Zebrafish Klf11b is Required to Maintain Cell Viability by Inhibiting p53-Mediated Apoptosis.

Krüppel-like factor 10 (KLF10) regulates various cellular functions, such as proliferation, differentiation and apoptosis, as well as the homeostasis of several types of tissue. In the present study, we attempted a loss-of-function analysis of zebrafish Klf11a and Klf11b, which constitute human KLF10 homologs. Embryos injected with klf11b-morpholino (MO) showed developmental retardation and cell death, whereas klf11a-MO-injected embryos showed normal development. In klf11b-MO-injected embryos, a dramatic increase in the amount of zebrafish p53 mRNA might be the cause of the increase in that of bax. The degree of apoptosis decreased in the klf11b-MO and p53-MO co-injected embryos. These findings imply that KLF10 is a negative regulator of p53-dependent transcription, suggesting that the KLF10/p53 complex may play an important role in apoptosis for maintenance of tissue homeostasis during embryonic development. © Copyright 2022 The Korean Society of Developmental Biology.

INTRODUCTION

Krüppel-like factors (KLFs) encode C2H2-type zinc finger proteins that regulate various cellular functions, such as proliferation, differentiation, and apoptosis in the development (Black et al., 2001; Subramaniam et al., 2010). In mammals, 14 KLF genes have been identified as transcription factors; these belong to the Sp1 superfamily (Black et al., 2001). Human KLF10 was initially isolated from fetal osteoblasts treated with tumor growth factor-β (TGF-β); thus, its original name was TGF-β-inducible gene 1 (TIEG1) (Subramaniam et al., 1995). Human KLF11 (originally termed TGF-β-inducible gene 2) was identified by BLAST using the EST database for rat TIEG sequences (Cook et al., 1998). Overexpression of either KLF10 or KLF11 caused inhibition of cell proliferation and induction of apoptosis (Subramaniam et al., 2010). Mouse KLF10 knockout (KO) caused an increase in the predisposition of epidermal tumorigenesis and development of epidermal inclusion cysts after 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) treatment (Song et al., 2012). Although the increased number of BrdU-positive cells in KLF10 KO mice supports the hypothesis that cell proliferation is inhibited by human KLF10, the relation between tumorigenesis in KLF10 KO mice and apoptosis in human KLF10-overexpressing cells remains unclear. As KLF10 KO mice exhibited a lower incidence of tumors during DEN-induced hepatic carcinogenesis (Heo et al., 2015), we suspect that a loss-of-function analysis in another model animal may aid understanding of the function of KLF10.

The Zebrafish Klf family is subdivided into four groups, consistent with the classification used for their mammalian homologs (Xue et al., 2015). Zebrafish Klf9, Klf10, Klf11a, Klf11b, Klf13, and Klf16 belong to members of group 4, which may facilitate transcriptional regulation by interacting with putative binding sites of Sin3A (Xue et al., 2015). Although a previous study showed that the respective losses of zebrafish Klf3 (group 3) and zebrafish Klf6a (group 2) led to an increased number of erythroid progenitor cells (EPC) and a smaller number of erythrocytes (Xue et al., 2015), these observations may be insufficient for a comprehensive analysis of the KLF family during embryogenesis in vertebrates. Moreover, they cannot provide new insights into the functions and mechanisms of KLF family during maintenance of cell homeostasis.

In the present study, we attempted a loss-of-function analysis of zebrafish homologs of human Klf10 by using antisense MO. Zebrafish Klf11a and Klf11b were isolated and characterized as homologs of human KLF10. Investigation of zebrafish Klf11b function in maintenance of cell homeostasis showed possible interactions between klf11b and p53. Our findings suggest that the relationship between Klf11b and p53 may play an important role during embryonic development in zebrafish.

MATERIAL AND METHODS

Zebrafish maintenance

Zebrafish were maintained as described by Yeo et al. (2004). The embryos were staged in accordance with the method of Kimmel et al. (1995). AB* wild-type and Tg[islet1:GFP] (Higashijima et al., 2000) were used.

Isolation of cDNAs and RT-PCR

The sequences of zebrafish klf11a (NM001044941), klf11b (NM001077604), EF1a (NM131263), p53 (U60804), Bax (BC055592), and caspase 3 (NM131877) were identified in GenBank. Total RNA of Con-MO-, klf11a-MO-, and/or klf11b-MO-injected embryos were employed for RT-PCR. PCR was performed using the primers as described in Table 1. PCR products, so called cDNA, were subcloned into the pGEM-T vector, and then verified by sequencing.

