Schuetz et al., 2006). At the bases of cilia, the WT-3xGFP activity is via assembly of the H3K4MT complex rather than nuclear localized as sharp puncta, whereas the 26-334-3xGFP had a more localization. Interestingly, the K7Q variant did not precisely localize diffuse localization. The GFP signal appeared more dispersed, to the bases of cilia. The K7Q localization was even more diffuse suggesting that the β-propeller is not sufficient for precise localization than that of the 26-334 construct, suggesting that the K7Q missense at the bases of cilia. This is specifically interesting because the mutation might interfere with the ability of WDR5 to localize to the patient’s missense mutation K7Q lies in the N-terminal tail (first 25 bases of LRO cilia. base pairs) of WDR5, which is dispensable for the assembly of In conclusion, our results emphasize the importance of patient- H3K4MT complex but required for precise ciliary localization (see driven gene discovery tightly coupled with developmental biology, below). Therefore, it is possible that that patient phenotype arises which together can be a powerful strategy to elucidate disease from the function of WDR5 at the bases of cilia, rather than its role in mechanisms. Recent studies in CHD and autism clearly point to a the assembly of a H3K4MT complex. role for chromatin modifiers in disease pathogenesis, but the In general, N-terminal constructs that include the first 25 amino molecular mechanisms remain unclear (De Rubeis et al., 2014; acids localize to the bases of cilia (including 1-117 and 1-201, Zaidi et al., 2013; Carneiro et al., 2011). In our search for the DEVELOPMENT

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Fig. 3. WDR5 has chromatin-dependent and -independent roles in ciliogenesis in the LRO. (A) In situ hybridization for foxj1 in the Xenopus LRO in uninjected controls and wdr5 morphants at stage 16. Embryos were bisected to reveal expression of foxj1 in the entire GRP (left column, dorsal to the top, ventral view so right margin of the LRO is to the left; the margin of the GRPs is marked by white lines). GRPs were again bisected within the LRO (dashed white lines) to reveal foxj1 expression within the tissue [right column, dorsal to the top; arrows mark the ventral surface (indicated by the dashed white line boxes at the bottom) where the LRO resides, showing loss of foxj1 in the ventral tissue]. A, anterior; D, dorsal; L, left; P, posterior; R, right; V, ventral. (B) Percentage of embryos that have abnormal foxj1 expression in control and wdr5 morphant Xenopus LROs. (C) Percentage of embryos that have abnormal pitx2 expression. Embryos were injected at one-cell stage with wdr5 MO or wdr5 MO+Xenopus foxj1 mRNA. (D) A Z-stack confocal image of Xenopus LRO, visualizing WDR5-3xGFP (green) and cilia (red). The green channel is taken at the plane of the nuclei; the red channel is taken at the apical plane to reveal cilia. Cilia are detected using Arl13b-mCherry expression. For WDR5, the look-up table (LUT) is green; for Arl13b-mCherry, the LUT is red. (E) A Z-stack confocal image of the Xenopus LRO visualizing WDR5-3xGFP (green) and cilia (red). Both the green and red channels are taken at the apical surface of the Xenopus LRO cells, showing WDR5-3xGFP localizing at the ciliary bases. Cilia are marked with Arl13b-mCherry. For WDR5-3xGFP, the LUT is green; for Arl13b-mCherry, the LUT is red. (F) Percentage of embryos that have abnormal pitx2 expression. Embryos were injected at one-cell stage with wdr5 MO+WT WDR5 mRNA or wdr5 MO+S91K-WDR5 mRNA. (G) WDR5 localizes to the ciliary base in hRPE cells. Cilia and basal bodies are marked with anti-acetylated (Ac.) α-tubulin and γ- tubulin antibodies, respectively. For Ac. α-tubulin, the LUT is yellow; for WDR5, the LUT is red; for γ-tubulin, the LUT is magenta. Experiments were repeated three to four times. ‘n’=number of embryos. ★P<0.05 and ★★★P<0.0005. underlying molecular mechanism for WDR5 in CHD using MATERIALS AND METHODS Xenopus, we uncovered an H3K4-dependent, and an unexpected Animal husbandry H3K4-independent, cilia role in the LRO. Importantly, this cilia X. tropicalis were housed and cared for in our aquatics facility according to phenotype could have important clinical implications for patient established protocols that were approved by the Yale Institutional Animal management. Care and Use Committee. DEVELOPMENT

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Fig. 4. Domain analysis of WDR5. Localization of different deletion constructs for WDR5-3xGFP at the ciliary base and nuclei of the Xenopus LRO. For WDR5-3xGFP, the LUT is green; for Arl13b-Cherry, the LUT is hot magenta.

