Calnexin is required for zebrafish posterior lateral line development

Int. J. Dev. Biol. 57: 427-438 (2013) doi: 10.1387/ijdb.120166sl

Calnexin is required for zebrafish posterior lateral line development I-CHEN HUNG1, BOR-WEI CHERNG1, WEN-MING HSU5,6,* and SHYH-JYE LEE1,2,3,4,5,* 1 Institute of Zoology, 2Department of Life Science, 3Center for Biotechnology, 4Center for Systems Biology, 5Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei and 6Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, R.O.C.

ABSTRACT The lateral line is a mechanosensory system in fish and amphibians to detect local water flow and pressure. Development of the posterior lateral line (PLL) originates from the migrating PLL primordium (PLLP). The PLLP deposits neuromasts on the trunk during migration to the tail. Molecular dissection revealed that PLL development is associated with genes mediating cell adhesion, morphogenesis, neurogenesis and development, but the regulatory signaling network is far from completion. To further investigate candidate regulatory genes for lateral line development, we found using whole-mount in situ hybridization that calnexin, an endoplasmic reticular (ER) calcium-binding protein gene, is expressed in PLL neuromasts. Knockdown of calnexin using antisense morpholino oligonucleotides resulted in a dose-dependent reduction in neuromasts and hair cells of the PLL. Using a transgenic claudin b:gfp line, we observed a notably reduced PLLP size, but no significant migration defect in calnexin morphants. Finally, we discovered that the reduced PLLP is associated with a reduction in cell proliferation and an increase in ER stress-dependent apoptosis. These results suggest that calnexin is essential for neuromast formation during lateral line development in the zebrafish.

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Fig. 1. Sequence analysis of zebrafish calnexin. (A) Multiple alignments of calnexin amino acid sequences of the rat (NM_172008.2), mouse (NM_007597.3), human (NM_001746.3), Xenopus laevis (NM_001085946.1), zebrafish (NM_213448.1), fruit fly (NM_170407.2), and nematode (NM_066775.3) by ClustalX. Identical amino acids across all and some species are respectively shaded in black and gray. (B) Identity table of calnexin amino acid sequences. Values represent percentages of identity between two species. (C) Phylogenic tree analysis of calnexins. The horizontal length indicates the estimated time that the sequence diverged from related family members. Multiple sequences were examined with ClustalX, using bootstrapping (500 reiterations), and the output tree was plotted by the Neighbor-joining method. The scale bar indicates the distance in units of fractions of amino acids differing between two sequences. (D) Synthetic analysis of calnexins. The resident chromosome for each calnexin is indicated. The orientation and comparable length of each gene are shown by arrows. Genes neighboring calnexin are also shown. Ch, chromosome; Mb, megabase.

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Fig. 2. Spatial and temporal expression patterns of calnexin in developing zebrafish embryos. RT-PCR analyses of calnexin were performed using cDNAs from embryos at designated stages (A) and in different adult tissues (B). A 107-bp calnexin fragment was amplified, and a 524-bp ef1_ fragment was also amplified as an internal control. (C-U’) Whole-mount in situ hybridization by a calnexin antisense riboprobe was performed in embryos at designated stages. All photographs shown are in lateral view except for F and G which are in dorsal view; (H-U’) anterior to the left, dorsal to the top. Arrows indicate the notochord (G) and hatching gland (H, I). Arrowheads point to selected neuromasts (K-M); Large scale view of primordium (N) and neuromast (O). (P-S) Co-expression assay eya1 and canx. Embryos were fixed at designated times and subjected to WISH against indicated genes. Arrows point to primordia in panels P and R and neuromasts in panels Q and S, respectively. Enlarged images are shown in panels P’-S’. hpf, hours post-fertilization; dpf, days post-fertilization; Prim, primordium; PA, pharyngeal arch; SB, swim bladder.

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Fig. 3. Calnexin morpholino oligonucleotide (MO) target sites and potency examination. (A) A partial mRNA map of calnexin is presented. Black and gray boxes represent part of the coding sequence (CDS) with an ATG translation initiation site and untranslated region (UTR), respectively. The calnexin MO1- and MO2-binding sites are indicated as shown. The potency of MO1 to reduce calnexin expression was shown by coinjecting 150 pg of the pCS2+-CANX-GFP-XLT plasmid with (B,C) or without (D, E) calnexin MO1; then it was cultured, observed under a bright (B,D) or dark field through an FITC filter (C,E), and photographed at 10 h post-fertilization (hpf). The percentage (%) of green fluorescent protein (GFP)-expressing embryos for each treatment is shown as the mean ( standard error in (F). Different letters at the top of each column indicate a significant difference between groups at p < 0.05. Scale bar: 200 +m. The number within each column is the sample size.