Table 1.

Primer list

Primer name	Sequence
klf11b RT_F	5’-GGA GCT CGG ATG GAG GTC TGT-3’
klf11b RT_R	5’-ATT GCA GAC ATA GTT GCG CCT-3’
klf11a RT_F	5’-TGC GGA AAC TTC AGG CAC AGC GGT-3’
klf11a RT_R	5’-ATG AAA CGG CGC TCA CAG ACA GGA-3’
EF1a RT_F	5‘-TAT CAC CAT TGA CAT TGC TCT-3’
EF1a RT_R	5‘-CAT CTC CGG ATT TGA GAG CCT-3’
p53 RT_F	5’-GTG CAT GTC CAG GCA GAG ACA-3’
p53 RT_R	5’-TGC TGC TTA CAA AAC AAC CGA-3’
Bax RT_F	5’-GGA GTT ATC ACC ACA GCA TTG-3’
Bax RT_R	5’-CTG CTG CCA CGC CAC CTC ATA-3’
Cas3 RT_F	5’-ATG GTC GTG AAA GGA TCC CAG-3’
Cas3 RT_R	5’-TCT CCA ATC TCA CTG ATT TAA-3’

Whole-mount in situ hybridization

Whole-mount in situ hybridization was performed as described by Yeo et al. (2004). Antisense riboprobes were transcribed from cDNAs for zebrafish klf11a, klf11b, p53, Bax, and caspase-3. Photos were taken with a differential interference contrast microscope (Axiopan2; Carl Zeiss, Oberkochen, Germany).

Confocal imaging and cell transplantation

Immunohistochemistry was performed as described by Yeo et al. (2004). Anti-phospho-histone H3 was used as the primary antibody (Upstate). AlexaFluor 568-conjugated goat anti-rabbit IgG was used as secondary antibody (Molecular Probes). Photos were taken with a confocal laser scanning microscope (LSM700; Carl Zeiss).

Cell transplantation was performed as described by Jung et al. (2012). Cells from klf11b-MO- and fluorescein-dextran-injected embryos, as well as from klf11a-MO- and rhodamine-dextran-injected embryos, were used as donors. Cell transplants were performed at the mid-blastula stage. At 10 h after transplantation, embryos were fixed with 4% PFA and mounted using Vectashield with DAPI (Vector Labs). Confocal images were taken at the 15-somite stage with a confocal laser scanning microscope (LSM700; Carl Zeiss).

Morpholino (MO) injection

Microinjection was performed as described by Yeo et al. (2004). Spliced klf11a-MO (5’-TAG AGT CAC TCA CCA GTG ATG AGA G-3’; Gene Tools, Philmath, OR, USA), 4-bp mismatched klf11a control-MO (5’-TAG AGA CAC TGT CCA GTC ATG AGA G-3’; Gene Tools), spliced klf11b-MO (5’-GGT CAT GCA CTG CAG GTA AAC AGG A-3’; Gene Tools), 3-bp mismatched klf11b control-MO (5’-GGT CAA GCA CAC CAG GTA AAC AGG A-3’; Gene Tools), and p53-MO (5’-GCG CCA TTG CTT TGC AAG AAT TG-3’; Gene Tools) (2–4 ng) were injected into fertilized zebrafish eggs.

Northern blot

Total RNA was extracted from embryos at the 1-cell stage, as well as at 6-, 12-, and 24-hpf using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA). Their isolation was performed as described by manufacturer. Total RNA of Con-MOs-, klf11a-MO-, and/or klf11b-MO-injected embryos were used for Northern blot analyses. DIG-labelled zebrafish klf11a, klf11b, EF1a, p53, Bax, and caspase-3 were used as probes. Anti-Dig secondary antibodies were detected with an ECL kit (KPL, Boca Raton, FL, USA).

TUNEL assay

The TUNEL assay was performed as described in Song et al. (2012). Zebrafish embryos injected with Con-MOs or klf11b-MO were fixed with 4% PFA overnight and then dehydrated with methanol. Apoptotic cells were detected using the In Situ Cell Death Detection Kit, in accordance with the manufacturer’s instructions (Roche, Basel, Switzerland).

