Gao Y et al. (2015), Kruppel-like factor family genes are expressed ...

XB-ART-51023

Dev Dyn 2015 Oct 01;24410:1328-46. doi: 10.1002/dvdy.24310.

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Kruppel-like factor family genes are expressed during Xenopus embryogenesis and involved in germ layer formation and body axis patterning.

Gao Y , Cao Q , Lu L , Zhang X , Zhang Z , Dong X , Jia W , Cao Y .

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BACKGROUND: Kruppel-like factors (Klfs) are a family of transcription factors consisting of 17 members in mammals, Klf1-Klf17, which are involved in fundamental cellular physiological procedures, such as cell proliferation, differentiation, and apoptosis. However, their functions in embryonic development have been poorly understood. Our previous study has demonstrated that the pluripotency factor Klf4 participates in germ layer formation and axis patterning of Xenopus embryos by means of the regulation of key developmental signals. In the present study, we further investigated comprehensively the expression and functions of the klf family genes, klf2, klf5, klf6, klf7, klf8, klf11, klf15, and klf17, during the embryogenesis of Xenopus laevis. RESULTS: Spatio-temporal expression analyses demonstrate that these genes are transcribed both maternally and zygotically in Xenopus embryos, and during organogenesis and tissue differentiation, they are localized to a variety of placodes and tissues. Gain and loss of function studies manifest that Klf factors play different roles in germ layer formation and body axis patterning. Moreover, each Klf factor exhibits distinct regulatory effects on the expression of genes that are essential for germ layer formation and body axis patterning. CONCLUSIONS: These results suggest that Klf factors are involved in the fine-tuning of these genes during early embryogenesis.

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Species referenced: Xenopus laevis
Genes referenced: cer1 chrd dkk1 foxa4 foxi1 gsc klf10 klf11 klf12 klf13 klf15 klf17 klf2 klf3 klf4 klf5 klf6 klf7 klf8 klf9 lhx1 nodal nodal5 odc1 prnp sox17a sox2 sox3 tbl1x tbxt trim33 vegt wnt8a wnt8b
???displayArticle.morpholinos??? klf11 MO2 klf15 MO1 klf2 MO2 klf5 MO1 klf6 MO1 klf7 MO1 klf8 MO2

