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Lnx2b, an E3 ubiquitin ligase, in dorsal forerunner cells and Kupffer's vesicle is required for specification of zebrafish left–right laterality Lnx2b, an E3 ubiquitin ligase, in dorsal forerunner cells and Kupffer's vesicle is required...
Kim, Min Jung; Rhee, Myungchull; Ro, Hyunju
2014-09-03 00:00:00
DEVELOPMENTAL BIOLOGY Animal Cells and Systems, 2014 Vol. 18, No. 5, 333–339, http://dx.doi.org/10.1080/19768354.2014.968205 Lnx2b, an E3 ubiquitin ligase, in dorsal forerunner cells and Kupffer’s vesicle is required for specification of zebrafish left–right laterality a b* b* Min Jung Kim , Myungchull Rhee and Hyunju Ro a b Department of Biological Sciences, Sookmyung Women’s University, Seoul 140-742, Republic of Korea; Department of Biological Sciences, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea (Received 18 September 2014; accepted 18 September 2014) The establishment of left–right (LR) axis in zebrafish embryos relies on numerous genes expressed in the cluster of dorsal forerunner cells (DFCs) that form Kupffer’s vesicle (KV), the transient cilia-rich organ with functional similarity to mouse node in the case of LR axis determination. Even though several genes in the DFCs and KV have been identified to be implicated in LR body patterning so far, the underlying regulatory mechanisms in particular dependent upon ubiquitin (Ub)– proteasome system have not yet been identified. In this study, we report that Lnx2b, a RING domain containing E3 Ub ligase, specifically expressed in migratory DFCs and developing KV, plays a critical role in the establishment of LR laterality. Depletion of Lnx2b using antisense morpholino oligonucleotides (MOs) inhibited the left-side biased expression of southpaw and resulted in the randomization of the heart jogging and looping in zebrafish embryos. A DFCs-specific Lnx2b loss of function approach showed that the randomization of LR patterning caused by the depletion of Lnx2b was not simply due to the early dorsoventral body patterning defects or the MO toxicity, but the loss of its function in the DFCs and KV. Collectively, our data showed that Lnx2b is the first analyzed E3 Ub ligase, which is involved in LR laterality during zebrafish embryogenesis. Keywords: zebrafish; LR asymmetry; dorsal forerunner cells; Kupffer’s vesicle; Lnx2b Introduction axis specification. In zebrafish, motile cilia reside in Kupffer’s vesicle (KV), the functional equivalent of the The complexity of multicellular organism arises from mammalian node. Similar to the mouse node, KV a fluid- mechanisms that establish different body axis through filled organ with motile cilia, is a transiently formed consecutive events during early embryonic development. during early somitogenesis (Essner et al. 2005; Matsui & In spite of individual random variations, the external Bessho 2012). In zebrafish, a group of non-involuting bilateral symmetry of the vertebrate is nearly immaculate. dorsal forerunner cells (20~30 DFCs), which locates However, various internal organs such as the heart, the adjacent to the embryonic shield, migrates toward the stomach, the pancreas, and spleen are asymmetrically vegetal pole to form the KV at the end of gastrulation localized; interestingly, laterality defects affect more than stage (tail bud stage; Cooper & D’Amico 1996; Matsui & 1 in 8000 live human births (Peeters & Devriendt 2006). Bessho 2012). Since the defects in either DFCs/KV There are currently two plausible models which explain specification or abnormal ciliogenesis in the KV caused how the initial symmetry of the vertebrate breaks. The failures of LR asymmetry patterning at later develop- first is termed as the “morphogen hypothesis”. It suggests mental stages (Matsui & Bessho 2012), the genes critical that directional beating of the nodal cilia leads to a for the specification and the maintenance of the function unidirectional transport of morphogens to the left side of of DFCs/KV must have been tightly regulated during KV node (Nonaka et al. 1998; Okada et al. 1999; Tanaka et al. organogenesis. 