Mol. Cells 2017; 40(4): 271-279
Published online March 28, 2017
https://doi.org/10.14348/molcells.2017.2308
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: zebrakim@cnu.ac.kr
Ran-binding protein family member, RanBP9 has been reported in various basic cellular mechanisms and neuropathological conditions including schizophrenia. Previous studies have reported that RanBP9 is highly expressed in the mammalian brain and retina; however, the role of RanBP9 in retinal development is largely unknown. Here, we present the novel and regulatory roles of RanBP9 in retinal development of a vertebrate animal model, zebrafish. Zebrafish embryos exhibited abundant expression of
Keywords mind bomb, notch, RanBP9, retinogenesis, zebrafish
Notch signaling is an evolutionarily conserved mechanism which controls a broad spectrum of cell fates and developmental processes through cell–cell interaction and lateral inhibition. Notch signaling is initiated when the extracellular domain of the ligand interacts with the Notch receptor, resulting in proteolytic cleavage of the Notch intracellular domain, NICD. Subsequent nuclear translocation of NICD regulates the transcription of various target genes. Defects in Notch signaling have been implicated in a wide variety of developmental defects and diseases (Selkoe and Kopan, 2003).
We previously identified Mind bomb, a key component of the Notch pathway, via positional cloning of zebrafish neurogenic mutants (Itoh et al., 2003). Mutations in
In this study we identified a Mind bomb-binding protein, Ran-binding protein 9 (RanBP9), using the yeast two-hybrid screening method. RanBP9 is a member of the Ran-binding protein family and is encoded by the gene,
In our study, the binding of RanBP9 with Mind bomb was confirmed by co-immunoprecipitation
For the construction of bait, the mid-region of the Ankyrin repeat domain (from amino acids 410–774) of the MIB cDNA (Genbank Accession No. AY147849) was amplified by PCR using the primers, 5′-CAGAATTCGTGGTGGAAGGGGT TGGCGCTCGGGT-3′ (Forward Primer) and 5′-GATGCATTGCTAATGGCTGATCCTG-3′ (Reverse Primer). The amplified PCR fragment was then cloned into the EcoRI site of the pGBT9 vector (encoding the GAL4 DNA-binding domain), pGBT9/Ank-MIB. The cloned vectors were transformed into the yeast host strain, AH109, which was then mated with the Y187 yeast cells. The cells were pre-transformed using the Human Matchmaker cDNA Library (Clontech). The pre-transformed cDNA library in the yeast Y187 strain was screened for MIB-interaction partners. The toxicity of baits on the AH109 yeast cells and their transcriptional activation were tested and screened. Positive colonies were allowed to grow on Trp-, Leu-, His-, and Ade-depleted, minimum synthetic dropout medium (SD) for yeast, and were selected by β-Galactosidase quantitative assay. Plasmid DNAs isolated from the yeast cells were used as templates for further PCR amplification and sequencing. From DNA sequencing analysis, we identified six clones for RanBP9, one clone for SNX5 (Yoo et al., 2006), one clone for FIH-1 (So et al., 2014), and clones for other genes.
The zebrafish RanBP9 was isolated from its genome by performing the RT-PCR, using specific primers, 5′-TAGCTGGCAAGCTGTTGGTATCGAC-3′ as the forward primer and 5′-GGCCTCTGTCACGTGGTCGTTTAA-3′ as the reverse primer, with the total RNA isolated from different developmental stages of the zebrafish. The amplified full-length RanBP9 fragment was then cloned into the pGEM-T Easy vector and later confirmed by sequencing. The full-length RanBP9 was then cloned into the pCS2+ expression vector. To make capped mRNA, vectors were linearized using respective digestive enzyme and transcribed with SP6 RNA polymerase using the mMESSAGE mMACHINE SP6
Also, to study the evolutionary relationship between the RanBP9 from the zebrafish and other organisms, the translated amino acid sequences were aligned with the ‘ClustalOmega’ Protein Alignment Tool, (
Synthetic capped RNA for RanBP9 was transcribed
DIG-labelled antisense RNA probe for RanBP9 and MIB were synthesized from corresponding linearized plasmids using SP6 RNA polymerase following manufacturer instructions (Ambion). Fluorescein labeled probes for gata1 and flk-1 were synthesized as previously described. Whole mount in situ hybridization was done as previously described with little modifications. For sectioning, the embryos were soaked in 30% sucrose at 4°C overnight, and later embedded in 1.5% agarose and 5% sucrose. The 8 μm thick sections were cut using a cryostat microtome. For paraffin sectioning, the embryos were dehydrated in ethanol and washed in isopropanol for 10 min. The embryos were, later equilibrated in xylene for 15 min and transferred to fresh solution of xylene. Embryos were transferred to Xylene: Paraffin (1:1), at 60°C for 20 min, replaced with paraffin, 3 times at 60°C after every 20 min. After complete equilibration of the embryos with paraffin, the embryos were oriented and molten paraffin was poured and solidified. Trimmed paraffin blocks were sectioned at 8 μm thickness, using a histostat microtome. Later, the dry sections were soaked in xylene to remove the paraffin, loaded with the Canada balsam mounting medium and covered with cover slips.