RESULTS

Expression of zebrafish klf11a and klf11b

Two zebrafish homologs of human KLF10 were identified and characterized in silico using GenBank and the Sanger zebrafish genome assembly (www.ensembl.org). The predicted amino acid (AA) sequence of human KLF10 had approximately 54% and 49% similarity to predicted sequences of zebrafish Klf11b and Klf11a, respectively, whereas the predicted sequence of human KLF11 had approximately 57% and 57% similarity to predicted sequences of zebrafish Klf11a and Klf11b, respectively. Therefore, zebrafish Klf11a was classified as a homolog of human KLF11, while zebrafish Klf11b was classified as a homolog of human KLF10.

To examine the spatial and temporal expression of zebrafish homologs of human KLF10/11, we performed whole-mount in situ hybridization using klf11a and klf11b genes at various stages (Fig. 1). A very low level of klf11b mRNA was detected ubiquitously in zebrafish embryos at the gastrula stage (data not shown). At 12 h post-fertilization (hpf), mRNA expression of klf11b was concentrated in the hindbrain primordia of the neural plate (Fig. 1A). This expression in the neural plate was maintained in the hindbrain at 24 hpf (Fig. 1B). At this stage, klf11b mRNA was highly expressed in the olfactory bulb, lens, and somites, while weaker expression levels were detected in the nervous system (Figs. 1B and C). Transcripts of klf11b were also present in the pronephric duct (PND) and lateral primordia (Figs. 1C–E). At 72 hpf, transcripts of klf11b were consistently observed in the PND, with weaker expression levels seen in the nervous system (Fig. 1F). Northern blot analysis of zebrafish klf11 homologs showed maternal expression of their transcripts (Fig. 1G). Although transcripts of klf11a were difficult to detect by whole-mount in situ hybridization until 12 hpf, Northern blot analysis showed that klf11a mRNA was present at low levels from 12 hpf through 24 hpf (Fig. 1G). In contrast to the relatively broad expression of klf11b at 24 hpf, klf11a was expressed in spatially restricted domains of telencephalon, as well as in the anterior lateral line at low levels (Figs. 1H and 1I). At 72 hpf, transcripts of klf11a were persistently observed in telencephalon, with strong expression in the nervous system (Fig. 1J).

Fig. 1.

Expression of zebrafish klf11a and klf11b.

Dorsal views (A, B, and I), lateral views (C–F, H, and J), and anterior to the left. (A–F), Expression of klf11b mRNA at 12-h post-fertilization (hpf) (A), 24-hpf (B–E), and 72-hpf (F). Transcripts of klf11b are detected in the hindbrain (bracket), olfactory bulb (OB, arrowheads), lens (asterisks), lateral primordial cells (LP, dotted line), and pronephric ducts (PND, arrows). (G), Northern blot analysis for zebrafish klf11a, klf11b, and elongation factor-1alpha (EF1α) mRNA at different stages, as indicated (1-cell stage and 6–24 hpf). In total, 20 μg of total RNA from the embryo are applied to each lane. The bottom panel shows the corresponding 18S and 28S rRNA bands. (H–J), Expression of klf11a mRNA at 24-hpf (H, I) and 72-hpf (J). Transcripts of klf11a are detected in the ventral telencephalon (Tv, open arrowheads) and the anterior lateral line (aLL, open arrows). OpP, optic primordium; OtP, otic primordium; OV, otic vesicle. Scale bars, 100 μm (A–F, H–J).

