The E3 ubiquitin ligase Hace1 is required for early embryonic development in Xenopus laevis
© The Author(s). 2016
Received: 12 November 2015
Accepted: 25 August 2016
Published: 21 September 2016
HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) regulates a wide variety of cellular processes. It has been shown that one of the targets of HACE1 is the GTP-bound form of the small GTPase Rac1. However, the role of HACE1 in early development remains unknown.
In situ hybridization revealed that Xenopus laevis hace1 is specifically expressed in the ectoderm at the blastula and gastrula stages and in the epidermis, branchial arch, kidney, and central nervous system at the tailbud stage. Knockdown of hace1 in Xenopus laevis embryos via antisense morpholino oligonucleotides led to defects in body axis elongation, pigment formation, and eye formation at the tadpole stage. Experiments with Keller sandwich explants showed that hace1 knockdown inhibited convergent extension, a morphogenetic movement known to be crucial for body axis elongation. In addition, time lapse imaging of whole embryos during the neurula stage indicated that hace1 knockdown also delayed neural tube closure. The defects caused by hace1 knockdown were partly rescued by knockdown of rac1. Moreover, embryos expressing a constitutively active form of Rac1 displayed phenotypes similar to those of embryos with hace1 knocked down.
Our results suggest that Xenopus laevis hace1 plays an important role in early embryonic development, possibly via regulation of Rac1 activity.
KeywordsHACE1 Early development Xenopus laevis
HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) was identified as a gene located in an affected region of chromosome 6q21 in human Wilms’ tumor . It has been shown that HACE1 expression is reduced in multiple human tumors, and forced expression of HACE1 in human tumor cell lines inhibits cell growth in vitro and in vivo . Moreover, Hace1 −/− mice develop spontaneous late-onset cancer . Therefore, HACE1 is considered to be a tumor suppressor gene. HACE1 protein is a HECT family E3 ligase and is known to regulate multiple cellular processes. The most investigated target of HACE1 is Rac1. HACE1 specifically recognizes GTP-bound Rac1, an active form of Rac1, and promotes its degradation through the ubiquitin-proteasome pathway . Rac1 degradation by HACE1 is important in regulating cell migration and reactive oxygen species (ROS) production [4, 5]. In addition, Rab GTPases are binding partners of HACE1 . HACE1 regulates Golgi membrane dynamics during the cell cycle by interacting with Rab proteins . One Rab GTPase Rab11a is activated by HACE1-mediated ubiquitination and subsequently promotes recycling of the β2-adrenergic receptor . In addition, HACE1 has protective actions against oxidative stress in the brain and hemodynamic stress in the heart [8, 9]. Thus, these findings have demonstrated HACE1 functions in diverse cellular processes. However, the roles of HACE1 during vertebrate embryonic development have not been previously reported in any model organism.
Here, we show that Xenopus laevis hace1 is mainly expressed in the ectoderm and plays an important role in embryonic development. Hace1-depleted Xenopus laevis embryos displayed defects in multiple developmental processes. Analysis with Keller sandwich explants revealed that Xenopus laevis hace1 is essential for convergent extension.
Xenopus laevis hace1 is mainly expressed in the ectoderm during early embryogenesis
To investigate a role of HACE1 in early embryonic development, we first cloned a Xenopus laevis HACE1 homolog (accession number AB894419) with a 2739 bp open reading frame. Compared with the reported sequence of Xenopus laevis hace1 in GenBank (accession number NM_001093608), the Xenopus laevis hace1 sequence that we obtained had five silent point mutations and a 96 bp deletion. These silent mutations may be single nucleotide polymorphisms. The 96 bp deletion may be caused by alternative splicing, because the human HACE1 gene deposited in GenBank (accession number NM_020771) has a similar deletion. We thus concluded that the obtained Xenopus laevis hace1 clone is a splicing variant. Human HACE1 protein shares 90 % and 87 % amino acid identity with our Xenopus laevis Hace1 protein and the previously reported protein, respectively. Hence, we used our clone in this study.
Knockdown of hace1 causes shortening of the body axis and inhibition of eye and pigment formation
Hace1 is required for convergent extension
Hace1 depletion causes a delay in neural tube closure
Additional file 1: Movie S1. Neural tube closure of an uninjected embryo.