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Fig. 4. Knockdown of calnexin dose-dependently inhibited the formation of neuromasts. (A) Diagram of a fish neuromast (modified from (Ma and Raible, 2009). (B) A zebrafish neuromast was labeled by WGA 488 (green) and DASPEI (red) to respectively reveal its cupula and hair cells. Two large black patches are pigments. (C-I) Embryos were treated without or with indicated morpholino oligonucleotide (MO; ng/embryo) of a standard control MO (stdMO, 5 ng), calnexin MO1 or calnexin MO2, incubated until 3 days post-fertilization, stained with WGA 488, and observed and photographed under epifluorescence microscopy. calnexin mRNAs (25 pg) was added for MO rescue (G). Photographs are shown in lateral view with the anterior toward the left and posterior to the right. Only left side trunk neuromasts are indicated by white arrowheads. Trunk neuromasts are designated L1¾ L5, Lll1, and ter as shown in untreated larvae (C, Ctrl). The vague green fluorescent spots are out-of-focus neuromasts on the opposite side of the trunk. Numbers of left side neuromasts were counted (I). Different letters at the top of each column indicate a significant difference between groups at p < 0.05. Scale bar: 200 am. The number within each column is the sample size.

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Fig. 5. Knockdown of calreticulin causes a reduction in posterior lateral line neuromasts. (A) A partial mRNA map of calreticulin is presented with indicated MO binding sites. The potency of MO1 to reduce calreticulin expression was shown by co-injecting 150 pg of the pCS2+-CRT-GFP-XLT plasmid with (B,C) or without (D,E) calreticulin MO1; then it was cultured, observed under a bright (C,E) or dark field (B,D), and photographed at 10 hpf. The percentage (%) of green fluorescent protein (GFP)-expressing embryos for 5ng calreticulin MO1 or calreticulin MO2 for each treatment is shown as the mean ( standard error in (F,G), respectively. Embryos were treated, observed and shown as described previously. Numbers of neuromasts on the left side of the trunk in all treatments were counted and are shown in (H). Different letters at the top of each column indicate a significant difference between groups at p < 0.05. Scale bar: 200 +m. The number within each column is the sample size.

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Fig. 6. Knockdown of calnexin dose-dependently inhibited neuromast hair-cell formation. Embryos were treated with different dosages of morpholino and calnexin mRNA are stained with DASPEI instead of WGA488 at 72 hpf to reveal neuromast hair cells. Representative photographs of one neuromast are shown in (A). Total hair-cell numbers of the L1¾L3 trunk neuromasts in all treatments were counted and are shown in (B). Scale bar: 25 +m. The number within each column is the sample size.

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Fig. 7. Calnexin morpholino oligonucleotide (MO)-induced lateral line defects are independent of p53. Embryos were treated without (Ctrl) or with 5 ng of the calnexin MO1 (MO) in the presence or absence of 2.5 or 5 ng of p53 MO until 3 day post-fertilization (dpf). Numbers of trunk neuromasts (A) and neuromast hair cells (B), and the survival rate (C) are shown. Treated embryos were also stained with the vital dye, acridine orange, at 36 hpf to observe apoptotic cells and photographed under epifluorescent microscopy. Representative photographs for all treatments are shown in lateral view with the anterior to the left and posterior to the right (D-F). Scale bar: 200 +m. The number within each column represents the sample size.

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Fig. 8. Loss of calnexin reduced primordium size but did not affect migration of the posterior lateral line primordium (PLLP). (A-D) Transgenic claudin b:gfp embryos were treated with 5 ng std MO and with 3.75 ng of the calnexin MO1 and imaged at 30 and 36 hpf. Embryos were observed and photographed under epifluorescence microscopy. Scale bar: 200 +m. (E) Ratios of the migration path of the PLLP to body length at 30 and 36 hpf are shown. (F-K) Transgenic claudin b:gfp embryos were treated without or with MO1, cultured until 32 hpf, stained with DAPI and examined under confocal microscopy for GFP (excitation: 455-490 nm; emission 500-540 nm) and DAPI signal (excitation: 350 nm; emission 470 nm), respectively. Representative confocal photographs of different severity of PLLP defects are shown in (F-H) and the percentages of embryos with different severities are shown in (I). Photographs of control embryos (J) and MO1 morphants (K) stained with DAPI are shown. (L) The average cell numbers in PLLPs are shown. * *Indicates that the value significantly differs from that of control embryos (p < 0.001).Scale bar: 50 +m. Photographs are shown in lateral view with the anterior to the left and posterior to the right.