Zebrafish klf11b morphant shows cell death

Our observation that transcripts of both klf11a and klf11b are maternally expressed suggested that they may be involved in control of early embryogenesis. As an initial step in analysis of the role of zebrafish homologs of human KLF10/11, we attempted to knock down klf11a and klf11b by injecting their MOs (ATG-MOs, which bind to a sequence on, or close to, the ATG start site of mRNA) into the embryos. A mixture of ATG-MOs caused severe developmental defects that interfered with analysis of morphological abnormalities after the gastrula stage (data not shown). Thus, we designed splice-site MOs to inhibit zygotic transcripts (Fig. 2). Reverse-transcription (RT)-PCR showed that embryos injected with 4 pg of klf11b-MO or klf11a-MO had abnormal amplicons of 1,728 base pairs (bp) or 1,721 bp at the 15-somite stage, respectively (Fig. 2A). Three bp-klf11b mismatch-MO was used as a control, and embryos injected with this MO did not generate alternate transcripts (Lane 3 in Fig. 2A). However, 4 bp-klf11a mismatch-MO slightly blocked pre-mRNA splicing of klf11a (Lane 2 in Fig. 2A). Abnormalities could be observed in embryos injected with a 4-pg mixture of klf11b-MO and klf11a mismatch-MO at the 15-somite stage, although these embryos developed normally through the end of gastrulation (Fig. 2C). By 16 hpf, all klf11 morphants could be separated into one of two groups: (i) moderate, characterized by cell death in the retina (Figs. 2C–2E); or (ii) severe, characterized by cell death in retina, as well as somites (Figs. 3B and 3C). The morphant phenotype was 54% moderate and 46% severe (n=52, Fig. 3). Embryos injected with 4 pg of klf11b mismatch-MO and klf11a mismatch-MO (Con-MOs) (Fig. 2B, n=50), as well as those injected with klf11a-MO and klf11b mismatch-MO (Fig. 2D, n=51), developed normally until 16 hpf (Fig. 2, Fig. 3).

Fig. 2.

Effects of zebrafish klf11a and klf11b morpholinos (MOs).

(A), Reverse transcription-polymerase chain reaction (RT-PCR) analysis shows klf11a transcripts (981 bp), klf11b transcripts (996 bp), and EF-1α transcripts (986 bp) from embryos injected with corresponding splice MOs and control MOs (Con-MOs; klf11a mismatch-MO and klf11b mismatch-MO). Unspliced RNA of klf11b (1,728 bp) is detected in embryos injected with klf11b-MO and klf11a mismatch-MO, as well as those injected with klf11a-MO and klf11b-MO (arrow in upper panel). Unspliced RNA of klf11a (1,721 bp) is detected in embryos injected with klf11a-MO and klf11b mismatch-MO, as well as those injected with klf11a-MO and klf11b-MO (arrow in lower panel). (B–E), Phenotypic analysis of klf11a and klf11b morphants. Lateral views, anterior to the left. The mixture of klf11a mismatch-MO and klf11b mismatch-MO (Con-MOs) was injected into zebrafish eggs as a control (B, n=50). At the 15-somite stage, degenerative and developmental defects are observed in embryos injected with klf11b-MO and klf11a mismatch-MO (C, n=21), as well as in embryos injected with klf11a-MO and klf11b-MO (E, n=31), but not in embryos injected with klf11a-MO and klf11b mismatch-MO (D, n=51). (F–I), Cell transplantation analysis reveals the cell-autonomous function of Klf11b. Confocal images show a klf11a-MO-and rhodamine-dextran-injected cell in the red channel (F) and a klf11b-MO- and FITC-dextran-injected cell in the green channel (G). DAPI-stained genomic DNA is shown in the blue channel (H) and the merge image is displayed (I). Scale bars, 200 μm (B–E) and 10 μm (F–I).

Fig. 3.

Phenotypic analysis of klf11a and klf11b morphants.

Lateral views, anterior to the left. The mixture of klf11a mismatch-MO and klf11b mismatch-MO (Con-MOs) was injected into zebrafish eggs as a control (A and A’, n=50). At the 15-somite stage, degenerative and developmental defects (arrowheads) are observed in embryos injected with klf11b-MO and klf11a mismatch-MO (B and B’, n=21), as well as in embryos injected with klf11b-MO and klf11a-MO (C and C’, n=31), but not in embryos injected with klf11a-MO and klf11b mismatch-MO (D and D’, n=51). Scale bars, 200 μm. MO, morpholino.

To assess whether Klf11a and Klf11b function in a cell-autonomous manner, we performed cell transplantation experiments (Figs. 2F–2I). At 3.5 hpf, cells from donor embryos that had been injected with klf11b-MO and FITC-dextran were mixed with cells from embryos that had been injected with klf11a-MO and rhodamine-dextran, and then transferred into wild-type embryos (Fig. 2I). At 16 hpf, klf11a-MO-injected cells were normally developed (Fig. 2F), whereas atypical nuclei were observed in the green channel (Figs. 2G–2H). This observation suggested that cells injected with klf11b-MO might undergo cell death, and that Klf11b functions in a cell-autonomous manner.