Additional file 2: Movie S2. Neural tube closure of an embryo injected with HACE1 MO.
Phenotypes of hace1 morphants are specific for loss of hace1
Developmental defects in hace1 morphants are largely caused by an excess of active Rac1
We next asked whether the defects observed in hace1 morphants could be rescued by reducing Rac1 activity. We used Rac1 MO to reduce the total Rac1 protein level. Immunoblotting analysis confirmed that Rac1 MO reduced the protein level of endogenous Rac1 by more than half (Fig. 6b). We injected HACE1 MO with control MO or Rac1 MO into embryos and fixed them at the tadpole stage (stage 40–41). Embryos coinjected with HACE1 MO and Rac1 MO displayed milder defects in eyes and pigments than did embryos coinjected with HACE1 MO and control MO, although these double MO-injected embryos displayed more severe phenotypes than embryos injected with HACE1 MO alone, possibly because of overdosing of MOs (Fig. 6c–e). In addition, embryos coinjected with HACE1 MO and Rac1 MO had a longer body length than did embryos coinjected with HACE1 MO and control MO (Fig. 6f, g). These results suggest that the defects in hace1 morphants are at least partly caused by an excess of active Rac1.
To further test this possibility, we expressed rac1-V12, a constitutively active mutant of rac1, in the presumptive neural plate to examine the effect of excessive active Rac1 on neurulation. Nearly half of the embryos injected with rac1-V12 mRNA displayed a phenotype similar to that of embryos injected with HACE1 MO, such as a shortened body axis (Fig. 6h, compare with Fig. 2b, middle), thus supporting our idea that developmental defects in hace1 knockdown embryos are at least partly due to excessive active Rac1. Because it has previously been reported that exogenous expression of rac1-V12 in the mesoderm (1 or 2 ng of rac1-V12 mRNA into the marginal zone of embryos) results in gastrulation defects , we carefully observed the effect of rac1-V12 on gastrulation and showed that injection of rac1-V12 mRNA in our experimental condition (30 pg of mRNA was injected into the animal region of two dorsal blastomeres at 4-cell stage) did not affect gastrulation (data not shown).
Excess of active Rac1 causes a delay in neural tube closure
To further characterize the phenotype of the embryos injected with rac1-V12 mRNA, we performed time-lapse imaging and quantified the delay in neural tube closure. Embryos in which rac1-V12 mRNA was injected into the right side showed a significant delay in neural tube closure in the injected side, as compared with uninjected embryos (Fig. 4b–d and see also Additional file 1: Movie S1 and Additional file 3: Movie S3; uninjected embryos and rac1-V12 mRNA-injected embryos, respectively). These results support our hypothesis that hace1 regulates Xenopus laevis neural tube closure through Rac1 degradation.
Additional file 3: Movie S3. Neural tube closure of an embryo injected with Rac1-V12 mRNA.
hace1 has a role in regulating differentiation to neural ectoderm
In this study, we investigated the role of the E3 ubiquitin ligase Hace1 in early embryonic development. We showed that Xenopus laevis hace1 is expressed in neural tissues and the kidney. High-throughput in situ hybridization in mouse embryos has revealed that Hace1 is expressed in neural tissues in mice at embryonic day 11.5 (E11.5), E15.5, and postnatal day 7 . In humans, HACE1 is expressed in adult tissues including the heart, brain, and kidney . These data suggest that HACE1 expression in neural tissues is common in vertebrates.
Hace1 depletion in Xenopus laevis embryos led to a shortened body axis. The process of body axis elongation is known to involve convergent extension [10, 16, 17]. Our analysis with Keller sandwich explants showed that knockdown of hace1 inhibited convergent extension. These results suggest that Xenopus laevis hace1 may control body axis elongation through regulation of convergent extension. Convergent extension occurs in both dorsal mesodermal and posterior neural tissues. Whereas convergent extension in the dorsal mesoderm mainly regulates gastrulation, convergent extension in the neural ectoderm mainly regulates neurulation. It has been shown using transplantation experiments that inhibition of convergent extension in only the neural ectoderm leads to a shortened body axis . Because knockdown of hace1 did not affect gastrulation, inhibition of convergent extension in the neural ectoderm by hace1 depletion might lead to a shortened body axis.