Paraffin from the paraffin section slides were removed using xylene, rehydrated in a graded ethanol series (100%, 95%, 85%, 70%) for 3 min, and washed in double distilled water. Later the slides were stained with Harris’ Alum Hematoxylin (Harleco) with 4% acetic acid at room temperature for 5 min. The slides were again washed with double distilled water for 2 min, followed by dipping 8 times in acid-alcohol (1% HCl in 70% ethanol) for 30 s. Subsequently, the sections were rinsed in double distilled water for 2 min and dipped 20 times in 0.5% ammonium sulphate (w/v) for 1 min, and again rinsed with double distilled water for 2 min. Later, the sections were stained with eosin Y (1 mg/ml eosin Y in 95% ethanol + 2.8 μl/ml acetic acid) at room temperature for 3 min. The sections were, then, rehydrated in a graded ethanol series (70%, 85%, 95%, 100%) for 3 min. Sections were soaked in xylene and left at room temperature to slightly dry before loading on with Canada balsam. Slides were viewed using a Leica DM5000B microscope under white light.
Transfectable COS7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% heat inactivated fetal bovine serum and antibiotics. The cDNA of the zebrafish RanBP9 was sub-cloned into pCS2+GFP via
At 24–48 h post-transfection of plasmids into the COS7 cells, the cells were washed in Phosphate Buffered Saline (PBS) and fixed in 4% paraformaldehyde with 3% sucrose at 4°C for 30 min. The fixed cells were then incubated in blocking solution (3% skim milk and 0.1% TritonX-100 in PBS) at 4°C overnight, and later stained with respective primary antibody in 3% skim milk in PBS at room temperature for 1 h. Eventually, the cells were incubated with a mouse antibody conjugated with Alex549 at room temperature for 30 min. For DNA staining, the cells were stained with Hoechst 33342 (10 μg/ml) for 2 min. After thorough rinsing with 0.1% TritonX-100 in PBS for three times, the cells were mounted on glass slides and analyzed with a fluorescent microscope (Zeiss).
Yeast two-hybrid screening was performed to identify putative interacting members or partner proteins of Mind bomb (Mib)-mediated Notch signaling pathways, using the Ankyrin (Ank) mid-region of the Mib as bait (Fig. 1A). We previously isolated the
To better understand the roles of RanBP9 in developing zebrafish embryos, we first tested spatiotemporal distribution of
The physical interaction between RanBP9 and Mib was tested by co-immunoprecipitation of the proteins expressed in culture cells (Fig. 3). RanBP9 was co-transfected with Mib into the COS7 cells. Interestingly, RanBP9 tagged at the C- terminal (RanBP9-Flag) did not show any interaction with Mib (Fig. 3B, lane 2). The CRA motif at the C-terminus of RanBP9 protein, which is comprised of approximately 100 amino acids, was found to be important for the interaction of RanBP9 with fragile X mental retardation protein (FMRP), but its functional significance has yet to be determined (Menon et al., 2004). However, RanBP9 tagged at the N-terminal (Myc-RanBP9) was co-immunoprecipitated with Mib (Fig. 3B, lane 5). Its interaction was higher with wild type Mib than a mutant form of Mib (ta52b), point mutation at the ring finger domain (M1013R), although the expression level of ta52b was higher than wild type Mib in transfected cells (Fig. 3B). As a positive control we used Moe/Epb41l5 which is known to bind to the N-terminal fragment of Mib, but not to the Ankyrin mid-region and the C-terminal domain (Matsuda et al., 2016). Moe showed higher expression level than that of Mib proteins. The interaction between RanBP9 and Mib was further tested by examining their subcellular localization in cultured cells (Fig. 4). The RanBP9 was localized both in the nucleus and the punctate structures of the cytoplasm (Fig. 4B). Interestingly, in the cytoplasmic punctate structures, Mib was co-localized with the RanBP9 (Figs. 4A and 4C), suggesting that RanBP9 associates with some roles in the Notch signaling pathway. Previously, we showed that Mib is localized in the early endosomes and involved in Notch ligand Delta processing (Yoo et. al., 2006).