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Fig. 9. Loss of calnexin inhibited cell proliferation and enhanced apoptosis in the posterior lateral line primordium (PLLP). (A) Transgenic claudin b:gfp embryos were treated without and with 3.75 ng of the calnexin MO1 and collected at 32 hpf. Embryos were labeled with BrdU to reveal proliferating cell and the PLLP was shown by immunostaining against GFP (green). Embryos were examined under confocal microscopy. Photographs are shown in each channel and superimposed images are shown at the bottom. (B) The proliferation rate was estimated by the average number of BrdU-positive cells per 0.001 mm2 of PLLP. (C) Nuclei of treated embryos were stained with DAPI in blue and apoptotic cells were revealed by a TUNEL staining in green. The average numbers of apoptotic cells in the PLLP are shown in (D). (E) Embryos were treated as above and subjected to real-time PCR analysis against designated endoplasmic reticular stress-related genes at 36 hpf. (F) WISH against bip was performed and photographed to show the posterior (left) and PLLP (right) regions. Black dotted lines outline the PLLP region. * The value significantly differs from that of control embryos (p < 0.05). Photographs are shown with the anterior to the left. Scale bar: 50 +m.

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7$%/( PRIMERS USED IN THIS STUDY Primers sets for gene cloning and RT-PCR Oligo Name

Accession No.

Direction

Oligo`s sequence 5` > 3`

Cloning of full-length Calnexin calnexin

NM_213448

forward

5’-ATGGAACTAAAAATGCGCCTTTGCGTGGC GCTGCTCTCTCT-3’

reverse

5’-AAAGAATTCATGGGGGAAGAGGGATGTTG-3’

forward

5’- AAAAAGCTTCGGACTATGGAGCTGAAGATGC-3’

RT-PCR for detecting gene expression calnexin

ef1˞

NM_213448

reverse

5’-AAAGAATTCATGGGGGAAGAGGGATGTTG-3’

forward

5’-CAAGGAAGTCAGCGCATACA-3’

reverse

5’-TGATGACCTGAGCGTTGAAG-3’

forward

5’- CAGATCTGGCCAAAATGCGG-3’

reverse

5’- CTACAGCTCGTCCTTCTCTTCGG-3’

Accession No.

Direction

Oligo`s sequence 5` > 3`

NM_001017750

forward

5’- CTCCATCATGAAGTGCGACGT-3’

NM_200009.2

Whole-mount in situ hybridization probes calnexin bip

Same as above NM_213058.1

Real time primer sets Oligo Name Genes for internal control actin1D ef1D

NM_200009.2

reverse

5’- CAGACGGAGTATTTGCGCTCA-3’

forward

5’- CTGGAGGCCAGCTCAAACAT-3’

reverse

5’- ATCAAGAGTAGTACCGCTAGCATTAC-3’

forward

5’-TGTGGACTCACTGTCACCAAA-3’

ER related genes atf6

NM_001110519.1

reverse

5’-TGGTTGGAGAGGTTTGGCTTT-3’ 5’-TAAGAACCGCTTCAGCCATGA-3’

ER p57

NM_001199737.1

forward reverse

5’- CGTATTTGTCTCCTTTGGCTGTT-3’

bip

NM_213058.1

forward

5’-CGAAGAAGCCAGATATCGATGA-3’

reverse

5’-ACGGCTCTTTTCCGTTGAAG-3’

ire1

NM_001020530.1

forward

5’-ATGCTGCTGTTGCTGGTTTGC-3’

reverse

5’-TACTCGGGAACCTGTATAATG-3’

xbp1

NM_131874.1

forward

5’-AAGTCCTCCTGATATCGGGAAAA-3’

reverse

5’-TGAAGAGGCTTGATTCGGTATCAT-3’

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