Zebrafish Klf4 maintains the ionocyte progenitor population by regulating epidermal stem cell proliferation and lateral inhibition

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition.

In zebrafish embryos, ionocytes and keratinocytes are derived from common precursors in the ventral non-neural ectoderm and express a dominant-negative form of p63 (ΔNp63) at the late gastrula stage [7]. ΔNp63 was shown to be a direct target of BMP signaling, which is essential to promote ectodermal cell differentiation into epidermal cells [8]. The functional consequences of ΔNp63 were revealed in a study that showed reduced expression of gata2 nonneural marker and enhanced expression of neuroectoderm markers (six3.1 and pax2.1) during gastrulation, suggesting an early role for ΔNp63 as a repressor of neural specification in the ventral ectoderm. In addition, ΔNp63 morphants were reported to have lost fin fold and pectoral fins, which was attributed to defective p53 inhibition. Thus, ΔNp63 also plays a late role, after 20 hours post fertilization (hpf), in the maintenance of epidermal proliferation [8,9]. Although ionocyte progenitors transiently express ΔNp63 from the bud to the 14 somite stage, knockdown of ΔNp63 does not abolish proliferation or differentiation of ionocytes [7,10], indicating that p63 is unlikely to be a master regulator of proliferation control in ionocyte progenitors.
Delta-Notch-mediated lateral inhibition determines whether cells from the p63 + ventral ectoderm will become ionocytes or keratinocytes [7,10]. Epidermal cells expressing high levels of Dlc ligand become ionocyte progenitors, and Dlc binding to Notch1a/3 receptors on neighboring epidermal cells prevents them from adopting the same cell fate. Without dlc expression, the neighboring cells develop into keratinocytes [10]. Lateral inhibition is widely known to be regulated by signaling strength, and a recent study on Drosophila sensory organ precursor (SOP) cells showed that cellular proliferation also plays a crucial role in determining lateral inhibition-controlled tissue patterning [11]. In addition to Notch signaling strength and proliferation rates, there are several other potential mechanisms by which lateral inhibition may be modulated. These mechanisms include effects on the extent of the lateral inhibition domain, or control of delta expression levels after cell division. These multiple control processes may be influenced by a master regulator that differentially modulates epidermal stem cells and ionocyte progenitors during lateral inhibition, however, such a master regulator has not yet been identified.
After the progenitor cell fate is determined by lateral inhibition, expression of two winged helix/forkhead transcription factors (foxi3a and foxi3b) can be observed in ionocyte progenitors during late gastrulation. Knockdown of foxi3a abolishes the development of ionocytes, including HR and NaR cells, indicating a requirement for Foxi3a in the specification and differentiation of ionocytes. Furthermore, a positive feedback regulatory loop between Foxi3a and Foxi3b is thought to control specification into different ionocyte subtypes [10]. This loop creates individual expression profiles for the two proteins that differentially regulate downstream determination factors, such as glial cell missing 2 (gcm2), to specify ionocyte progenitors into HR cells or NaR cells [12,13]. Thus, a self-organized and evenly distributed pattern of ionocytes emerges in the epidermal tissue, and factors that influence ionocyte progenitor proliferation would be expected to affect all types of matured ionocytes.
Mammalian krüppel like factor 4 (KLF4) is a zinc-finger transcription factor [14] that is composed of an N-terminal activation domain, a central repression domain, and three zincfinger DNA binding motifs at the C-terminus. The protein is expressed and functions in a variety of tissues, including intestinal epithelium and skin [15,16]. Interestingly, Klf4 -/mice die shortly after birth because of defects in the skin barrier function [17]. An epidermal permeability barrier consists of several layers, including the outer stratum corneum, which is composed of a cornified envelope and lipid bilayers; it is the development of the cornified envelope that is selectively affected in Klf4 -/mice [17,18]. Mechanistically, KLF4 positively regulates expression of Sprr2a, which encodes a proline-rich protein in the cornified envelope, and nine different Keratin genes, which form keratin filaments. Thus, KLF4 is known to be essential for terminal differentiation of skin epidermis [17,19].
KLF4 also acts as an oncogene or tumor suppressor in a context-dependent manner [20,21]. The tumor suppressor activity is related to induction of cell cycle arrest via transcriptional upregulation of the CDKN1A gene, which encodes p21 Cip1 . Correspondingly, gastric epithelia in mice with Klf4 deficiency exhibit low levels of p21 Cip1 expression, resulting in increased proliferation [22,23]. Conversely, KLF4 may act as an oncogene by binding to the promoter of p53 and suppressing transcription of the gene [24]. In cells expressing RAS V12 , retroviral delivery of KLF4 promoted proliferation through repression of p53 and inactivation of p21 Cip1 via Cyclin D1 inhibition [24]. In addition, KLF4 was shown to regulate embryonic stem cell selfrenewal by directly enhancing Nanog expression to prevent differentiation [25]. Thus, the cellular functions of KLF4 are multifaceted, and it is still not clear how KLF4 regulates the balance of epidermal proliferation-differentiation.
Here, we report novel roles for zebrafish Klf4 in the maintenance of the ionocyte progenitor population by regulating epidermal stem cell proliferation and modulating dlc-mediated lateral inhibition. In order to examine ionocyte development in zebrafish embryos, we generated dlc transgenic lines that recapitulate endogenous dlc expression. Strikingly, dlc + ionocyte progenitor cells were absent in mutant lines with a defective KLF binding motif on the dlc promoter, which was identified by chromatin immunoprecipitation (ChIP). Furthermore, we found that Klf4 is expressed universally in p63 + epidermal stem cells located in the ventral ectoderm from 90% epiboly. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63 + stem cells, resulting in decreased numbers of p63 + stem cells, dlc + p63 + ionocyte and dlc -p63 + keratinocyte progenitors. These decreased numbers led to subsequent decreases in the densities of HR and NaR ionocytes, as well as col1a1a + keratinocytes. We found that Klf4 modulated the ionocyte progenitor population through multiple mechanisms, including p53-mediated effects on proliferation of p63 + epidermal stem cells and ionocytes, regulating the range of lateral inhibition domain, repressing dlc expression and affecting dlc + progenitor clustering.