To test the roles of RanBP9 in embryonic development, gene knockdown experiments were performed using morpholino antisense oligonucleotides (Fig. 5). A morpholino oligonucleotide was designed to target the exon splice donor site of RanBP9 and produce premature translation termination, which was confirmed by RT-PCR (Fig. 5D). Morpholino injected embryos exhibited defects in brain development and swim-bladder inflation (Fig. 6A). By acridine orange staining method, we identified specific cell death in the brain, especially in the optic tectum, in 54 hpf morphants (Fig. 6B). Because RanBP9 is also expressed in the developing retina, we concentrated on examining retinal development in the
To understand the molecular mechanisms of RanBP9 in retinal cell development, we tested the expression of several molecular markers in the retina of
Furthermore, the expression of a cell cycle modulator,
In this study, we report of the molecular interaction between RanBP9 and Mind bomb. Mind bomb is a key component of the Notch signaling pathway, which plays a critical role in neural development. RanBP9 is known to interact with multiple proteins and modulate various processes including synaptic plasticity, actin cytoskeleton remodeling and mitogen-activated protein kinase cascade (Haase et al., 2008). It has also been shown to simultaneously inhibit cell-adhesive processes and enhance amyloid β generation by accelerating APP, LRP, and β1-integrin endocytosis (Woo et al., 2012); and inhibit agonist-induced Mu opioid receptor internalization (Talbot et al., 2009). Reports have demonstrated through genetic epistasis experiments the interaction of RanBP9 with fragile-X mental retardation protein (Menon et al., 2004), as well as disrupted in schizophrenia 1 (DISC1) which is associated with autism, schizophrenia and bipolar disorder (Bae et al., 2015). However, previous study with neo-natal RanBP9 null mice showed suckling problems caused by defects in brain development and subsequent premature death (Palavicini et al., 2013).
The retinal phenotype of RanBP9 knock-down zebrafish was different from that of the Mib knock-out zebrafish. As RanBP9 interacts with multiple proteins, Mib also binds to several other proteins, such as Snx5, Fih-1, Usp1, Usp9, Trabid, and Moe (Matsuda et al., 2016; So et al., 2014; Tseng et al., 2014; Yoo et al., 2006). Notch signaling controls a broad spectrum of developmental processes in various different kinds of tissues. There are multiple homologues of Notch receptors and Notch ligands in zebrafish, which are differentially regulated in a temporal and spatial expression pattern. To our knowledge, no knock-out animal of these Notch signaling components have shown morphological phenotypes similar to that of the Mib knock-out; this being in spite of defects in Notch signaling in specific tissue types of the KO animal. We also identified another Mib member, Mib2, which showed normal development and was completely viable in its homozygous mutant (Koo et al., 2007). Mib-like E3 ubiquitin ligases, Neuralized and Neuralized-like 1, are additionally involved in the Notch signaling (Koutelou et al., 2008). There was also no detectable phenotype in the double knock-out of Neuralized1 and Neuralized2 animals (Koo et al., 2007). Thus we can speculate all these multiple components may have different combination of complementary functions in different cell types and tissues.