2005; Raya & Izpisúa Belmonte 2006, Babu & Roy Ubiquitin (Ub) is a small protein highly conserved 2013). The other is the “two-cilia hypothesis”, which from yeast to human. Covalently linked Ub chain argued that the beating of motile cilia on pit cells creates a outgrowth can be initiated on the lysine residues in leftward fluid flow whose local fluid current can be substrate molecules through consecutive enzymatic reac- detected by immotile cilia on the perinodal crown cells tions in which Ub is activated by E1-activating enzyme, (Pennekamp et al. 2002; McGrath et al. 2003; Raya & transferred to an E2-conjugating enzyme and then cova- Izpisúa Belmonte 2006, Babu & Roy 2013). Those two lently attached on the substrate via an isopeptide bond hypotheses are based upon the function of cilia on the selected by an specific E3 Ub ligase (Deshaies & Joazeiro node in mammals or its functional equivalent in teleost. 2009; Anuppalle et al. 2013; Skaar et al. 2013). Even The zebrafish embryo is an excellent system to study though the Ub-conjugating system was initially reported the molecular processes that govern the left–right (LR) as part of an energy dependent protein destruction system, *Corresponding authors. Email: mrhee@cnu.ac.kr; rohyunju@cnu.ac.kr © 2014 Korean Society for Integrative Biology 334 M.J. Kim et al. ubiquitylation was also involved in non-proteolytic roles, Morpholino microinjection such as endocytosis, signal transduction, and DNA repair Morpholino oligonucleotides (MOs) purchased from (Welchman et al. 2005; Kirkin & Dikic 2007). Although Gene Tools, LLC were dissolved and diluted in Diethyl Ub dependent post-translational modification is undoubt- pyrocarbonate (DEPC)-treated 0.1 M KCl solution. edly important for the vertebrate body axis specification, Approximately 2.5 nl of MO dissolved solution was so far the studies on E3 Ub ligases involved in LR axis pressure injected into the yolk of 1–4 cell or 256–1K orientation have been very limited. cell stage embryo. p53 MO was co-injected with other Previously, we found that a multi-Post synaptic density MOs to inhibit possible off-target effects (Robu et al. protein, Drosophila disc large tumor suppressor and Zonula 2007). The followings represent the sequence of MOs used in our assay: occludens-1 protein (PDZ) domain-containing E3 Ub ligase Lnx2b (zebrafish homologue of Ligand of Numb lnx2b UTR MO, 5′-CCTACGCCTCTTTCACAGCTCA Protein X-2; Nie et al. 2002) is involved in dorsoventral CAA-3′ (DV) as well as anterior–posterior axis specification via lnx2b splicing MO, 5′-GTAAGTGATGCAATACCATCT destabilizing Bozozok transcriptional repressor or, by TCGC-3′ acting as a molecular scaffold, stabilizing canonical Wnt p53 MO, 5′-GCGCCATTGCTTTGCAAGAATTG-3′. transcriptional repressor complex through the recruitment of corepressor proteins to TCF3 (Ro & Dawid 2009, 2010, RT-PCR 2011). In this study, we propose that Lnx2b specifically First strand of cDNA was generated with 3 µg of total expressed in migratory DFCs and developing KV plays an RNA from embryos at tail bud stage using the Superscript important role in the establishment of LR axis. Depletion of First Strand Synthesis System (Invitrogen). The following Lnx2b in the DFCs/KV using morpholinos caused rando- primers were used for amplifying lnx2b fragment from the mized expression of southpaw (spaw) and resulted in heart first-strand cDNA: forward primer, 5′-CTGGATTGGAA looping and jogging defects. To our knowledge, Lnx2b is ATATTTGGTTGCTTCTCATC-3′ and reverse primer, 5′- the first identified E3 Ub ligase, which is not only CCAAATCCTCGCCTGGATCTTCACG-3′. specifically expressed in DFCs/KV but also involved in LR asymmetry patterning. Results and discussion Lnx2b is expressed in migrating DFCs as well as Materials and methods developing KV Zebrafish embryos Previously we revealed that lnx2b is expressed in the Embryos were obtained by natural spawning from wild- caudal domain during early zebrafish embryogenesis (Ro & Dawid 2009). To address the detailed expression pattern type (WT) (AB*) fish line. of lnx2b, whole mount in situ hybridization was carried out to characterize the lnx2b expression domain more precisely. As shown in Figure 1A and B, ventral-to-dorsal Whole mount in situ hybridization graded lnx2b transcripts were detected with strong expres- The embryos were collected at the indicated stage and sion in the migrating DFCs at the late gastrula stage of fixed for 8–10 hours in 4% paraformaldehyde in phos- zebrafish embryos (arrowhead in Figure 1B, 80% epi- phate buffered saline (PBS) and then dechorionated using body). At the tail bud stage, the lnx2b was expressed in syringe needle. Digoxigenin-labeled antisense riboprobes the KV besides the central lumen (Figure 1B and C). The (lnx2b, cardiac myosin