Zebrafish Klf4 regulates the proliferation of p63 + stem cells and dlc + ionocyte progenitor cell number
Previously, we demonstrated that zebrafish Klf4 plays an evolutionarily conserved role in regulating the differentiation of intestinal goblet cells, much like its counterpart, KLF4, in mouse [26]. Because mouse KLF4 also plays an essential role in the terminal differentiation of skin epidermis, we investigated whether zebrafish klf4 is expressed in the epidermis and affects the development of ionocytes in embryos. Immunofluorescence was conducted using an anti-zebrafish Klf4 antibody, and Klf4 was found to be universally expressed in the epiblast of the deep cell layer (DEL), yolk syncytial layer (YSL), and enveloping layer cells (EVL) during gastrulation. Moreover, Klf4 staining was observed in both ventral and dorsal ectoderm, and EVL cells during early somite stages (Fig 1A).
Epidermal stem cells are marked by p63 [27], and are known to give rise to ionocyte progenitors [10]. We examined the distribution pattern of p63 and Klf4 by double immunofluorescence in stages ranging from late gastrulation to early somite. p63 expression was first observed in the ventral ectoderm of 90% epiboly and bud stage embryos, where it was highly colocalized with Klf4 (93.8 ± 8.6% in 90% epiboly and 85.5 ± 15.5% in bud, mean ± SEM) ( Fig  1B). Expanded p63 expression in the dorsal ectoderm was found in 5s and 10s embryos, and some Klf4 positive cells did not colocalize with p63 epidermal stem cells in the ventral ectoderm region at the stages screened ( Fig 1B). Those cells that only express Klf4 are not expected to be ionocyte precursors, because p63 was consistently colocalized with ionocyte precursors until at least the 14s stage [10]. In addition, colocalization of Klf4 protein and dlc mRNA was observed beginning at 90% epiboly until the bud stage, and all dlc + cells also stained positive for Klf4 (Fig 1Ca-h). At the 5s stage, some dlc + cells in the epidermal ionocyte domain (yolk ball) were not labeled by Klf4 staining (Fig 1Ci-l). dlc + ionocyte progenitor number reached a maximum at bud stage and was decreased at the 5s stage (Fig 1Cm). This result suggests that the selection of dlc + ionocyte progenitors via lateral inhibition occurs at the bud stage and that dlc degradation is initiated between the bud and 5s stage.
Since mammalian KLF4 is known to regulate embryonic stem cell self-renewal, we wondered whether zebrafish Klf4 might modulate p63 + epidermal stem cell proliferation and dlc + ionocyte progenitor number. Thus, we knocked out klf4 by CRISPR-Cas9 genomic editing [28]. Four sgRNAs were designed to target exon 4 at a position 5'-upstream of the first zinc finger motif, but only one of the sgRNAs efficiently generated a new mutant strain, which was named klf4 d5i1 and contained an indel consisting of a 5-bp deletion and a 1-bp insertion. This mutant produces a truncated Klf4 protein that consists of 328 amino acids, 27 of which are misframed, and no functional zinc finger motif (Fig 2A). We labeled 80% epiboly klf4 d5i1 F3 embryos with BrdU and fixed the embryos at the bud stage. Immunofluorescence staining was performed, using anti-BrdU and anti-p63 antibodies, in combination with fluorescence in situ hybridization, using a dlc antisense RNA probe (Fig 2B). Each klf4 d5i1 F3 embryo was genotyped after imaging. The p63 + epidermal stem cell number was reduced by 13.1% in klf4 d5i1 heterozygous mutants (klf4 +/+ 1222.5 ± 41.7 cells vs. klf4 +/-1062.3 ± 49.2 cells; t-test, p = 9.5 × 10 −4 ) and 23.5% in klf4 d5i1 homozygous mutant embryos (klf4 +/+ 1222.5 ± 41.7 cells vs. klf4 -/-935 ± 43.6 cells; t-test, p = 5.8 × 10 −9 ) compared to sibling wild-type controls at the bud stage (Fig 2Bf,k and 2Ca). These reductions were attributable to a decreased proportion of p63 + BrdU + epidermal stem cells in klf4 d5i1 heterozygous and homozygous mutant embryos at the bud stage (Fig 2Bi,n and 2Ca), suggesting that Klf4 is required to maintain the proliferation rate of p63 + epidermal stem cells.
We also knocked down klf4 by antisense morpholino oligomers (klf4 MO1 and klf4 MO2), which have been previously validated for specificity and efficacy [26]. Significant decreases in the total number of p63 + epidermal stem cells, which may be attributed to reduced proliferation rate, were identified in klf4 morphants compared to control embryos at the bud stage (S1Ca Fig). Similar declines in dlc -p63 + keratinocyte progenitor cell number and proliferation rate were detected in klf4 morphants compared to control embryos (S1Cb Fig). A substantially reduced number of dlc + p63 + ionocyte progenitor cells was found in klf4 morphants, however the proliferation rate was not altered (S1Cc Fig). We also identified a small number (< 5) of dlc + p63cells in control and klf4 morphants, which were probably the result of erroneous labeling (S1Cd Fig). Together, these results indicate that Klf4 regulates the proliferation rate of p63 + epidermal stem cells and dlc -p63 + keratinocyte progenitor cells, as well as dlc + p63 + ionocyte progenitor cell number.
Co-injection with klf4-7mm mRNA completely rescued the cell densities of foxi3a + expressing ionocytes in 24 hpf morphants, while co-injection with LacZ mRNA had no such effect (S4A Fig). In addition, Klf4 protein was scarcely detected by immunofluorescence in bud embryos injected with klf4 MOs as compared with control embryos (S4B Fig). Immunofluorescence with antibodies against Na + , K + -ATPase or H + -ATPase further confirmed that klf4 knockdown significantly reduced the densities of NaR and HR cells in a dose-dependent manner at 72 hpf, as compared to uninjected wild types or embryos injected with control MOs (S5 Fig). These results demonstrate that Klf4 affects the differentiation of NaR and HR ionocytes as well as col1a1a + keratinocytes by regulating cell densities of their progenitors.