The loss-of-function phenotype of
Proliferation of retinoblast cells to glial cell differentiation is a complex process comprising various events such as cell cycle exit (Dyer and Cepko, 2001). Cyclin kinase inhibitors (CKIs) have been shown to initiate cell cycle exit in various contexts. In the present study, inhibition of
In conclusion, our data suggest that the novel Mib-interacting protein, RanBP9, is critically required for neural development during embryogenesis of zebrafish. Based on the knockdown experiments and expression analysis, it can additionally be inferred that RanBP9 plays a significant role in the generation of various retinal cell types. RanBP9 may be particularly important for the modulation of Notch target gene expression and the proliferation and differentiation of neural cells by delaying cell cycle exit. In future experiments, it would be of interest to study these novel roles further in
(A) Schematic representation of Mib bait and RanBP9 prey proteins. The Mib bait contains the middle region of ankyrin repeats from amino acids 410 to 744. Multiple clones of RanBP9 prey were identified. Here, RanBP9 polypeptides starting from 136th or 158th amino acid were represented. (B) Protein sequence alignment between human, mouse and zebrafish RanBP9 homologues. (C) Comparison of domain similarity (%) between human and zebrafish RanBP9. PRD, proline-rich domain; SPRY, domain in sp1A and Ryanodine receptor; LisH, Lissencephaly type-1-like homology motif; CTLH, C-Terminal to the LisH; and a CRA motif.
Whole-mount
(A) Each cell lysate transfected with a combination of expression vectors was pulled down with anti-Myc antibody and the western blot was stained with anti-Myc antibody. Top panels show lysates without co-immunoprecipitation. (B) Cell lysates were pulled down with anti-Myc antibody and the western blot was stained with anti-Frag antibody. ta52b (M1013R) and tfi101 (C1009S) are mutant forms of Mib, having point mutation in the 3rd ring finger. Moe was used as a positive control for Mib binding. Arrows indicate the size for Frag-Mib and Frag-Mib/ta52b.
COS7 cells were transiently transfected with expression vectors encoding Mib-GFP and HA-RanBP9. (A) Mib-GFP was detected in the cytoplasm in a punctuate pattern. (B) HA-RanBP9 was localized in a similar punctuate pattern in the cytoplasm, in addition to its localization in the nucleus. Transfected cells were fixed and stained with anti-HA antibody. The yellow color and arrows in the merged image in (C) indicate co-localization of Mib and RanBP9 in the cytoplasmic punctuate structures. Hoechst dye staining (blue) shows the nucleus.
(A) 14 exons of
(A) Control (Cont) and
(A) Whole-mount
Retinal sections were stained with an antibody against Glutamine synthetase, which is used as a molecular marker for the Müller glia cells. Glial cell differentiation is also affected in
Mol. Cells 2017; 40(4): 271-279
Published online April 30, 2017 https://doi.org/10.14348/molcells.2017.2308
Copyright © The Korean Society for Molecular and Cellular Biology.
Kyeong-Won Yoo1, Maivannan Thiruvarangan1, Yun-Mi Jeong1, Mi-Sun Lee1, Sateesh Maddirevula1, Myungchull Rhee1, Young-Ki Bae2, Hyung-Goo Kim3, and Cheol-Hee Kim1,*
1Department of Biology, Chungnam National University, Daejeon 34134, Korea, 2Comparative Biomedicine Research Branch, Research Institute, National Cancer Center, Goyang 10408, Korea, 3Department of OB/GYN, Department of Neuroscience and Regenerative Medicine, Augusta University, GA 30912, USA
Correspondence to:*Correspondence: zebrakim@cnu.ac.kr
Ran-binding protein family member, RanBP9 has been reported in various basic cellular mechanisms and neuropathological conditions including schizophrenia. Previous studies have reported that RanBP9 is highly expressed in the mammalian brain and retina; however, the role of RanBP9 in retinal development is largely unknown. Here, we present the novel and regulatory roles of RanBP9 in retinal development of a vertebrate animal model, zebrafish. Zebrafish embryos exhibited abundant expression of
Keywords: mind bomb, notch, RanBP9, retinogenesis, zebrafish
Notch signaling is an evolutionarily conserved mechanism which controls a broad spectrum of cell fates and developmental processes through cell–cell interaction and lateral inhibition. Notch signaling is initiated when the extracellular domain of the ligand interacts with the Notch receptor, resulting in proteolytic cleavage of the Notch intracellular domain, NICD. Subsequent nuclear translocation of NICD regulates the transcription of various target genes. Defects in Notch signaling have been implicated in a wide variety of developmental defects and diseases (Selkoe and Kopan, 2003).