light chain 2 [cmlc2], and spaw) stereotypical horseshoe like expression pattern of lnx2b were synthesized with linearized template DNA using around the anterior KV was observed at 5-somite stages appropriate RNA polymerase following the manufac- (Figure 1D and E), and the expression pattern was turer’s instructions (Roche). Proteinase K treatment maintained similarly until 10-somite stages (Figure 1G (10 µg/ml) was performed for 1–3 min depending on and H). It should be noted that although the expression embryonic stages. The hybridized riboprobes were pattern of lnx2b in KV is similar to that of genes that were detected using pre-absorbed anti-digoxigenin-AP Fab previously reported to be critical in LR body patterning, fragments (Roche) diluted (1:2000) in blocking solution such as spaw and charon, it takes on the inverse (PBS, 0.1% Tween-20, 5% sheep serum, and 0.2% orientation (Long et al. 2003; Hashimoto et al. 2004). blocking reagent [Roche]). After staining with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/ Lnx2b is required for LR laterality BCIP) substrate (Roche), the embryos were dehydrated in 100% MeOH for 10 min, rinsed in PBST (PBS + 0.1% Since we identified enriched expression of lnx2b in the Tween-20), and then examined under a microscope. DFCs/KV, we set out to test whether the Lnx2b is required Animal Cells and Systems 335 other lnx2b targeting MOs, relatively small amounts of UTR MO (2.5 ng) were sufficient to completely inhibit protein production (Ro & Dawid 2009). To analyze the laterality information in lnx2b morphants, we initially evaluated the expression pattern of cmlc2 which is specifically expressed in the developing heart (Yelon et al. 1999). In uninjected control embryos, the heart is jogged in the left side at 26 hpf (hour post fertilization) with 100% penetrance (26/26, Figure 2A). However, the heart localization became randomized in the lnx2b UTR MO (2.5 ng) injected embryos, of which 40% also showed cardia bifida or loss of cmlc2 expression (left jogging, 6/35; right jogging, 4/35; no jogging, 11/35; cardia bifida, 6/35; no expression, 8/35; Figure 2B–C). The early cardiac laterality defects observed in the lnx2b morphants resulted in the later alteration of cardiac looping (normal looping, 12/64; reverse looping, 8/64; no looping, 33/64; cardia bifida, 3/64; no expression, 8/64; Figure 2E–H). Even though the data revealed that Lnx2b plays an important role in modulating normal cardiac jogging and looping, it is unclear whether the heart laterality defects of the morphants were caused by early alteration of LR asymmetry cues, like spaw laterality in lateral plate mesoderm (LPM) during early somitogenesis stage from the depletion of functional Lnx2b. Since the hallmark of LR patterning is the existence of Nodal transcripts in the left LPM, which is evolutionally well conserved in all vertebrates that have been analyzed so far, we examined the expression pattern of spaw which Figure 1. lnx2b transcripts are detected in migrating DFCs and belongs to the nodal-related factors and is expressed only KV. (A) The level of lnx2b transcripts shows dorsal-to-ventral in the left LPM during early to late zebrafish somitogen- gradient at 80% epiboly stage. Lateral view, dorsal is right. (B) esis (Matsui & Bessho 2012). Nodal flow mediated by Vegetal view of (A). Arrowhead indicates migrating DFCs. (C) Zebrafish embryo at tail bud stage. Arrowhead indicates KV. spaw has been reported to amplify preexisting laterality Lateral view, anterior is left, dorsal is up. (D) Posterior view of information in zebrafish embryos (Raya & Izpisúa Bel- (C). Arrowhead indicates KV. Dorsal is up. (E) Lnx2b transcripts monte 2006; Matsui & Bessho 2012; Babu & Roy 2013). become concentrated in the posterior region at 5-somite stage. Uninjected WT embryos at 15-somite stage displayed the Lateral view, anterior is left, dorsal is up. (F) Posterior view of left LPM biased spaw expression with 100% penetrance (E). Dashed line indicates KV restricted expression pattern of lnx2b. (G) lnx2b expression in the posterior region is still (19/19; Figure 3A). On the contrary, alterations in maintained at 10-somite stage. (H) Posterior view of (H). KV- expression of spaw were observed in the embryos injected specific expression of lnx2b is demarcated by dashed line. Dorsal with high dose of lnx2b UTR MO (2.5 ng) (left biased is up. NT, notochord. Scale bar, 200 μT, expression, 8/32; right