Fig 3. Altered klf4 expression at bud or 5s stages affects cell density of ionocyte progenitors expressing dlc, foxi3a or foxi3b. (A)
The cell density of dlc + ionocyte progenitors was reduced in embryos injected with both klf4 MO1 and MO2 (c) as compared to uninjected wild type (a) or control embryos injected with klf4 5mmMO2 (b) at bud stage. Cell densities of dlc + ionocyte progenitors were quantified from the indicated area in wild-type embryos and embryos injected with klf4 5mmMO2 or both klf4 MO1 and MO2 (d). The density of dlc + ionocyte progenitors was increased in klf4-overexpressing embryos (g) compared to LacZoverexpressing (f) or uninjected wild-type (e) embryos. Cell density of dlc + ionocyte progenitors was quantified in wild-type embryos and LacZ or klf4 mRNAinjected embryos (h). (B) The density of foxi3a + ionocyte progenitors was reduced in embryos injected with both klf4 MO1 and MO2 (c), as compared to uninjected wild-type (a) or control embryos injected with klf4 5mmMO2 (b) at 5s stage. Cell densities of foxi3a + ionocyte progenitors were quantified from the indicated area in wild-type embryos and embryos injected with klf4 5mmMO2 or both klf4 MO1 and MO2 (d). The density of foxi3a + ionocyte progenitors was increased in klf4overexpressing embryos (g), as compared to LacZ-overexpressing (f) or uninjected wild-type (e) embryos. Cell densities of foxi3a + ionocyte progenitors were quantified in wild-type embryos and LacZ-or klf4-injected embryos (h). (C) The density of foxi3b + ionocyte progenitors was reduced in embryos injected with both klf4 MO1 and MO2 (c) as compared to uninjected wild-type (a) or control embryos injected with klf4 5mmMO2 (b) at 5s stage. Cell density of foxi3b + ionocyte progenitors was quantified from the indicated area in wild-type embryos and embryos injected with klf4 5mmMO2 or both klf4 MO1 and MO2 (d). The density of foxi3b + ionocyte progenitors was increased in klf4-overexpressing embryos (g) compared to LacZ-overexpressing (f) or uninjected wild-type (e) embryos. Cell density of foxi3b + ionocyte progenitors was quantified in wild-type embryos and LacZ or klf4 mRNA injected embryos (h). Embryos are shown in lateral view. Statistical significance was determined by Student's t-test. � p < 0.05; ��� p < 0.001. Scale bar, 100 μm. Error bars indicate standard error. https://doi.org/10.1371/journal.pgen.1008058.g003

Fig 4. Perturbation of klf4 expression affects the densities of NaR and HR cells at 24 hpf. (A)
The density of atp1a1a.1 + -Na + -K + -ATPase-rich (NaR) cells was reduced in yolk extensions of embryos injected with both klf4 MO1 and MO2 (c) as compared to uninjected wild types (a) and control embryos injected with klf4 5mmMO2 (b). The density of atp1a1a.1 + -NaR cells was increased in yolk extensions of embryos injected with klf4 (g) mRNA, as compared to embryos injected with the same amount of LacZ (f) mRNA, or uninjected wild type (e) embryos. Quantification of results from (a-c) and (e-g) are shown in (d) and (h), respectively. (B) The density of atp6v1aa + -H + -ATPase-rich (HR) cells was reduced in yolk extensions of embryos injected with both klf4 MO1 and MO2 (c) as compared to uninjected wild types (a) and control embryos injected with klf4 5mmMO2 (b). The density of atp6v1aa + -HR cells was increased in yolk extensions of embryos injected with klf4 (g) mRNA, as compared to embryos injected with the same amount of LacZ (f) mRNA, or uninjected wild-type (e) embryos. Quantification of results from (a-c) and (e-g) are shown in (d) and (h), respectively. Statistical significance was determined by Student's t-test. ��� p < 0.001. Scale bar, 300 μm. Error bars indicate standard error. https://doi.org/10.1371/journal.pgen.1008058.g004 Klf4 modulates epidermal stem cell proliferation epidermal stem cells to a control level, indicating that reduced p63 + epidermal stem cell proliferation is due to klf4 deficiency (Fig 5Aj-l). Because p53 is known to regulate the G1/S cell cycle checkpoint by transactivation of CDKN1A/p21 expression [29], and mammalian KLF4 was shown to repress transcription of p53, we evaluated p53 and cdkn1a/p21 expression in klf4 morphants [30]. Upregulated expression levels of p53 and cdkn1a/ p21 were found by RT-qPCR in klf4 morphants compared to control embryos at the 5s stage. Upregulation of p53 and cdkn1a/p21 was prevented in 5s embryos co-injected with klf4-7mm but not klf4ΔC-7mm mRNA, demonstrating that upregulation of p53 and cdkn1a/p21 is dependent on decreased klf4 expression (Fig 5B and 5C). A lack of cdkn1a/p21 upregulation was further observed in 5s embryos co-injected with klf4 MOs and p53 MO (Fig 5C). The decreased percentage of p63 + BrdU + epidermal stem cells was also completely rescued in embryos co-injected with klf4 MOs and p53 MO or cdkn1a MO (Fig 5Ah,i,l). Injection of p53 MO or cdkn1a MO also restored p63 + epidermal stem cell number and percentage of p63 + BrdU + epidermal stem cells in klf4 d5i1 heterozygous mutant embryos to levels comparable to klf4 +/+ sibling wild types at bud stage (S6 Fig). In addition, no apoptosis was observed in ventral ectoderm of klf4 morphants compared to wild-type and control embryos at 5s stage (S7 Fig).
In order to investigate whether Klf4 regulates p63 + epidermal stem cell proliferation in a cell-autonomous manner, we produced chimeric embryos by transplanting FITC dextranlabeled wild-type blastomeres into klf4-morphant hosts or FITC dextran-labeled klf4-morphant blastomeres into wild-type hosts. Chimeric embryos were labeled with BrdU at 80% epiboly and fixed at bud stage. Immunofluorescence was conducted with anti-FITC, anti-p63 and anti-BrdU antibodies. The difference in percentage of FITC + BrdU + p63 + cells in wild-type hosts transplanted with klf4-morphant cells compared to klf4-morphant hosts transplanted with wild-type blastomeres (S8 Fig) was greater than the difference in percentages of BrdU + p63 + cells detected in klf4-morphant embryos (Fig 5l). This unequal difference between the percentages of FITC + BrdU + p63 + and BrdU + p63 + cells may be due to variations in wildtype response to morpholino injection. Nevertheless, this result demonstrates that Klf4 cellautonomously regulates epidermal stem cell proliferation by repressing p53 expression. Thus, in klf4-deficient embryos, p53 activity is not inhibited and activates cdkn1a/p21 expression to prevent cell cycle progression.