To assess embryos injected with klf11b-MO, we performed whole-mount in situ hybridization with p53, Bax, and caspase-3, all of which are involved in cell death in zebrafish (Fig. 4). Based on phenotypic analysis (Fig. 2C), embryos injected with klf11b-MO were fixed at the 5-somite stage (Figs. 4A–4F). Zebrafish p53 was ubiquitously expressed in Con-MO-injected embryos (Fig. 4A). However, its transcripts were dramatically increased in klf11b-MO-injected embryos (Fig. 4B). Compared to control embryos (Fig. 4C), the expression of bax, a direct target of p53, also increased (Fig. 4D). These data were consistent with the hypothesis that overexpressed Bax accelerates apoptotic cell death and counters the death repressor activity of Bcl-2 (Oltval et al., 1993). The expression of caspase-3 was examined at the 5-somite stage (Fig. 4E), which showed that overexpression of zebrafish caspase-3 induced apoptosis and served to modulate the pro-apoptotic signal during zebrafish development (Yabu et al., 2001). The expression level of caspase-3 remained unchanged, or slightly reduced, in the klf11b-MO-injected embryos compared to that in Con-MO-injected embryos (Figs. 4E and 4F). Northern blot analysis confirmed increased expression levels of p53 and bax, as well as unchanged expression levels of caspase-3, in the klf11b-MO-injected embryos (Fig. 4G). These findings suggested that the phenotype of Klf11b knockdown embryos might be rescued by reduced expression of p53. To test this, we performed the TUNEL assay in klf11b-MO- and/or p53-MO-injected embryos at 26 hpf (Figs. 4H–4K). Apoptotic cells were observed in embryos injected with Con-MO (Fig. 4H, n=50), as well as in embryos injected with klf11b-MO (Fig. 4I, n=23). However, the number of apoptotic cells was dramatically reduced in embryos co-injected with klf11b-MO and p53-MO (Fig. 4K, n=34), as well as in embryos injected with p53-MO (Fig. 4J, n=29). MO-mediated knockdown of p53 in klf11b-MO-injected embryos rescued the apoptotic phenotype, implying that the function of Klf11b was equivalent to that of p53 or was upstream of p53 in the p53-dependent apoptotic pathway.

Fig. 4.

Cell death in zebrafish klf11b morphants.

Dorsal view (A–F, H–K), anterior to the left. Whole-mount in situ hybridization shows expression of p53 (A, B), Bax (C, D), and Cas3 (E, F) in the klf11b mismatch-MO-(Con-MO) (A, C, E) and klf11b-MO-injected embryos (B, D, F) at the 5-somite stage. (G), Northern blot analysis of zebrafish p53, Bax, Cas3, and EF1α mRNA. In total, 20 μg of total RNA from Con-MO-injected embryos and klf11b-MO injected embryos are applied to each lane. (H–K), TUNEL assay was performed on Con-MO-(H), klf11b-MO-(I), p53-MO-(J), and a mixture of klf11b-MO- and p53-MO-injected embryos (K) at 28 hpf. Apoptotic cells (blue dots) are observed in embryos injected with klf11b-MO (I, n=23), as well as those injected with Con-MO (H, n=50), but small numbers in embryos injected with p53-MO (J, n=29) or co-injected with klf11b-MO and Con-MO (K, n=34). Scale bars, 200 μm (A–F, H–K). MO, morpholino.

Zebrafish klf11b morphant reduces the number of proliferative cells

To determine which cell types are most strongly affected after knockdown of klf11b function, Tg[islet1-GFP] embryos were injected with klf11b-MO, and then labeled with an antibody that recognized phosphorylated histone H3 on Ser-10 (PH3), an established marker for chromosome condensation during mitosis (Fig. 5, Hendzel et al., 1997; Higashijima et al., 2000). In the Tg[islet1-GFP] line, GFP mRNA is expressed in a majority of cranial motor neurons, in a manner similar to that of islet-1 mRNA, beginning at 21 hpf (Higashijima et al., 2000). At 36 hpf, GFP-positive cranial motor neurons were well organized spatiotemporally in the central nervous system (CNS) of Con-MO-injected embryos (Fig. 5A), whereas morphological and spatial disorganization of GFP-positive cells was observed in the CNS of klf11b-MO-injected embryos (Fig. 5D). Although the number of GFP-positive cells was difficult to determine, the number of PH3-positive cells significantly decreased in klf11b-MO-injected embryos (Fig. 5E; 122.3±9.3, n=6), compared to that in the Con-MO-injected embryos (Fig. 5B; 342.8±8.7, n=4). These observations implied that reduction of proliferating precursors may be caused by apoptosis.