We previously identified Mind bomb, a key component of the Notch pathway, via positional cloning of zebrafish neurogenic mutants (Itoh et al., 2003). Mutations in
In this study we identified a Mind bomb-binding protein, Ran-binding protein 9 (RanBP9), using the yeast two-hybrid screening method. RanBP9 is a member of the Ran-binding protein family and is encoded by the gene,
In our study, the binding of RanBP9 with Mind bomb was confirmed by co-immunoprecipitation
For the construction of bait, the mid-region of the Ankyrin repeat domain (from amino acids 410–774) of the MIB cDNA (Genbank Accession No. AY147849) was amplified by PCR using the primers, 5′-CAGAATTCGTGGTGGAAGGGGT TGGCGCTCGGGT-3′ (Forward Primer) and 5′-GATGCATTGCTAATGGCTGATCCTG-3′ (Reverse Primer). The amplified PCR fragment was then cloned into the EcoRI site of the pGBT9 vector (encoding the GAL4 DNA-binding domain), pGBT9/Ank-MIB. The cloned vectors were transformed into the yeast host strain, AH109, which was then mated with the Y187 yeast cells. The cells were pre-transformed using the Human Matchmaker cDNA Library (Clontech). The pre-transformed cDNA library in the yeast Y187 strain was screened for MIB-interaction partners. The toxicity of baits on the AH109 yeast cells and their transcriptional activation were tested and screened. Positive colonies were allowed to grow on Trp-, Leu-, His-, and Ade-depleted, minimum synthetic dropout medium (SD) for yeast, and were selected by β-Galactosidase quantitative assay. Plasmid DNAs isolated from the yeast cells were used as templates for further PCR amplification and sequencing. From DNA sequencing analysis, we identified six clones for RanBP9, one clone for SNX5 (Yoo et al., 2006), one clone for FIH-1 (So et al., 2014), and clones for other genes.
The zebrafish RanBP9 was isolated from its genome by performing the RT-PCR, using specific primers, 5′-TAGCTGGCAAGCTGTTGGTATCGAC-3′ as the forward primer and 5′-GGCCTCTGTCACGTGGTCGTTTAA-3′ as the reverse primer, with the total RNA isolated from different developmental stages of the zebrafish. The amplified full-length RanBP9 fragment was then cloned into the pGEM-T Easy vector and later confirmed by sequencing. The full-length RanBP9 was then cloned into the pCS2+ expression vector. To make capped mRNA, vectors were linearized using respective digestive enzyme and transcribed with SP6 RNA polymerase using the mMESSAGE mMACHINE SP6
Also, to study the evolutionary relationship between the RanBP9 from the zebrafish and other organisms, the translated amino acid sequences were aligned with the ‘ClustalOmega’ Protein Alignment Tool, (
Synthetic capped RNA for RanBP9 was transcribed
DIG-labelled antisense RNA probe for RanBP9 and MIB were synthesized from corresponding linearized plasmids using SP6 RNA polymerase following manufacturer instructions (Ambion). Fluorescein labeled probes for gata1 and flk-1 were synthesized as previously described. Whole mount in situ hybridization was done as previously described with little modifications. For sectioning, the embryos were soaked in 30% sucrose at 4°C overnight, and later embedded in 1.5% agarose and 5% sucrose. The 8 μm thick sections were cut using a cryostat microtome. For paraffin sectioning, the embryos were dehydrated in ethanol and washed in isopropanol for 10 min. The embryos were, later equilibrated in xylene for 15 min and transferred to fresh solution of xylene. Embryos were transferred to Xylene: Paraffin (1:1), at 60°C for 20 min, replaced with paraffin, 3 times at 60°C after every 20 min. After complete equilibration of the embryos with paraffin, the embryos were oriented and molten paraffin was poured and solidified. Trimmed paraffin blocks were sectioned at 8 μm thickness, using a histostat microtome. Later, the dry sections were soaked in xylene to remove the paraffin, loaded with the Canada balsam mounting medium and covered with cover slips.