Klf4 directly binds to the dlc promoter to modulate dlc-mediated lateral inhibition
The haploinsufficiency of klf4 d5i1 was found in p63 + stem cells but not in their direct downstream dlc + ionocyte progenitors, suggesting there may be additional regulatory mechanisms in dlc + cells. To investigate whether Klf4 binds directly to the dlc promoter in vivo, we used JASPAR, a sparse matrix multiplication benchmark for JAVA/F90/C, to identify four potential KLF binding motifs located in the 5 0 upstream region of the dlc promoter. We then used Klf4 or Myc antibodies to immunoprecipitate cross-linked chromatin from 5s stage wild-type embryos or embryos injected with klf4-Myc mRNA. The KLF binding motif, located at -756 to -747 bp, was significantly enriched in the immunoprecipitated chromatin, as measured by qPCR ( Fig 6A).
https://doi.org/10.1371/journal.pgen.1008058.g005 Moreover, we cloned the entire dlc coding gene, including nine exons and eight introns, as well as 7505 bp upstream of the transcription initiation site into a mini-Tol2-mCherry-based vector and established a stable Tg(dlc11k:mCherry) transgenic line. Expression of mCherry in the epidermal ionocyte domain, cranial ganglia, somites and presomitic mesoderm regions of Tg(dlc11k:mCherry) F1 embryos recapitulated expression patterns of endogenous dlc at the 5s stage (Fig 6Bb-e). To create a mutated KLF binding motif between -756 and -747, we cloned 296 bp of dlc exon 1 and 2840 bp upstream of the transcriptional initiation site of the dlc promoter containing a wild-type or a mutated KLF binding motif into a mini-Tol2-mCherrybased vector and established two stable transgenic lines, namely wild-type Tg(dlc3k:mCherry) and mutated Tg(dlc3kM:mCherry). mCherry expression was observed in the epidermal ionocyte domain and in nonspecific ectoderm covering the entire trunk of Tg(dlc3k:mCherry) embryos (Fig 6Bf,g). This observation suggests that the sequence between -7505 and -2840 bp upstream of dlc promoter is involved in proper patterning of dlc expression in the cranial ganglia, somite and presomitic mesoderm. In addition, more mCherry-expressing cells were detected in the epidermal ionocyte domains of Tg(dlc3k:mCherry) embryos compared to Tg (dlc11k:mCherry) embryos. This difference may be attributed to a shorter half-life for fulllength Dlc-mCherry fusion protein based on ubiquitination and degradation events. In contrast, no mCherry expression was detected in the epidermal ionocyte domain, and low level mCherry expression was observed in the presomitic mesoderm region of the mutant Tg(dlc 3kM:mCherry) transgenic line at the 5s stage (Fig 6Bh,i). Furthermore, increased mCherry protein was detected in klf4-overexpressing Tg(dlc3k:mCherry) but not Tg(dlc3kM:mCherry) transgenic embryos compared to LacZ-overexpressing Tg(dlc3k:mCherry) transgenic embryos at bud stage (Fig 6Ca-g). Taken together, these findings suggest that Klf4 binds directly to the KLF binding motif at -756 to -747 bp to modulate dlc transcription.
Mutation of the -756 to -747 upstream KLF binding motif abolished mCherry expression in the ionocyte domain, while knockdown of klf4 only resulted in decreased cell density of dlc + ionocyte progenitors at the bud stage (Figs 6Bh and 3Ac,d). These differing observations imply that additional transcription factors may bind to the KLF binding motif or act as essential cofactors for dlc expression in the ionocyte expression domain. The transcription factor, Suppressor of Hairless (Su(H)), interacts with the intracellular domain of Notch to activate downstream genes, while Su(H) DBM contains a point mutation in the DNA binding domain and acts as dominant negative to inhibit Notch signaling [31]. To examine the influence of Notch signaling on dlc expression in ionocyte progenitors, we further injected dominant-negative X-Su(H) DBM RNA into 1-cell zygotes of Tg(dlc3k:mCherry) or Tg(dlc3kM:mCherry) transgenic lines and evaluated mCherry + or endogenous dlc + ionocyte progenitor cell numbers (S9 Fig). We observed significant increases in cell numbers for both mCherry + and endogenous dlc + ionocyte progenitors in X-Su(H) DBM -injected Tg(dlc3k:mcherry) embryos with Notch inhibition. However, no mCherry + ionocyte progenitors were detected in X-Su(H) DBM RNA-injected Tg(dlc3kM:mCherry) embryos, despite the increased number of endogenous dlc + ionocyte progenitors. These results further demonstrate that the -756 to -747 KLF binding motif on dlc promoter is essential for lateral inhibition, and this motif might be additionally regulated by unknown transcription factor(s) that act downstream of Su(H).
In order to further investigate whether Klf4 acts as an activator or suppressor of dlc expression, we generated two chimeric constructs, which contained either a VP16 activator or an Engrailed repressor domain linked to a NLS sequence and Klf4 zinc finger DNA binding domain. At the 5s stage, similar foxi3a + ionocyte cell densities were detected in embryos injected with 50 pg VP16-klf4 or 130 pg LacZ mRNA compared to embryos injected with 130 pg klf4 mRNA, suggesting that Klf4 is not likely to function as activator (S10H Fig). However, abnormal embryonic development and decreased foxi3a + ionocyte cell density were identified in embryos injected with 50 pg engrailed-klf4 mRNA (S11 Fig). Based on preliminary tests of different doses, we injected a very low amount (0.1 pg) of engrailed-klf4 mRNA and observed a significant increase in foxi3a + ionocyte cell density at the 5s stage compared to LacZ-overexpressing embryos, which was similar to that seen in klf4-overexpressing embryos (S10D Fig). Together, these results suggest that Klf4 functions as repressor of dlc expression.