Notably, Hace1 −/− mice do not display any developmental defects , whereas knockdown of hace1 in Xenopus laevis led to severe developmental defects. This discrepancy might be explained by the possibility that other proteins with roles redundant with that of HACE1 may compensate for HACE1 function during embryonic development of Hace1 −/− mice. For instance, the activity of Rac1, one of the targets of HACE1, is negatively regulated by GAP and other E3 ubiquitin ligases targeting Rac1, such as IAPs [19–21]. Ectodermal tissues in mouse embryos might express these proteins to provide robustness to loss of Hace1 protein. Most recently, it has been reported that knockdown of hace1 using a splice-blocking MO causes ROS production in zebrafish . Although this report has not described the effects of the MO on zebrafish embryonic development, the gross morphology of embryos injected with the MO is apparently normal. Whereas a splice-blocking morpholino inhibits only zygotic expression but not maternal expression, a translation blocking morpholino, which was used in our study, inhibits both maternal expression and zygotic expression. This might explain the difference between our results and those from the previous study in zebrafish.
hace1 depletion in Xenopus laevis embryos led to diverse developmental defects, including a shortened body axis and the inhibition of eye and pigment formation. These defects were at least partly rescued by rac1 inhibition. In addition, we showed that overexpression of active Rac1 in the presumptive neural plate led to a delay in neural tube closure and a number of subsequent developmental defects, which are similar to hace1 morphants. The migration of neural crest cells, which give rise to pigment cells, is disturbed by constitutively active or dominant negative forms of Rac1 . Therefore, the inhibition of pigment formation may result from impaired neural crest migration. Additionally, conditional knockout of Rac1 in the surface ectoderm results in a failure of neural tube closure in mice . Thus, fine tuning of Rac1 activity may be important for neural tube closure. However, the detailed molecular mechanism by which excessive active Rac1 affects neural tube closure is unclear. Rac1 regulates a variety of biological processes, including cytoskeleton remodeling and PCP signaling regulation [24, 25]. PCP signaling plays an important role in neural tube closure , and there may be crosstalk between RhoA and Rac1 signaling in the regulation of actin cytoskeleton . It would be interesting to investigate the presence of the crosstalk and, if it is present, to clarify the regulation mechanism of the PCP pathway through RhoA and Rac1 signaling during neural tube closure.
In summary, our study show that the E3 ubiquitin ligase Hace1 plays an important role in early developmental processes in Xenopus laevis. Detailed analyses of downstream targets of Hace1 should be performed in future studies.
Molecular cloning and plasmid construction
Primers were designed on the basis of the Xenopus laevis hace1 sequence (GenBank accession NM_001093608) as follows: F, 5′-GGTAGATCTATGGAGAGAGCAATGGAGCAACTC-3′; R, 5′-GGTAGATCTTTATGCCATTGTGTAGCCGTAGCT-3′, and the Xenopus laevis rac1 sequence (GenBank accession NM_001095863) as follows: F, 5′- CGGAAGATCTGTAGGGAGAGCAAAGAAGAGGGAGGGAG-3′; R, 5′- TCCGGAATTCAGGGACAGAAGAAAAGATGGCATGTGGG-3′. PCR was performed with complementary DNAs derived from embryos at stage 2 for hace1 and stage 13 for rac1. The entire amplified coding sequences were cloned into the expression vector pCS2+. A constitutively active form of Xenopus rac1 was constructed by replacing glycine 12 with valine by site-directed mutagenesis.
Whole mount in situ hybridization
Whole-mount in situ hybridization was performed on albino Xenopus laevis embryos according to a standard protocol  using a robot (InSituPro, Intavis). The digoxigenin-labeled probes were synthesized from hace1-pCS2+.