biased expression, 6/32; bilateral expression, 8/32; no expression, 10/32; Figure 3B–E). However we could not induce any significant randomized for the LR laterality determination using previously expression of spaw by injecting relatively high dose of SP defined translational- (UTR MO) and splicing blocking MO (10 ng) designed to target the third lnx2b splicing (SP MO) morpholinos targeting Lnx2b (Ro & Dawid donor site (left biased expression, 20/22; right biased 2009). To test the efficacy of the SP MO, reverse expression, 1/22; bilateral expression, 1/22; Figure 3F–H). transcription polymerase chain reaction (RT-PCR) was These unexpected results were presumably due to the carried out on total RNA prepared from tail bud stage remnant of lnx2b mRNA, which survived SP MO treat- embryos injected with different dosage of SP MO. The ment or the contribution of maternally deposited lnx2b amounts of normal lnx2b transcripts from the morphants mRNA. To evaluate the MO efficacy, we coinjected lnx2b were reduced in a SP MO-dose-dependent manner (Figure SP MO (10 ng) with small amount of lnx2b UTR MO 3M). However, we failed to eliminate lnx2b transcripts (0.5 ng) whose quantity was not enough to elicit LR even by injecting high dose of SP MO (10 ng). Since the laterality defects, and found that approximately 26% of efficacy of UTR MO, meanwhile, was much higher than embryos showed undetectable expression of spaw in both 336 M.J. Kim et al. Figure 2. lnx2b morphants show cardiac jogging and looping defect. (A–D) At 26 hpf, the heart was stained with cmlc2 riboprobes. (A) WT embryos showed normal left-jogged heart. (B–D) lnx2b MO (UTR MO, 2.5 ng) injected embryos at 1–4 cell stage exhibited cardiac jogging defect (B, C) including cardia bifida phenotype (D). (E–H) Cardiac looping was analyzed at 60 hpf with cmlc2 riboprobes. (E) WT embryos with normal heart looping. (F–H) There was inversed cardiac looping in various degrees (F, G) or no looping at all (H) in the lnx2b morphants. Scale bar, 200 μm. left and right LPM (8/31) (Figure 3I–L). As such, we observed in the embryos injected with lnx2b MO at 1–2 concluded that the lack of the randomized phenotype in cell stage. We concluded the clearance of spaw expression embryos injected with lnx2b SP MO might be mainly due in both LPM in the lnx2b morphants with the introduction to the inappropriate blockage of splicing event by the of MO at 1–2 cell stage is presumably due to the early DV SP MO. specification defects which are similar to our previous reports (Ro & Dawid 2009, 2010). Collectively these data suggest that the embryonic function of Lnx2b in DFCs Lnx2b in DFCs and KV has critical roles for LR and KV may suppress the expression of spaw in the right laterality determination LPM instead of triggering its expression in the left LPM. Since Lnx2b has functions in early embryonic develop- The canonical Wnt pathway has been implicated in KV mental stage, overall knockdown of Lnx2b led to severe formation and LR asymmetry patterning, as the depletion of embryonic dorsalization with truncation of posterior Wnt3a and Wnt8 in zebrafish induced constricted KV, tissues (Figure 4A and B; Ro & Dawid 2009), preventing shorten cilia and LR patterning defects (Lin & Xu 2009). appropriate evaluation of LR laterality, we introduced However, the Wnt/beta-catenin signaling pathway should lnx2b UTR MO (2.5 ng) specifically into the DFCs be suppressed earlier, during DFC migration, since the (Amack & Yost 2004) to avoid the general developmental ectopic induction of canonical Wnt signaling in the DFCs defects induced by global Lnx2b depletion. The embryos resulted in LR asymmetry defects (Schneider et al. 2008). in which the MO was delivered to the yolk by injection at Recently other reports support this notion that Nkd1, a Wnt/ the 256–1000 cell stages showed generally normal mor- beta-catenin antagonist expressed in migrating DFCs clus- phology at 15-somite stage compared with the morphants DFC ter and around KV, has a critical role in LR laterality by injected with MO at the 1–2 cell stage (UTR MO , DFC limiting Wnt/beta-catenin signaling (Van Raay et al. 2007; Figure 4C–H). Over the 28% of UTR MO injected Schneider et al. 2010). Thus, given the previously identified embryos (17/60) exhibited bilateral expression of spaw, role of Lnx2b as a negative molecular scaffold of canonical while only small portion of the morphants showed the Wnt signaling (Wnt/beta-catenin