Zebrafish Klf4 maintains the ionocyte progenitor population by modulating Dlc-mediated lateral inhibition
Klf4 modulation of dlc + ionocyte progenitor cell number may be regulated by direct binding of Klf4 to the dlc promoter (Fig 6). Several potential explanations may account for the alteration of dlc + ionocyte progenitor number that resulted from perturbation of klf4 expression. The first potential explanation is that the cell size may be altered. To examine this possibility, we compared cell diameters after making two assumptions: (1) the ionocyte domain is a twodimensional single cell layer, and (2) dlc expression levels do not change cell size. We compared the normalized dlc + cell diameters of klf4 morphants and klf4-overexpressing embryos (S12A Fig). An approximately 5.2% larger cell diameter was measured in klf4 morphants compared to control embryos, leading us to estimate that 9.7% fewer cells should be found in the ionocyte domain. On the contrary, in klf4-overexpressing embryos, a 7.9% smaller cell diameter was observed, suggesting that 17.9% more cells should be contained in the ionocyte domain. However, the estimated cell density differences do not quantitatively match the observed reductions in dlc + ionocyte progenitor numbers (Fig 2Cc). The second possibility is that the output densities of lateral inhibition were changed by perturbation of klf4 expression. The average normalized nearest spacing between dlc + cells ranged from 1.29 to 1.54 cell diameters in both klf4 morphants and klf4-overexpressing embryos, which is close to that found in in vitro synthetic lateral inhibition circuits [32]. This observation suggests that the range of ionocyte lateral inhibition is relatively short in comparison to Drosophila SOP [11,33]. Furthermore, the normalized nearest spacing between dlc + cells is not different between klf4 morphants and klf4-overexpressing embryos (S12B Fig). Thus, the output densities of lateral inhibition seem to be unaffected by klf4 knockdown or overexpression. The third possibility is that Klf4 controls the range in which precursor cells participate in lateral inhibition. The angle between two vectors that extend from the embryo centroid as the vertex to two points on the embryo edges which flank the dlc + cell domain was measured [10]. This measurement is proportional to the total area of the ionocyte domain, and was 12.1% smaller in klf4 morphants and 36.3% larger in klf4-overexpressing embryos compared to controls (S12C Fig).
We anticipate that a combination of the effects on cell diameter (S12A Fig) and domain size (S12C Fig) is required to account for the experimentally determined differences in cell number that are presented in Fig 2Cc and Fig 3A. True quantitative comparisons are not possible due to the variation of embryo batches and sensitivity of in situ detection methods. However, future studies with live time-lapse analysis may be sufficient to fully describe the morphology of alterations induced by perturbation of klf4 expression. Nevertheless, we uncovered multiple routes by which Klf4 modulates ionocyte development, including controlling proliferation rates of epidermal stem cells, modulating precursor cell numbers prior to lateral inhibition, and influencing the range of ionocyte domain.
In addition to the three possibilities discussed above, we observed some large dlc + cell clusters in klf4 overexpressing embryos that were not observed in klf4 mutant or morphants. When we analyzed the clustering effect in klf4 morphants and klf4-overexpressing embryos, there was no difference in the percentage of dlc + connected pairs between klf4 morphants and control embryos, however, klf4-overexpressing embryos showed a significantly increased percentage of dlc + connected pairs (S12D Fig). In both control and klf4 morphants, the average maximum dlc + cluster size in an embryo was 2.1 cells, but in klf4-overexpressing embryos, the average maximum dlc + cluster size was significantly increased to 4.1 cells with a highest observed value of 9 cells (S12E and S12F Fig). Furthermore, the dlc + cell clustering phenotype does not appear to be temporary, because we detected increased foxi3a + cluster size (ranging from 4 to 6 cells) in klf4-overexpressing embryos, compared to LacZ-overexpressing embryos (2 cells) at 24 hpf (S12G Fig). Since injection of p53 or cdkn1a MO rescued proliferation of p63 + epidermal stem cells in klf4 morphants at bud stage (Fig 5), we wondered whether injection of p53 or cdkn1a MO could rescue the reduction in differentiated atp6v1aa + ionocyte cell density in klf4 morphants. Although the p53 MO-mediated rescue of atp6v1aa + ionocyte cell density did not reach statistical significance, injection of downstream cdkn1a MO did produce a significant rescue effect on atp6v1aa + ionocyte cell density of klf4 morphants at 24 hpf (S13 Fig). This result suggests that cdkn1a expression is necessary to produce the reduction in differentiated atp6v1aa + ionocyte cell density in klf4 morphants.

Klf4 maintains the ionocyte progenitor population by regulating proliferation of epidermal stem cells
In the present study, we uncovered a novel role for Klf4 in zebrafish epidermis development. In zebrafish embryos, dlc + ionocyte progenitors are specified and differentiate from epidermal stem cells during late gastrulation [10]. We showed that Klf4 is expressed in p63 + epidermal stem cells beginning at 90% epiboly (Fig 1). Knockout or knockdown of klf4 reduced epidermal stem cell proliferation, resulting in fewer stem cells, which in turn reduced the number of differentiated dlc + p63 + ionocyte progenitors (Fig 2, S1 Fig). We further demonstrated that zebrafish Klf4 regulates the epidermal stem cell population by repressing p53 expression. A significant reduction in the percentage of BrdU + epidermal stem cells was also observed in klf4 morphants and was accompanied by increased expression levels of p53 and cdkn1a/p21. Coinjection of klf4 MOs with either p53 MO or klf4-7mm mRNA reversed cdkn1a/p21 upregulation and restored the percentage of BrdU + epidermal stem cells to control level. Co-injection of cdkn1a MO also completely rescued the proportion of BrdU + epidermal stem cells, owing to the fact that cdkn1a/p21 is an essential downstream target gene of P53 in cell cycle regulation [29]. Similar rescue effects on the percentage of BrdU + p63 + epidermal stem cells were detected in klf4 d5i1 heterozygous mutant embryos after injection of p53 MO or cdkn1a MO (S6 Fig). In addition, injection of cdkn1a MO restored atp6v1aa + differentiated ionocyte cell density in klf4 morphants at 24 hpf (S13 Fig), indicating that the decreased dlc + p63 + progenitor cell number and reduced cell density of differentiated ionocytes in klf4 morphants could be attributed to upregulation of p53 and cdkn1a/p21 expression. Maintenance of epidermal stem cell proliferation also requires an intact klf4 C-terminal DNA binding domain, suggesting that Klf4 may directly suppress p53 expression (Fig 5). Mammalian KLF4 was previously shown to suppress p53 expression through direct binding to a specific element within the p53 promoter. Moreover, this repression of p53 expression is one feature that transforms KLF4 from a tumor suppressor to an oncogene [24]. Therefore, zebrafish Klf4 may have a conserved function as a suppressor of p53 expression; further study will be required to analyze potential KLF binding motifs within the zebrafish p53 promoter.