Xenopus laevis embryos were obtained by in vitro fertilization and cultured in 0.1 × MBS at 15 or 22 °C. Embryos were staged according to Nieuwkoop and Faber . Antisense morpholino oligonucleotides (MOs) and mRNAs were injected into the animal region of one or two dorsal blastomeres at the four-cell stage in 4 % Ficoll in 0.1× MBS. In vitro synthesis of capped mRNA was performed using the mMESSAGE mMACHINE (Ambion). MOs were purchased from Gene Tools. The MO sequences were as follows: HACE1 MO, 5′- GAGTTGCTCCATTGCTCTCTCCATC-3′; HACE1 MO2, 5′- AGAGGCTCAGCAGTTCCTAAGCAGT-3′; Rac1 MO, 5′-CCACACATTTAATGGCCTGCATGGC-3′; and a standard control oligo (Control MO), 5′-CCTCTTACCTCAGTTACAATTTATA-3′. The animals were bred and handled with care, according to a published laboratory manual . Xenopus laevis experiments complied with the Regulation on Animal Experimentation at Kyoto University and were approved by the Animal Experimentation Committee of Kyoto University.
Embryos were lysed in a buffer consisting of 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 2 mM EGTA, 25 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 1 % Nonidet P-40, 10 mM NaF, 1 mM vanadate, 1 mM DTT, and 1× Protein Inhibitor Cocktail (Sigma). Extracts were then centrifuged, and supernatants were collected. Anti-Myc (A14, Santa Cruz), anti-GFP (JL8, Clontech), anti-Rac1 (23A8, Millipore) or anti-α-tubulin (DM1A, Sigma) antibodies were used as primary antibodies, and anti-mouse IgG HRP-conjugated (1:10,000; GE healthcare) and anti-rabbit IgG HRP-conjugated (1:10,000; GE healthcare) were used as secondary antibodies.
Quantification of body length
Embryos were fixed in MEMFA [100 mM MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSO4, 3.7 % formaldehyde] at stage 40–41. Embryos were placed on agarose gel, and images were collected. The body length was measured along the dorsal midline using the AxioVision (Zeiss) measure tool.
Keller sandwich explant analysis
Keller sandwich explant experiments were performed as previously described . MOs and mRNA were injected into the animal region of two dorsal blastomeres at the four-cell stage. Injected embryos were grown to stage 10.5 for preparation of Keller sandwich explants. Explants were cultured in Sater’s modified blastocoel buffer containing 0.1 % BSA until uninjected embryos reached stage 19. The length and width of explants were measured using cellSens software (Olympus).
Time-lapse analysis of neural tube closure
Embryos injected with MOs or mRNAs were grown to stage 13 and placed dorsal side up in a 60 mm culture dish. This dish contained 2 % agarose with a groove made by an acrylic comb. Images were collected with an Olympus SZX16 microscope, and time-lapse movies were obtained using cellSens software. For quantitative analysis of the delay in neural tube closure, the maximum distance between the dorsal midline and the left (i) or right (ii) inner edge of the neural fold was measured at the time point 60 min after start of time-lapse imaging, as shown in Fig. 4c. At the time point 60 min after start of time-lapse imaging, embryos reached stage 17. The inner edge of the neural fold was easily visible, owing to pigmentation.
Quantitative RT-PCR analysis
Total RNA isolation and cDNA synthesis were performed as previously described . The gene expression levels were normalized to those of odc1 (ornithine decarboxylase 1). The sequences of primers used were as follows: Xenopus laevis rac1 (NM_001095863) (F, CATGCACATGTCAAGCCAGTTC; R, ATGGCAAGTCCCTGAGGATAGG); Xenopus laevis rac2 (NM_001092288) (F, ACCAGTAAACTTGGGCTTGTGG; R, CTCATAAGATGCCGGACTCACC); Xenopus laevis odc1 (NM_001086698) (F, TGAAAGTGGCAAGGAATCACCC; R, GATACGATCCAGCCCATCACAC).
HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1
Inhibitors of apoptosis protein
Reactive oxygen species
We thank members of the Nishida laboratory for technical advice and helpful discussions.
This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to MK) and the Takeda Science Foundation (to MK).
Availability of data and materials
All supporting data are presented in the main manuscript and supplementary data files.
AI, FY, TS, and TE designed experiments. AI, FY, and TE performed experiments. AI, FY, TS, EN, and MK wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Xenopus laevis experiments complied with the Regulation on Animal Experimentation at Kyoto University and were approved by the Animal Experimentation Committee of Kyoto University.
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