Fig. 5.

KLF10 negatively regulates p53 activity as well as transcription of p53.

(A), Human KLF10 (hKLF10) abolish activity of the human p53 promoter in transiently transfected HEK 293 cells (mean±SD, n=3 independent transfections, * p<0.01). HEK 293 cells were co-transfected with p53-Luc reporter (2 μg) and increasing amounts of myc-hKLF10 (0, 0.5, 1, and 2 μg) plasmid DNAs. (B), Human KLF10 modulates p53-dependent transcription. HEK 293 cells were co-transfected with PG13-Luc reporter (2 μg) and increasing amounts of myc-hKLF10 (0, 0.5, 1, and 2 μg) plasmid DNAs (mean±SD, n=3, ** p<0.01). Lower panel, Western blot analysis of myc-hKLF10, p53 and alpha-acetylated Tubulin (αTb) were performed to determine dose dependency of myc-hKLF10 in HEK 293 cells. (C), Upper panel, endogenous p53 interacts with hKLF10. HEK 293 cells transfected with Myc epitope-tagged full-length hKLF10 were subjected to immunoprecipitation (IP) with anti-Myc antibody. The immunocomplexes were analyzed by Western blot analysis with antibodies against p53 and Myc tag. Lower panel, pulldown assay of [35S] methionine-labeled p53 ([35S]Met-p53) were performed as described in materials and methods, by using purified GST and GST-hKLF10 fusion proteins. (D), Interaction between p53 and hKLF10 in the mammalian two-hybrid assay. The luciferase reporter pG5-Luc was co-transfected with the pairs of VP16-hKLF10 and p53-Gal4 into HEK 293 cells (1 μg of each). The experiment was repeated with a similar result (mean±SD, n=3, *** p<0.01). KLF, Krüppel-like factors.

DISCUSSION

We identified and characterized zebrafish Klf11b as a homolog of human KLF10. Structural comparisons showed that zebrafish Klf11 homologs are highly conserved, with respect to human KLF10 and KLF11. Both zebrafish klf11a and klf11b mRNA are maternally expressed. During zebrafish development, klf11a is expressed in the ventral telencephalon and anterior lateral line, while klf11b is highly expressed in the olfactory blub, lens, PNDs, lateral primordia, and somites. However, only klf11b transcripts can be detected at the gastrulation stage. This might be the cause of epiboly retardation in klf11b morphants, as well as the source of their degenerative phenotype after this stage. Although structural preservation of zebrafish Klf11 homologs strongly suggests conservation of KLF homolog functions during evolution, the function and expression according to specific spatiotemporal patterns may have varied among individual subtypes.

Structural alignment showed that three zinc-finger domains of human KLF10 had approximately 79% and 84% similarity to zebrafish Klf11a and Klf11b, respectively, whereas human KLF11 had approximately 88% and 93% similarity to zebrafish Klf11a and Klf11b, respectively. Although zebrafish Klf11b is classified as a homolog of human KLF10 based on comparison of the overall AA sequence, zinc-finger domains of zebrafish Klf11b showed greater similarity with those of human KLF11. This may be why zebrafish Klf11a and Klf11b were characterized as human KLF11 homologs. In the zebrafish genome assembly, another Klf homolog is present: Klf10, in chromosome 16. Human KLF10 and zebrafish Klf11b consist of 469 and 458 AAs, respectively, whereas zebrafish Klf10 only contains 218 AAs. Comparison of predicted AA sequences showed that the zinc-finger domain (approximately 132 AAs) of zebrafish Klf10 had approximately 69%, 71%, 68%, and 68% similarity to zebrafish Klf11a, zebrafish Klf11b, human KLF10, and human KLF11, respectively. However, neither whole-mount in situ hybridization nor RT-PCR could detect zebrafish Klf10 transcripts (data not shown), suggesting that zebrafish Klf10 might be a pseudogene.