Paraffin from the paraffin section slides were removed using xylene, rehydrated in a graded ethanol series (100%, 95%, 85%, 70%) for 3 min, and washed in double distilled water. Later the slides were stained with Harris’ Alum Hematoxylin (Harleco) with 4% acetic acid at room temperature for 5 min. The slides were again washed with double distilled water for 2 min, followed by dipping 8 times in acid-alcohol (1% HCl in 70% ethanol) for 30 s. Subsequently, the sections were rinsed in double distilled water for 2 min and dipped 20 times in 0.5% ammonium sulphate (w/v) for 1 min, and again rinsed with double distilled water for 2 min. Later, the sections were stained with eosin Y (1 mg/ml eosin Y in 95% ethanol + 2.8 μl/ml acetic acid) at room temperature for 3 min. The sections were, then, rehydrated in a graded ethanol series (70%, 85%, 95%, 100%) for 3 min. Sections were soaked in xylene and left at room temperature to slightly dry before loading on with Canada balsam. Slides were viewed using a Leica DM5000B microscope under white light.
Transfectable COS7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% heat inactivated fetal bovine serum and antibiotics. The cDNA of the zebrafish RanBP9 was sub-cloned into pCS2+GFP via
At 24–48 h post-transfection of plasmids into the COS7 cells, the cells were washed in Phosphate Buffered Saline (PBS) and fixed in 4% paraformaldehyde with 3% sucrose at 4°C for 30 min. The fixed cells were then incubated in blocking solution (3% skim milk and 0.1% TritonX-100 in PBS) at 4°C overnight, and later stained with respective primary antibody in 3% skim milk in PBS at room temperature for 1 h. Eventually, the cells were incubated with a mouse antibody conjugated with Alex549 at room temperature for 30 min. For DNA staining, the cells were stained with Hoechst 33342 (10 μg/ml) for 2 min. After thorough rinsing with 0.1% TritonX-100 in PBS for three times, the cells were mounted on glass slides and analyzed with a fluorescent microscope (Zeiss).
Yeast two-hybrid screening was performed to identify putative interacting members or partner proteins of Mind bomb (Mib)-mediated Notch signaling pathways, using the Ankyrin (Ank) mid-region of the Mib as bait (Fig. 1A). We previously isolated the
To better understand the roles of RanBP9 in developing zebrafish embryos, we first tested spatiotemporal distribution of
The physical interaction between RanBP9 and Mib was tested by co-immunoprecipitation of the proteins expressed in culture cells (Fig. 3). RanBP9 was co-transfected with Mib into the COS7 cells. Interestingly, RanBP9 tagged at the C- terminal (RanBP9-Flag) did not show any interaction with Mib (Fig. 3B, lane 2). The CRA motif at the C-terminus of RanBP9 protein, which is comprised of approximately 100 amino acids, was found to be important for the interaction of RanBP9 with fragile X mental retardation protein (FMRP), but its functional significance has yet to be determined (Menon et al., 2004). However, RanBP9 tagged at the N-terminal (Myc-RanBP9) was co-immunoprecipitated with Mib (Fig. 3B, lane 5). Its interaction was higher with wild type Mib than a mutant form of Mib (ta52b), point mutation at the ring finger domain (M1013R), although the expression level of ta52b was higher than wild type Mib in transfected cells (Fig. 3B). As a positive control we used Moe/Epb41l5 which is known to bind to the N-terminal fragment of Mib, but not to the Ankyrin mid-region and the C-terminal domain (Matsuda et al., 2016). Moe showed higher expression level than that of Mib proteins. The interaction between RanBP9 and Mib was further tested by examining their subcellular localization in cultured cells (Fig. 4). The RanBP9 was localized both in the nucleus and the punctate structures of the cytoplasm (Fig. 4B). Interestingly, in the cytoplasmic punctate structures, Mib was co-localized with the RanBP9 (Figs. 4A and 4C), suggesting that RanBP9 associates with some roles in the Notch signaling pathway. Previously, we showed that Mib is localized in the early endosomes and involved in Notch ligand Delta processing (Yoo et. al., 2006).