signaling) and its specific right LPM biased expression of spaw (3.3%, 2/60) without expression in DFCs/KV (Ro & Dawid 2011), it is plausible alteration of the expression in KV (Figure 4C–H). to propose that the Lnx2b in the DFCs/KV plays a key role However, the wiped-out expression of spaw in the LPM DFC for LR asymmetry determination through negatively was not detected in UTR MO injected embryos, as was Animal Cells and Systems 337 Figure 3. Depletion of Lnx2b randomizes spaw expression in the LPM at 15-somite stage. (A) WT embryos with spaw expression (*) in the left LPM. (B–E) lnx2b UTR MO (2.5 ng) injected embryos show randomized, bilateral, or wiped-out expression of spaw in the LPM. (F–H) Randomization of spaw expression in the LPM is not significant in the embryos injected with lnx2b SP MO (10 ng). (I–L) lnx2b SP MO (10 ng) together with low dose of UTR MO (0.5 ng), which alone could not elicit any discernable LR asymmetry defect, increases spaw expression defect in the left LPM. (M) The splicing blocking efficacy of lnx2b SP MO was dosage dependent. At least 10 ng of lnx2b SP MO was required for over 80% blockage of normal lnx2b splicing. Shifted PCR bands were not detected in this experiment, presumably due to the blocking of the splicing events using SP MO causing production of large pieces of DNA fragments not suitable for amplifying through PCR. Dashed lines indicate the midline barrier. Dorsal view, anterior is up, besides J and L. (J, L) Lateral view, anterior is left. Scale bar, 200 ant modulating Wnt signaling outputs. Further studies using phosphotyrosine-binding domain dependent manner (Dho other model organisms may elucidate evolutionary con- et al. 1998; Nie et al. 2002, 2004). Through high-through- served molecular and embryonic function of LNX family put screening, several putative interacting partners of LNX for the LR axis determination. were identified including Nkd2, an Nkd1 homologue The multi-PDZ domain E3 ligase LNX family was (Wolting et al. 2011; Guo et al. 2012). Nkd2, much like initially identified as a Numb interacting protein, and Nkd1, inhibits Wnt/beta-catenin signaling by binding to reported to induce polyubiquitylation and proteosomal and destabilizing Disheveled (Dvl), a highly conserved degradation of specific Numb isoforms (p66 and p77) in a scaffold protein family governing cell fate and cell polarity 338 M.J. Kim et al. Figure 4. Depletion of Lnx2b limited to DFCs/KV suffices to induce LR laterality defects. (A) WT embryos at 5-somite stage. (B) lnx2b morphants (UTR MO, 2.5 ng) show early embryonic dorsalization and truncated posterior tissues. (C) WT embryo at 15-somite stage shows normal left LPM biased spaw expression (*). (D) Lateral view of (C). Anterior is left, dorsal is up. Arrowhead indicates spaw expression in the KV. (E–H) lnx2b UTR MO (2.5 ng) was pressure injected into the yolk at 1K cell stage. Bilateral (E, F) or right LPM biased expression (G, H) of spaw is observed in the lnx2b morphants at 15-somite stage. Note that the spaw expression in the KV is not affected in lnx2b morphant. (F, H). (I) Graphical view of C–H. Dashed lines indicate the midline barrier. Scale bar, 200 μm. through modulating Wnt pathways (Wharton et al. 2001; the present study for the first time demonstrates how the Yan et al. 2001; Schneider et al. 2010). Thus, it would be specific loss of Lnx2b in DFCs/KV affects LR asymmetry interesting to test whether the LR laterality defects caused determination. Further insight into the mechanism of LR axis determination may be gained from uncovering of by Lnx2b depletion is due to the elevated level of Nkd Lnx2b molecular function and its substrate molecules. proteins, which may compromise Wnt signaling in the DFCs/KV by eliminating functional Dvl. Acknowledgement So far, Mahogunin ring finger-1 (MGRN1) is the only We thank Prof. Choi, S-Y (Chonnam National University) for the reported E3 Ub ligase that is involved in LR laterality valuable comments and thorough reading of our manuscript. specification defect in MGRN1 induces multiple embryonic malformation in rodents (Cota et al. 2006; Jiao et al. 2009; Srivastava & Chakrabarti 2014). Although MGRN1 was Funding known to be ubiquitously expressed throughout the entire This work was supported by research fund of Chungnam embryonic developmental process, it is unclear whether the National University. 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