The vertebrate body plan is accomplished by left-right asymmetric organ development and the heart is a representative asymmetric internal organ which jogs to the left-side. Kupffer’s vesicle (KV) is a spherical left-right organizer during zebrafish embryogenesis and is derived from a cluster of dorsal forerunner cells (DFCs). Cadherin1 is required for collective migration of a DFC cluster and failure of DFC collective migration by Cadherin1 decrement causes KV malformation which results in defective heart laterality. Recently, loss of function mutation of A-kinase anchoring protein 12 (
Kupffer’s vesicle (KV) is a spherical left-right organizer which appears transiently during an embryonic stage in zebrafish (1). KV originates from a cluster of dorsal forerunner cells (DFCs). Cadherin1 (Cdh1)-mediated adherens junctions sustain cell cluster formation between the adjacent DFCs and a cluster of DFCs actively migrates towards the vegetal pole to form KV (2). Then, migrated DFCs attach to the overlying surface epithelium and become polarized to construct a rosette-like structure which contains the lumen at the apical point (3). Finally, cilia-formed and fluid-filled KV expands the internal lumen and motile cilia generate fluid flow in a counterclockwise direction to evoke asymmetric signal(s) such as Nodal, Lefty, and Pitx2 (1).
The vertebrate body plan is accomplished by left-right asymmetric organ development. The heart is a representative asymmetric internal organ which jogs to the left-side and proper positioning during embryonic development is crucial for its function (4). Accordingly, about ~1% of newborn babies suffer from congenital heart disease (CHD), which has high mortality (5). Dextrocardia, a rare condition in which the apex of the heart is located on the right side of the body, comprises a CHD case with heterotaxy which is often accompanied by asymmetric defects such as a left-sided liver and a right-sided stomach (6).
A-kinase anchoring protein 12 (AKAP12) is a member of the AKAP family proteins, which bind to the regulatory subunit of protein kinase A (PKA) and holoenzyme localizes to specific locations within the cell. Besides PKA, AKAP12 displays diverse docking sites for protein kinase C, calmodulin, cyclins, β-1,4-galactosyltransferase, protein phosphatases, the non-receptor tyrosine kinase Src, and β2-adrenergic receptor. In addition, AKAP12 consists of three polybasic domains, four nuclear localization signals, and a nuclear exclusion domain. Therefore, AKAP12 plays various roles in many biological processes including cell migration, cell cycle regulation, barriergenesis, tumor progression, and wound healing (7). Our group previously reported that AKAP12 regulates the blood-brain and blood-retinal barrier (8, 9). This barriergenic property of AKAP12 is also applied to the repair of the central nervous system (CNS) after injury. AKAP12 is strongly expressed in the fibrotic scar during the CNS repair process where it mediates barrier functions (10). Moreover, we and other group reported that
Next, we investigated the spatiotemporal expression of
Specific expression of
To investigate whether the specific downregulation of
Next, we validated the notochordal expression of
Taken together, these data suggest that reduced
Fragmented DFC clusters are the symbolic phenotype of disrupted cell collectivity between DFCs which is maintained by Cdh1-based adherence junction (18). Thus, we evaluated Cdh1 expression in
The current study investigated the role of
AKAP12 was first identified as an autoantigen in myasthenia gravis, so it was named Gravin (7). In the present study, we investigated the specific role of
Our group reported that AKAP12 regulates junctional protein expression such as E-Cadherin, VE-Cadherin, Claudin-1, Occludin, and ZO-1 in diverse systems (8–10, 12). In zebrafish, Cdh1-mediated cell adhesion between adjacent DFCs is essential for their collective migration followed by KV morphogenesis (2, 18). In this regard, we also observed reduction in
Recent studies have revealed that asymmetric distribution of hypoxia contributes to dorsoventral axis establishment during embryogenesis of sea urchin and that retinoic acid (RA) is involved (21–23). Moreover, our group previously reported that partial oxygen pressure regulates AKAP12 expression and that RA induces AKAP12 expression in CNS injury repair (9, 24) and we hypothesized that gradation of such factors could regulate the spatiotemporal expression of
In addition to DFC collective migration, DFC numbers and ciliogenesis in KV are crucial for heart laterality (17). Our data indicated that proliferation of DFCs in
Given genetic evidences of
Tuebingen wild-type zebrafish and transgenic
The protocol of MO injection into zebrafish embryos was previously described (13). Briefly, splice-blocking MOs were injected into the yolk at one-cell stage for whole embryo knockdown or at 128–512-cell stage for DFC-specific knockdown as indicated. Translation-blocking MOs were used to rule out the off-target effects (
The protocols of qRT-PCR and RT-PCR were previously described (29). Total RNA was isolated from zebrafish embryos at indicated stages with TRIzol reagent (Invitrogen) and cDNA was obtained from 2 μg of total RNA using MMLV reverse transcriptase (Promega). qRT-PCR was then performed using StepOnePlus RT-PCR system (Applied Biosystems) with RealHelix qPCR kit (NanoHelix). Relative mRNA expression levels were calculated by the comparative 2−ΔΔCt method.
The protocol of ISH was previously described (16). Specific regions of
Measurement of KV lumen area was described previously (16). The data are presented as means ± SD and analyzed with Prism 5 (GraphPad Software, Inc.). The data in Fig. 4B and 4D were analyzed by two-tailed Student’s
The authors appreciated Emeritus Prof. Kyu-Won Kim (Seoul National University, Seoul, Korea) for mentoring and providing expertise on this study. J.-g.K. designed the research, performed experiments and cared for zebrafish; H.-H.K. analyzed data and helped write the manuscript; S.-J.B. wrote the manuscript and supervised the research. This work was supported by Basic Science Research Program (NRF-2017R1A6A3A11032239) through the NRF funded by the Korean Ministry of Education and the Medical Research Center Program (2014R1A5A20009936) through the NRF funded by the Korean Ministry of Science, ICT and Future Planning (MSIP).
The authors have no conflicting interests.