One of our especially intriguing discoveries, which stands in contrast to previous reports using different models, is that Klf4 maintains the ionocyte progenitor population by regulating epidermal stem cell proliferation [17,34]. Mammalian KLF4 suppresses keratinocyte proliferation by transcriptional activation of CDKN1A/p21 expression [35]. Nevertheless, some studies have shown that KLF4 can also facilitate cell proliferation [24,36,37]. For example, KLF4 plays an essential role in B cell development and in activation-induced B cell proliferation by regulating Cyclin D2 expression [36]. KLF4 also functions as an oncogene to promote proliferation of breast cancer and bladder cancer cells in the presence of RAS V12 -Cyclin-D1 signaling or the absence of p21 CIP1 [24,37]. Altogether, our findings further support the idea that KLF4 may exert distinct functions to regulate stem cell proliferation in a context-dependent manner.

Klf4 is a master regulator of cell proliferation-mediated tissue patterning in ionocyte development
The effects of cell proliferation on tissue patterning by lateral inhibition were largely ignored until two recent publications highlighted the issue. First, Akanuma et al. [38] showed that polarized localization of Dlc in developing zebrafish V2 neural progenitor cells determines an asymmetric fate of V2a and V2b daughter cells after cell division. Second, In Drosophila notum, Hunter et al. showed that Notch signaling-dependent cell cycle rate contributes to lateral inhibition-mediated microchaete patterning [11]. These findings demonstrated the essential role of the cell cycle in asymmetric fate and lateral inhibition-mediated tissue patterning. Similarly, we discovered that the proliferation rate of dlc + cells is lower than that of dlccells during ionocyte determination. Although the underlying mechanisms of this proliferation rate difference remain unclear, our data suggest that klf4 might be a crucial factor, since our lossof-function experiments showed closer proliferation rates between the two cell types (Fig 2Cb,  c, S1Cb,c Fig).
In the present study, we describe an important role for Klf4 in regulating epidermal stem cell proliferation and the ionocyte progenitor population, which consequently affects the patterning of ionocytes through dlc-mediated lateral inhibition. Thus, we propose a model to describe Klf4 function in the maintenance of the ionocyte progenitor population (Fig 7). In wild-type embryos, Klf4 represses p53 expression to prevent induction of cdkn1a/p21, thereby allowing proper proliferation of p63 + epidermal stem cells during late gastrulation. At the same time, Klf4 modulates Dlc-mediated lateral inhibition by repressing dlc expression via direct binding to the dlc promoter, thus maintaining proper ionocyte progenitor population and patterning. In klf4-deficient embryos, p53 expression is no longer suppressed and cdkn1a/ p21 expression is activated. cdkn1a/p21 inhibits epidermal stem cell proliferation, and as a consequence, the ionocyte progenitor population is restricted. Conversely, when klf4 is overexpressed, the ionocyte progenitor population is increased, and an aberrant lateral inhibition pattern is produced by dlc + cell clustering. These observations represent novel discoveries in tissue pattern formation by Delta-Notch signaling.

Ethics statement
All animal procedures were approved by the Academia Sinica Institutional Animal Care & Use Committee (AS IACUC) (Protocol ID: 15-12-918). All methods were performed in accordance with the approved guideline.
To generate klf4 full-length coding region with seven mismatched nucleotides at the N-terminus (klf4-7mm), PCR with 5 0 -ATGAGaCAaCCgCCaACcGAaTTcGATAGCATGGCACTG AGCGGAA-3 0 (mismatched bases are in lowercase) and 5 0 -TCACTAGTCTATAGATGGCG CTTCATGTG-3 0 (restriction site is underlined) primers was conducted, and PCR product In wild-type embryos, Klf4 represses p53 expression, preventing the induction of cdkn1a/p21, and thereby allowing proper p63 + epidermal stem cell proliferation (box 1). At a second level, Klf4 acts as a repressor, regulating Dlc-mediated lateral inhibition by binding to the dlc promoter to maintain proper dlc + ionocyte progenitor population and patterning during the initiation of ionocyte progenitor differentiation (box 2). The output patterns are illustrated in 2D cell schematics, and representative images of dlc mRNA in situ hybridization from klf4 morphants, wild-type and klf4-overexpressing embryos at bud stage are shown in box 3. Under klf4 deficiency, p53 expression is no longer repressed and cdkn1a/p21 is activated, which results in reduced proliferation of p63 + epidermal stem cell and larger/fewer dlc + progenitors after selection by lateral inhibition (box 3.1). When klf4 is overexpressed (box 3.3), more stem cells are selected as ionocyte progenitors and dlc + progenitor clusters develop.
https://doi.org/10.1371/journal.pgen.1008058.g007 was cloned into pGEMT vector. This construct was used as template and 5 0 -TCACCGGTATG AGaCAaCCgCCaACcGAaTTcGA-3 0 and 5 0 -TCACTAGTC TATAGATGGCGCTTCATG TG-3 0 (restriction sites are underlined) primers were used to conduct a second round PCR. PCR product was first digested with AgeI and blunted with Klenow fragment, followed by digestion with SpeI. Digested PCR product was then cloned into a T7TS vector digested with EcoRV and SpeI.
To create the dlc11k-mCherry plasmid, a 3230 bp long upstream region of the dlc gene from -7245 to -4016 bp was amplified by a first PCR using genomic DNA as template and 5 0 -ATA GGGCCCCATTTGAGAAGAGTGGGACA-3 0 and 5 0 -TCGCCTCACAGTAAGAAAGTCA CTGG-3 0 (restriction site is underlined) primers. A 4275 bp long upstream region of dlc gene from -4030 to +245 bp (+1 corresponding to transcription initiation site) was amplified by a second PCR using genomic DNA as template and 5 0 -CTTACTGTGAGGCGACAGTGCTA ACC-3 0 and 5 0 -TTTCCGCGGCTTTGCCTTCTTGTCTGCTA-3 0 primers. A third PCR was conducted to merge these two fragments, which comprise 7505 bp upstream of dlc gene. The products of the first and second PCRs were used as templates, and 5 0 -ATAGGGCCCCATTT GAGAAGAGTGGGACA-3 0 and 5 0 -TTTCCGCGGCTTTGCCTTCTTGTCTGCTA-3 0 were used as primers. The PCR product was then cloned into a miniTol2-mCherry vector digested with ApaI and SacII [41,42]. A 3307 bp dlc coding gene region from +219 to +3525 bp was amplified by a fourth PCR using genomic DNA as template and 5 0 -CGTTCAGTAGCAGACA AGAAGGCAAAG-3 0 and 5 0 -AACTCGAGTACCTGAGGAAGGACAGAA-3 0 primers. The final dlc 11k gene was combined by PCR using plasmid DNA containing 7.5 kb dlc upstream region and the fourth PCR product of 3.3 kb dlc coding gene as template, and 5 0 -ATAGGGC CCCATTTGAGAAGAGTGGGACA-3 0 and 5 0 -AACTCGAGTACCTGAGGAAGGACAGA A-3 0 primers. PCR product was then cloned into a miniTol2-mCherry vector digested with ApaI and XhoI.