Cell culture into one-cell or two-cell embryos as described previously (Khokha et al., hRPE cells were obtained from the Brueckner laboratory of Yale School of 2002). The following MOs were injected: wdr5 translation blocking Medicine and were contamination free. Cells were cultured in Dulbecco’s (2.5-4 ng 5′-CGGGTTTCTTTTCTTCTGTTGCCAT-3′), second wdr5 modified eagle medium/Ham’s F12 medium supplemented with 10% fetal translation blocking (15 ng/embryo 5′-TGCAAGAACAACTTGTGGCC- bovine serum. GGATA-3′) and scrambled control MO (4 ng 5′-CCTCTTACCTCAGTT- ACAATTTATA-3′). Alexa Fluor 488 (Invitrogen), mini-ruby (Invitrogen) or Microinjection of MOs and mRNA in Xenopus GFP (100 pg) were injected as tracers. We generated in vitro capped mRNA Embryos were produced by in vitro fertilization and raised to appropriate using a mMessage mMachine Kit (Ambion) and followed the stages in 1/9×MR+gentamycin according to established protocols (Khokha manufacturer’s instructions. Full-length human WDR5 was obtained et al., 2002; del Viso and Khokha, 2012). Staging of Xenopus tadpoles was from the IMAGE consortium collection (Thermo Fisher Scientific as previously described (Nieuwkoop, 1994). MOs or mRNA were injected IMAGE clone 3538255) and subcloned into the pCSDest vector using DEVELOPMENT