To test the roles of RanBP9 in embryonic development, gene knockdown experiments were performed using morpholino antisense oligonucleotides (Fig. 5). A morpholino oligonucleotide was designed to target the exon splice donor site of RanBP9 and produce premature translation termination, which was confirmed by RT-PCR (Fig. 5D). Morpholino injected embryos exhibited defects in brain development and swim-bladder inflation (Fig. 6A). By acridine orange staining method, we identified specific cell death in the brain, especially in the optic tectum, in 54 hpf morphants (Fig. 6B). Because RanBP9 is also expressed in the developing retina, we concentrated on examining retinal development in the
To understand the molecular mechanisms of RanBP9 in retinal cell development, we tested the expression of several molecular markers in the retina of
Furthermore, the expression of a cell cycle modulator,
In this study, we report of the molecular interaction between RanBP9 and Mind bomb. Mind bomb is a key component of the Notch signaling pathway, which plays a critical role in neural development. RanBP9 is known to interact with multiple proteins and modulate various processes including synaptic plasticity, actin cytoskeleton remodeling and mitogen-activated protein kinase cascade (Haase et al., 2008). It has also been shown to simultaneously inhibit cell-adhesive processes and enhance amyloid β generation by accelerating APP, LRP, and β1-integrin endocytosis (Woo et al., 2012); and inhibit agonist-induced Mu opioid receptor internalization (Talbot et al., 2009). Reports have demonstrated through genetic epistasis experiments the interaction of RanBP9 with fragile-X mental retardation protein (Menon et al., 2004), as well as disrupted in schizophrenia 1 (DISC1) which is associated with autism, schizophrenia and bipolar disorder (Bae et al., 2015). However, previous study with neo-natal RanBP9 null mice showed suckling problems caused by defects in brain development and subsequent premature death (Palavicini et al., 2013).
The retinal phenotype of RanBP9 knock-down zebrafish was different from that of the Mib knock-out zebrafish. As RanBP9 interacts with multiple proteins, Mib also binds to several other proteins, such as Snx5, Fih-1, Usp1, Usp9, Trabid, and Moe (Matsuda et al., 2016; So et al., 2014; Tseng et al., 2014; Yoo et al., 2006). Notch signaling controls a broad spectrum of developmental processes in various different kinds of tissues. There are multiple homologues of Notch receptors and Notch ligands in zebrafish, which are differentially regulated in a temporal and spatial expression pattern. To our knowledge, no knock-out animal of these Notch signaling components have shown morphological phenotypes similar to that of the Mib knock-out; this being in spite of defects in Notch signaling in specific tissue types of the KO animal. We also identified another Mib member, Mib2, which showed normal development and was completely viable in its homozygous mutant (Koo et al., 2007). Mib-like E3 ubiquitin ligases, Neuralized and Neuralized-like 1, are additionally involved in the Notch signaling (Koutelou et al., 2008). There was also no detectable phenotype in the double knock-out of Neuralized1 and Neuralized2 animals (Koo et al., 2007). Thus we can speculate all these multiple components may have different combination of complementary functions in different cell types and tissues.
The loss-of-function phenotype of
Proliferation of retinoblast cells to glial cell differentiation is a complex process comprising various events such as cell cycle exit (Dyer and Cepko, 2001). Cyclin kinase inhibitors (CKIs) have been shown to initiate cell cycle exit in various contexts. In the present study, inhibition of
In conclusion, our data suggest that the novel Mib-interacting protein, RanBP9, is critically required for neural development during embryogenesis of zebrafish. Based on the knockdown experiments and expression analysis, it can additionally be inferred that RanBP9 plays a significant role in the generation of various retinal cell types. RanBP9 may be particularly important for the modulation of Notch target gene expression and the proliferation and differentiation of neural cells by delaying cell cycle exit. In future experiments, it would be of interest to study these novel roles further in
(A) Schematic representation of Mib bait and RanBP9 prey proteins. The Mib bait contains the middle region of ankyrin repeats from amino acids 410 to 744. Multiple clones of RanBP9 prey were identified. Here, RanBP9 polypeptides starting from 136th or 158th amino acid were represented. (B) Protein sequence alignment between human, mouse and zebrafish RanBP9 homologues. (C) Comparison of domain similarity (%) between human and zebrafish RanBP9. PRD, proline-rich domain; SPRY, domain in sp1A and Ryanodine receptor; LisH, Lissencephaly type-1-like homology motif; CTLH, C-Terminal to the LisH; and a CRA motif.