Generation of klf4 mutants using CRISPR-Cas9 system klf4 mutant was generated using a CRISPR-Cas9 system. CCTop was used to design four sgRNAs targeting exon 4 [45]. Aligned complementary oligomers of individual sgRNA was cloned into BsmBI-digested pT7-gRNA [46]. sgRNA was synthesized using BamHI-linearized pT7-gRNA and MEGAshortscript T7 Transcription Kit (Ambion, Austin, TX, USA). klf4 sgRNA (250 pg) and Cas9 protein (500 ng; Tools, Taipei, Taiwan) were co-injected into 1-cell zygotes. Genomic DNA was isolated from pools of 10 injected embryos at 24 hpf. PCR was conducted using forward (5 0 -CGGCAGCCAGAAGAGAGAATAATGTC-3 0 ) and reverse (5 0 -TTAACACTACAACCGTCTCACTCAAATGC-3 0 ) primers, and amplified DNA was digested with T7 endonuclease I (T7E1) to evaluate deletion and insertion (indel) efficiency. Only one out of four sgRNAs showed high indel efficiency and the injected embryos were reared to adulthood. Injected fish were designated as the F0 generation. To detect the DNA sequence alterations induced by klf4 sgRNA, genomic DNA was isolated from clipped tail fin of adult F1 fish, T7E1 digestion was performed and DNA sequencing was conducted to determine whether F1 adult fish carried DNA sequence alterations. klf4 d5i1 F1 mutants containing a 5 bp deletion and 1 bp insertion in the sgRNA target site were crossed with wild-type fish to produce the F2 generation. A pair of primers (forward: 5 0 -GCTCATTTCCCCAGCCGAGG-3 0 and reverse: 5 0 -GTGTGTCCTGTGGTGGGCTTTCA-3 0 ) were used for genotyping of F3 heterozygous or homozygous mutant embryos. Since klf4 d5i1 homozygous embryos are viable, F4 homozygous adults were also maintained.
Whole mount immunofluorescence for Klf4 protein and fluorescence in situ hybridization for dlc mRNA was conducted on 3% H 2 O 2 permeable embryos. Whole-mount in situ hybridization using a digoxigenin-labeled dlc RNA probe was conducted first at 60˚C. After hybridization wash, embryos were blocked with 1% blocking reagent for 1 h before incubation with rabbit anti-Klf4 antibody (1:50) diluted in 1% blocking reagent at 4˚C overnight. After PBST (PBS + 0.1% tween 20) washes for 10 min four times, embryos were incubated with anti-rabbit Alexa-488 (1:200, Thermal Fisher Scientific) at room temperature for 3 h. Embryos were then washed with PBST and blocked with 2% blocking reagent for 1 h before incubation with anti-Digoxigenin-POD (1:500, Roche) diluted in 2% blocking reagent at 4˚C overnight. After PBST washes, embryos were incubated with TSA-Cy3 (1:50, Perkin Elmer) diluted in Amplification buffer at 28˚C for 1 h. Embryos were then washed with PBST, post fixation with 4% paraformaldehyde for 20 min, PBST washes and stored in 80% glycerol at 4˚C.
For labeling epidermal NaR and HR cells, 72 hpf-embryos were fixed with 4% paraformaldehyde at room temperature for 3 to 4 h. After two washes with solution (PBS + 0.1% triton X-100) for 5 min each time, embryos were permeabilized with 100% ice-cold acetone at -20˚C for 7 min. Embryos were then washed with dH 2 O and PBST several times, after which they were blocked with 10% serum for 1 h. Embryos were incubated with α5 monoclonal antibody against Na + -K + -ATPase (1:200, Developmental Studies Hybridoma Bank, Iowa, USA) or a polyclonal antibody against killifish H + -ATPase (1:200) [48] diluted with 10% serum at 4˚C overnight. After PBST washes, embryos then treated with anti-mouse Alexa 488 antibody (1:200) or anti-rabbit Alexa 568 antibody (1:200, Thermal Fisher Scientific) diluted in 10% serum at room temperature for 3 h. Embryos were washed with PBST and stored in 80% glycerol at 4˚C.
Immunofluorescence on chimeric embryos was conducted on fixed BrdU-exposed bud embryos that had been stored in 100% methanol at -20˚C. After rehydration and PBST washes, embryos were blocked with 2% blocking reagent for 1 h at RT. Embryos were then incubated with anti-fluorescein-POD in 2% blocking reagent (1:500) at 4˚C overnight. After several PBST washes and a rinse with Plus Amplification Diluent (Perkin Elmer), embryos were then incubated with TSA-fluorescein amplification reagent (1:100-1:150) in Plus Amplification Diluent at 28˚C for 1 h. After PBST washes, embryos were incubated in 2N HCl for 20 min and washed with PBST several times. After blocking in 1% blocking reagent for 1 h at RT, embryos were incubated at 4˚C overnight with rabbit anti-BrdU antibody (1:200; Abcam) that was diluted in 1% blocking reagent. After several PBST washes, embryos were incubated with antirabbit Alexa-647 (1:200; Thermo Fisher) in 0.5% blocking reagent at RT for 5 h. After PBST washes, embryos were incubated in mouse anti-p63 antibody (1:200) diluted in 1% blocking reagent at 4˚C for one or two days. After several PBST washes, embryos were incubated in mouse Alexa-568 in 0.5% blocking reagent (1:200; Thermo Fisher) at 4˚C overnight. Embryos were then washed with PBST and incubated in Hoechst 33342 in PBST (1:1000) for 30 min at RT. After PBST washes, 4% paraformaldehyde fixation and more PBST washes, embryos were embedded in 1% low-melting agar for confocal imaging.