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Gateway recombination techniques. We generated the K7Q mutation using Supplementary information PCR amplification and cloning into PCS2 vector. We injected 400 pg Supplementary information available online at human WT-WDR5, WDR5-GFP, WDR5-S91K, WDR5-3xGFP, WDR5- http://dev.biologists.org/lookup/doi/10.1242/dev.159889.supplemental K7Q-3xGFP, WDR5-26-334-3xGFP and 150 pg Xenopus foxj1 RNA for rescue of wdr5 morphants. Mutant constructs for domain analysis were References Amack, J. D. and Yost, H. J. (2010). Cardiac left–right asymmetry. In Heart generated using PCR amplification and cloning into PCS2 vector. Human WDR5 Development and Regeneration (ed. N. Rosenthal and R. P. Harvey), WT and the mutant constructs of were tagged with 3xGFP and were pp. 281-293. San Diego: Academic Press. injected (200 pg) into one-cell embryos to mark WDR5 in the LRO. We Ang, Y.-S., Tsai, S.-Y., Lee, D.-F., Monk, J., Su, J., Ratnakumar, K., Ding, J., Ge, injected 200 pg Arl13b-Cherry to mark LRO cilia. Y., Darr, H., Chang, B. et al. (2011). Wdr5 mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network. Cell RNA in situ hybridization 145, 183-197. X. tropicalis embryos were collected at various stages and in situ Basu, B. and Brueckner, M. (2008). Cilia multifunctional organelles at the center of vertebrate left-right asymmetry. Curr. Top. Dev. Biol. 85, 151-174. hybridization was performed according to standard protocols (Khokha foxj1 wnt8 dkk1 xnr3 otx2 Blum, M., Schweickert, A., Vick, P., Wright, C. V. and Danilchik, M. V. (2014). et al., 2002). We collected stage 10 embryos for , , , , Symmetry breakage in the vertebrate embryo: when does it happen and how does and myf5 expression, and stage 14 embryos for sox2 expression. For GRPs, it work? Dev. Biol. 393, 109-123. we collected stage 16 embryos for dand5, foxj1, shh, dnah9, xnr1 and rfx2, Boskovski, M. T., Yuan, S., Pedersen, N. B., Goth, C. K., Makova, S., Clausen, stage 19-21 for dand5 and lefty, and stage 28 for pitx2. GRPs were dissected H., Brueckner, M. and Khokha, M. K. (2013). The heterotaxy gene GALNT11 as described previously (Schweickert et al., 2007). Additional probe glycosylates Notch to orchestrate cilia type and laterality. Nature 504, 456-459. Burdine, R. D. and Schier, A. F. (2000). Conserved and divergent mechanisms in information is available upon request. left-right axis formation. Genes Dev. 14, 763-776. Cao, F., Chen, Y., Cierpicki, T., Liu, Y., Basrur, V., Lei, M. and Dou, Y. (2010). An Image analysis Ash2L/RbBP5 heterodimer stimulates the MLL1 methyltransferase activity Images were captured using a Zeiss 710 Live confocal microscope. Images through coordinated substrate interactions with the MLL1 SET domain. PLoS were processed in Fiji (National Institutes of Health), ImageJ (National ONE 5, e14102. Institutes of Health) or Adobe Photoshop. Quantification of GRP cilia Capdevila, J., Vogan, K. J., Tabin, C. J. and Izpisúa Belmonte, J. C. (2000). – length was performed using Volocity (Quorum Technologies) software on Mechanisms of left right determination in vertebrates. Cell 101, 9-21. Carneiro, K., Donnet, C., Rejtar, T., Karger, B. L., Barisone, G. A., Dıaz,́ E., 3D image stacks. The number of GRP cilia was measured using the Kortagere, S., Lemire, J. M. and Levin, M. (2011). Histone deacetylase activity is ‘Analyze particle’ function in Fiji. Final figures were made in Adobe necessary for left-right patterning during vertebrate development. BMC Dev. Biol. Illustrator. Cilia polarity was measured using previously described methods 11, 29. (Walentek et al., 2013). Chung, M.-I., Peyrot, S. M., Leboeuf, S., Park, T. J., Mcgary, K. L., Marcotte, E. M. and Wallingford, J. B. (2012). RFX2 is broadly required for ciliogenesis during Statistical analysis vertebrate development. Dev. Biol. 363, 155-165. Sample size (n) is defined as the number of embryos. Statistical analysis Couture, J.-F. and Skiniotis, G. (2013). Assembling a COMPASS. Epigenetics 8, 349-354. was performed using GraphPad Prism, JMP (SAS) and VassarStats Couture, J.-F., Collazo, E. and Trievel, R. C. (2006). Molecular recognition of (VassarStats.net) software. pitx2c, dand5, rfx2, dnah9, foxj1, xnr1, lefty histone H3 by the WD40 protein WDR5. Nat. Struct. Mol. Biol. 13, 698-703. and shh were compared using Chi-square analysis. All other comparisons Del Viso, F. and Khokha, M. (2012). Generating diploid embryos from Xenopus were made using Student’s t-tests after confirming the normal distribution of tropicalis. Methods Mol. Biol. 917, 33-41. the data. We randomly picked one-cell X. tropicalis embryos from De Rubeis, S., He, X., Goldberg, A. P., Poultney, C. S., Samocha, K., Cicek, fertilization as uninjected controls or for MO or RNA injections. A. E., Kou, Y., Liu, L., Fromer, M., Walker, S. et al. (2014). 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Development 133, 1205-1217. for the C-3xGFP construct; and Dr Michael Cosgrove for the human WT and S91K- Homsy, J., Zaidi, S., Shen, Y., Ware, J. S., Samocha, K. E., Karczewski, K. J., WDR5 constructs. Depalma, S. R., Mckean, D., Wakimoto, H., Gorham, J. et al. (2015). De novo Competing interests mutations in congenital heart disease with neurodevelopmental and other congenital anomalies. Science 350, 1262-1266. The authors declare no competing or financial interests. Khokha, M. K., Chung, C., Bustamante, E. L., Gaw, L. W., Trott, K. A., Yeh, J., Lim, N., Lin, J. C., Taverner, N., Amaya, E. et al. (2002). Techniques and probes Author contributions for the study of Xenopus tropicalis development. Dev. Dyn. 225, 499-510. Conceptualization: S.S.K., M.K.K.; Methodology: S.S.K.; Validation: S.S.K.; Formal Khokha, M. K., Yeh, J., Grammer, T. C. and Harland, R. M. (2005). Depletion of analysis: S.S.K.; Investigation: S.S.K.; Data curation: S.S.K.; Writing - original draft: three BMP antagonists from Spemann’s organizer leads to a catastrophic loss of S.S.K.; Writing - review & editing: S.S.K., M.K.K.; Visualization: S.S.K.; Supervision: dorsal structures. Dev. Cell 8, 401-411. M.K.K.; Project administration: M.K.K.; Funding acquisition: S.S.K., M.K.K. Kulkarni, S. S., Griffin, J. N., Date, P. P., Liem, K. F., Jr. and Khokha, M. K. (2018). WDR5 stabilizes actin architecture to promote multiciliated cell formation. Dev. Funding Cell 46, 595-610.e3. This work was supported by the National Heart, Lung, and Blood Institute (NHLBI) Li, Y., Klena, N. T., Gabriel, G. C., Liu, X., Kim, A. J., Lemke, K., Chen, Y., [Pilot Project as part of 5U01HL098188; K99/R00-5K99HL133606-02 to S.S.K.; Chatterjee, B., Devine, W., Damerla, R. R. et al. (2015). Global genetic analysis R33HL120783 to M.K.K.] and the National Institute of Child Health and Human in mice unveils central role for cilia in congenital heart disease. Nature 521, Development [R01HD081379 to M.K.K.]. M.K.K. is an Edward Mallinckrodt, Jr. 520-524. Foundation Scholar. The contents of this publication are solely the responsibility of the Lin, C. R., Kioussi, C., O’connell, S., Briata, P., Szeto, D., Liu, F., Izpisua- authors and do not necessarily represent the official views of the NHLBI. Deposited in Belmonte, J. C. and Rosenfeld, M. G. (1999). Pitx2 regulates lung asymmetry,

PMC for release after 12 months. cardiac positioning and pituitary and tooth morphogenesis. Nature 401, 279-282. DEVELOPMENT

7 RESEARCH REPORT Development (2018) 145, dev159889. doi:10.1242/dev.159889

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