Whole-mount
(A) Each cell lysate transfected with a combination of expression vectors was pulled down with anti-Myc antibody and the western blot was stained with anti-Myc antibody. Top panels show lysates without co-immunoprecipitation. (B) Cell lysates were pulled down with anti-Myc antibody and the western blot was stained with anti-Frag antibody. ta52b (M1013R) and tfi101 (C1009S) are mutant forms of Mib, having point mutation in the 3rd ring finger. Moe was used as a positive control for Mib binding. Arrows indicate the size for Frag-Mib and Frag-Mib/ta52b.
COS7 cells were transiently transfected with expression vectors encoding Mib-GFP and HA-RanBP9. (A) Mib-GFP was detected in the cytoplasm in a punctuate pattern. (B) HA-RanBP9 was localized in a similar punctuate pattern in the cytoplasm, in addition to its localization in the nucleus. Transfected cells were fixed and stained with anti-HA antibody. The yellow color and arrows in the merged image in (C) indicate co-localization of Mib and RanBP9 in the cytoplasmic punctuate structures. Hoechst dye staining (blue) shows the nucleus.
(A) 14 exons of
(A) Control (Cont) and
(A) Whole-mount
Retinal sections were stained with an antibody against Glutamine synthetase, which is used as a molecular marker for the Müller glia cells. Glial cell differentiation is also affected in
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(A) Schematic representation of Mib bait and RanBP9 prey proteins. The Mib bait contains the middle region of ankyrin repeats from amino acids 410 to 744. Multiple clones of RanBP9 prey were identified. Here, RanBP9 polypeptides starting from 136th or 158th amino acid were represented. (B) Protein sequence alignment between human, mouse and zebrafish RanBP9 homologues. (C) Comparison of domain similarity (%) between human and zebrafish RanBP9. PRD, proline-rich domain; SPRY, domain in sp1A and Ryanodine receptor; LisH, Lissencephaly type-1-like homology motif; CTLH, C-Terminal to the LisH; and a CRA motif.
|@|~(^,^)~|@|Spatiotemporal expression ofWhole-mount
(A) Each cell lysate transfected with a combination of expression vectors was pulled down with anti-Myc antibody and the western blot was stained with anti-Myc antibody. Top panels show lysates without co-immunoprecipitation. (B) Cell lysates were pulled down with anti-Myc antibody and the western blot was stained with anti-Frag antibody. ta52b (M1013R) and tfi101 (C1009S) are mutant forms of Mib, having point mutation in the 3rd ring finger. Moe was used as a positive control for Mib binding. Arrows indicate the size for Frag-Mib and Frag-Mib/ta52b.
|@|~(^,^)~|@|Subcellular co-localization of RanBP9 and Mib in COS7 cells.COS7 cells were transiently transfected with expression vectors encoding Mib-GFP and HA-RanBP9. (A) Mib-GFP was detected in the cytoplasm in a punctuate pattern. (B) HA-RanBP9 was localized in a similar punctuate pattern in the cytoplasm, in addition to its localization in the nucleus. Transfected cells were fixed and stained with anti-HA antibody. The yellow color and arrows in the merged image in (C) indicate co-localization of Mib and RanBP9 in the cytoplasmic punctuate structures. Hoechst dye staining (blue) shows the nucleus.
|@|~(^,^)~|@|Genomic structure of(A) 14 exons of
(A) Control (Cont) and
(A) Whole-mount
Retinal sections were stained with an antibody against Glutamine synthetase, which is used as a molecular marker for the Müller glia cells. Glial cell differentiation is also affected in