BrdU labeling, TUNEL, rescue and photography
Dechorionated embryos from 80% epiboly were incubated in egg water containing 10 mM BrdU and 15% DMSO for 20 min on ice and washed with egg water. BrdU treated embryos were allowed to grow to bud stage at 28˚C before fixation with 4% paraformaldehyde at 4˚C overnight. After washing with PBST, embryos were dehydrated through a methanol series and stored in 100% methanol at -20˚C overnight. Embryos were incubated with 3% H 2 O 2 in methanol for 30 min, rehydrated with a methanol series and washed with PBST. Whole-mount in situ hybridization using digoxigenin-labeled dlc antisense RNA was conducted first at 60 or 65˚C. After hybridization washes, embryos were blocked with 2% blocking reagent at room temperature for 1 h before incubation with anti-digoxigenin-POD antibody (1:500, Roche) diluted in 2% blocking reagent at 4˚C overnight. After PBST washes, embryos were incubated with TSA-Cy3 (1:50, Perkin Elmer) diluted in Amplification buffer at 28˚C for 1 h. Once the reaction was completed, embryos were washed with PBST and incubated in 2N HCl for 20 min. Following PBST washes and blocking in 1% blocking reagent at room temperature for 1 h, embryos were treated with diluted anti-rabbit BrdU antibody (1:200, Abcam) and antimouse P63 antibody (1:200) diluted in 1% blocking reagent at 4˚C overnight. Embryos were then washed with PBST and blocked in 1% blocking reagent at room temperature for 1 h before incubation with anti-rabbit Alexa-647 antibody (1:200, Thermal Fisher Scientific) and anti-mouse Alexa-488 antibody (1:200) diluted in 0.5% blocking reagent at room temperature for 5 h. After PBST washes, cell nuclei were stained with Hoechst 33342 (1:1000) in PBST for 30 min. Embryos were then washed with PBST, fixed with 4% paraformaldehyde, more PBST washes, and stored in 80% glycerol at 4˚C. Rescue experiments were conducted by co-injection of 3 ng each of klf4-MO1 and MO2 with 50 pg klf4-7mm mRNA, 50 pg klf4ΔC-7mm mRNA, 9-12 ng of p53 MO or cdkn1a MO into 1-2 cell zygotes and embryos were allowed to develop to 80% epiboly stage before BrdU incubation.
Images of embryos were taken using an AxioCam HRC camera on a Zeiss Axio Imager M1 microscope equipped with a DIC mode. High resolution fluorescent images were taken using a Leica TCS-SP5-MP confocal microscope (Leica, Wetzlar, Germany).
To investigate the role of the KLF binding motif within -756 to -747 on dlc gene expression, 25 pg of dlc11k:mCherry, dlc3k:mCherry, or dlc3kM:mCherry plasmid and 25 pg of transposase mRNA were injected into 1 cell zygotes. Injected embryos were allowed to grow to adulthood. Positive F0 transgenic fish was screened by expression of mCherry and later crossed with wild type fish to generate F1 generation. F2 embryos of three transgenic fish lines obtained by crossing with wild-type fish were then analyzed for mCherry expression patterns at 5s stage. F2 embryos of Tg(dlc3kM:mCherry) were genotyping confirmed by sequencing.

Generating chimeric embryos by transplantation
We produced chimeric embryos by transplantation as described [49]. A 3% solution of fluorescein-conjugated dextran (MW 10,000, Invitrogen) alone or mixed with klf4 MO1 and MO2 was injected into 1-2-cell zygotes. Approximately 50 to 150 blastomeres from wild-type or both klf4 MO1 and klf4 MO2-injected embryos were transplanted into klf4-morphant or wildtype hosts at a region above the blastoderm margin at a developmental stage between sphere and 40% epiboly.

Quantification of cell number and area
The number of stained ionocytes or keratinocytes was determined using ImageJ software as follows: (i) an image was loaded in ImageJ; (ii) 'Cell counter' was selected from the 'Analyze' item in the Plugins menu; (iii) 'Initialize' was selected; (iv) the software output cell number was recorded. The ionocyte/keratinocyte domain areas in the yolk ball of bud embryos and in the yolk ball or yolk extension of 24 hpf embryos were quantified using ImageJ software as follows: (i) a scale bar image of appropriate magnification was loaded in ImageJ; (ii) a line was drawn over the scale bar to determine the conversion factor between pixel number and length; (iii) from the Analyze menu, 'set scale' was selected to define parameters, including distance in pixels, known distance, pixel aspect ratio and unit of length; (iv) a 'polygon symbol' was used to draw the outline of the yolk ball or yolk extension; (v) from the Analyze menu, 'measure' was selected to determine area.

Statistical methods
Values are presented as mean ± s.e.m. unless otherwise noted. Two-tailed Student's t-test with unequal variance was performed in Microsoft Excel.  MO1 and klf4 MO2 (c, d), as compared to uninjected wild type (a) and control embryos injected with combined klf4 5mmMO1 and klf4 5mmMO2 (b). NaR cell density in yolk balls of uninjected wild type, embryos injected with combined klf4 5mmMO1 and klf4 5mmMO2, or the indicated amounts of combined klf4 MO1 and klf4 MO2 are shown (e). (B) H + -ATPase-rich (HR) cell density was reduced in yolk balls of embryos injected with different amounts of klf4 MO1 and klf4 MO2 (c, d), as compared to uninjected wild type (a) and control embryos injected with klf4 5mmMO1 and klf4 5mmMO2 (b). HR cell density in yolk balls of uninjected wild type, embryos injected with klf4 5mmMO1 and klf4 5mmMO2, or the indicated amounts of klf4 MO1 and klf4 MO2 is shown (e). Embryos are shown in lateral view. Significance was determined by Student's t-test. �� p < 0.01, ��� p < 0.001. Scale bar, 300 μm. Error bars indicate the standard error. The percentage of connected dlc + cell pairs was increased by klf4 overexpression. A connected pair is defined as the distance between 2 dlc + cells being less than 1.25-cell diameters. (E) Maximum dlc + cells cluster number of embryos is increased by klf4 overexpression. Cell clusters are defined by number of cells that form contiguous pairs. An isolated cell (nearest distance > 1.25-cell diameter long) is cluster number 1, a paired cell has cluster number 2, A cluster of three has cluster number 3, and so on. (F) Representative images from klf4 overexpression in (E). Three images of different embryos from control and klf4 mRNA overexpression groups were selected to show the maximum cluster numbers found. Arrowheads indicate dlc + cells; # = cluster number. All measurements were made from the same data sets in Fig 3A. (G) Representative images show foxi3a + cell clusters on the yolk ball in klf4-overexpressing embryos at 24 hpf. Embryo heads to the left. Statistical significance was determined by Student's t-test. NS, not significant; � p < 0.05; ��� p < 0.001. Error bars indicate standard deviation.