Northern blot analysis has shown that human KLF10 (TIEG1) transcripts are highly expressed in skeletal muscle, and moderately expressed in heart, placenta, and pancreas, whereas KLF11 (TIEG2) mRNA is ubiquitously expressed, with the highest levels occurring in the pancreas and skeletal muscle (Subramaniam et al., 1995; Cook et al., 1998). Human KLF11 is more prominently expressed in the pancreas, whereas KLF10 is expressed at higher relative levels in skeletal muscle (Cook et al., 1998). In zebrafish, whole-mount in situ hybridization showed that only klf11b transcripts are present in muscle, although both klf11a and klf11b are highly expressed in the brain of 3-day-old embryos. Taken together with the AA sequence comparison, the most likely classification of zebrafish Klf11b is as a homolog of human KLF10.

In the present study, we showed that a reduction in the number of proliferative precursor cells may be the result of cell death in zebrafish Klf11b morphants, suggesting that such cell death may be mediated by p53. Biochemical analysis might elucidate that a KLF gene might be a negative regulator of p53 transcription. Based on these observations, we propose three models: first, zebrafish Klf11b may function as a transcriptional repressor, with or without a corepressor, by binding to Sp1 elements of the p53 gene; second, zebrafish Klf11b may function as a corepressor by binding to p53 protein; and finally, DNA-binding Klf11b may mask the function of p53 as a transcriptional activator. Although interaction of the corepressor mSin3A with an alpha-helical motif of Klf11/ Klf10 supports one of our hypotheses (Zhang et al., 2001), previous studies have shown that overexpression of human KLF10 in cancer cell lines induced apoptosis, in a manner similar to that of the mitochondrial pathway (Tachibana et al., 1997; Ribeiro et al., 1999). Additionally, transgenic overexpression of KLF11 has been shown to result in a decreased number of proliferative cells, increased rate of apoptosis, and reduction in the size of the pancreas (Fernandez-Zapico et al., 2003). In human leukemia cells, overexpression of human KLF10 promoted chemically induced apoptosis through the mitochondrial pathway, as represented by upregulation of Bax, downregulation of Bcl-2, release of cytochrome C, and activation of caspase-3 (Jin et al., 2007). These studies indicate that loss of KLF10 function could promote carcinogenesis (Tachibana et al., 1997; Ribeiro et al., 1999; Fernandez-Zapico et al., 2003). Previous study provides some support for this hypothesis, based on treatment with carcinogens such as DMBA and TPA (Song et al., 2012). However, KLF10-deficient mice demonstrated increased numbers of apoptotic cells in DMBA/ TPA-treated epidermis, and lower incidence of tumors, than wild-type mice, following DEN-induced liver tumorigenesis (Song et al., 2012; Heo et al., 2015).

Although human KLF6 is essential for hepatic carcinoma cells to evade p53-mediated apoptosis in vitro (Sirach et al., 2007), our study provides the first observation concerning the relationship between KLFs and p53 in vivo. To better understand the function of KLFs, further studies are needed to address their roles in apoptosis, as well as in tumorigenesis. First, KLF10 and KLF11 double KO mice could help determine the functions of these proteins. In humans, KLF10 and KLF11 exhibit approximately 84% similarity, and their expression pattern is very similar in adult human tissues (Cook et al., 1998). Based on the principal of genetic redundancy, KLF10 and KLF11 double KO mice may show functions of KLFs during gastrulation, as the expression of mouse KLF11 is unclear during early embryonic development. As described above, zebrafish klf11b is expressed at the gastrulation stage, whereas klf11a is not, and the apoptotic phenotype is observed after gastrulation. Second, co-immunoprecipitation with the KLF11b and p53 antibody as well as isolation and characterization of genes downstream of the KLF11b/p53 complex could aid in characterizing their functions. Finally, examination of p53 stability in the absence of mammalian KLF10 could help determine its function. Mungamuri and colleagues reported that USP7 deubiquitinase protected SUV39H1 from MDM2-mediated ubiquitination in the absence of a p53 stimulus (Mungamuri et al., 2016).

In summary, we have shown that Klf11b morphants undergo apoptosis, which may be mediated by p53. Zebrafish Klf11b morphants could be used in genetic screening for identification of unknown genes, as well as for identification of small molecules involved in chemically induced cell death. This model will greatly facilitate understanding of the homeostasis underlying cell viability, which is mediated by the interaction between KLF and p53.