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	Figure 1. Genetic relationship between human and Xenopus Klf factors and temporal expression analysis of klf genes. A: Phylogenetic analysis using protein sequences of human KLF1 (Protein ID: Q13351), KLF2 (Protein ID: Q9Y5W3), KLF3 (Protein ID: P57682), KLF4 (Protein ID: O43474), KLF5 (Protein ID: Q13887), KLF6 (Protein ID: Q99612), KLF7 (Protein ID: O75840), KLF8 (Protein ID: O95600), KLF9 (Protein ID: Q13886), KLF10 (Protein ID: Q13118), KLF11 (Protein ID: O14901), KLF12 (Protein ID: Q9Y4X4), KLF13 (Protein ID: Q9Y2Y9), KLF14 (Protein ID: Q8TD94), KLF15 (Protein ID: Q9UIH9), KLF16 (Protein ID: Q9BXK1), KLF17 (Protein ID: Q5JT82), Xenopus laevis Klf2 (Genbank Accession number: NP_001080430), Xenopus tropicalis Klf3 (XP_002938694), Xenopus laevis Klf4 (AEK12773), Klf5 (Encoded by Gurdon EST transcript: Xl3.1-IMAGE: 6955854.5), Klf6a (Encoded by cDNA with Accession number: KP013621), Klf7 (NP_001080906), Klf8 (NP_001082615), Klf9 (NP_001079066), Klf10 (NP_001089340), Klf11 (NP_001086010.1), Xenopus tropicalis Klf12 (Ensembl: ENSXETP00000010232), Klf13 (NP_001093690.1), Xenopus laevis Klf15 (NP_001084413.1), and Klf17 (NP_001082133). Phylogenetic tree was generated using the program Clustal Omega at www.ebi.ac.uk. B: Comparison of colinearity of genes flanking KLF17 in human and klf17 in Xenopus tropicalis. Diagram was drawn according to the gene colinearity from the Ensembl genome browser (http://asia.ensembl.org). C: Temporal expression patterns of the klf genes during the embryogenesis of Xenopus laevis as detected with semi-quantitative RT-PCR. odc (ornithine decarboxylase) was used as loading control. gsc (goosecoid) was used as a control to show the absence of maternal transcription. RT-, reverse transcription without reverse transcriptase.Download figure to PowerPoint
	Figure 2. A–H: Spatial expression patterns klf2 (A), klf5 (B), klf6 (C), klf7 (D), klf8 (E), klf11 (F), klf15 (G), and klf17 (H) during Xenopus embryogenesis detected with whole mount in situ hybridization. a, animal view; aa, aortic arches; ad, adenohypophysial placode; an, anterior view, dorsal is up; ba, branchial arches; bl, blastopore lip; cg, cement gland; d, dorsal view, the anterior is up; da, dorsal anterior view; dv, dorsal vegetal view; ey, eye; fb, forebrain; fep, facial epibranchial placode; hb, hindbrain; he, heart anlage; hg, hatching gland; hm, hypaxial muscles; is, intersomitic vessels; l, lateral view, the animal pole is up; la, lateral view, the anterior is up and the ventral is to the left; ld, lateral dorsal view, the animal pole is up; le, lens; li, liver primordium; lu, lung primordium; mb, midbrain; nc, neural crest; np, neural plate; op, olfactory placode; ov, otic vesicle; pcv, posterior cardinal vein; pl, plexus; pn, pronephros; pop, presumptive olfactory placode; ppe, preplacodal ectoderm; prp, preplacodal region; pt, proctodeum; sd, primordia of stomach/duodenum; sm, somite; tg, trigeminal placode; v, ventral view, the anterior is up; vbi, ventral blood island; vg, vegetal view, the dorsal is up. (Xenbase curators note: gene expression is curated in separate images for each panel/ klf gene- see XB article page for isolated images).
	Figure 3. A–K: Spatial distribution of klf transcripts in st.8.5 and st.10 embryos detected with quantitative RT-PCR. Data are presented as relative levels of expression. Error bars: standard error of the mean. WE, whole embryos; An, animal explants; Eq, equatorial explants; Vg, vegetal explants; Dor, dorsal halves of embryos; Ven, ventral halves of embryos.Download figure to PowerPoint
	Figure 4. Effects of overexpression of Klf factors on embryonic development and germ layer formation. A–K: Injection of each 500 pg mRNA for Klf factors, as indicated in each panel, generated different effects on embryonic development. Lateral view is shown for uninjected control (Uninj.) and injected embryos. (Putative) dorsal is up and anterior is to the left. L: Ratios (percentages) of normal and defect embryos among the total numbers (n) of control (ctrl) embryos or embryos injected with Klf mRNAs. M: Analysis on the expression of t, sox17a and sox2 in control (ctrl) embryos and embryos injected with different Klf mRNA. Lateral view is shown for embryos with t expression, and animal pole is up. Both animal (a. view) and vegetal view (v. view) are shown for the embryos with sox17a expression. Dorsal view is shown for embryos with sox2 expression. N: Ratios (percentages) of the embryos with regular gene expression or misregulated gene expression among the total numbers (n) of control (ctrl) or injected embryos.Download figure to PowerPoint
	Figure 5. Analysis on gene expression in response to overexpression of Klf factors. A: Changes in expression of genes that are involved in germ layer formation in uninjected control embryos (ctrl) and embryos injected with mRNAs as indicated. a, animal view; l, lateral view, animal pole is up; la, lateral animal view; ld, lateral dorsal view, animal pole is up; v, vegetal view; vd, vegetal dorsal view, dorsal is up; vl, vegetal lateral view. B: Ratios (percentages) of the embryos with regular gene expression or misregulated gene expression among the total numbers (n) of control (ctrl) or injected embryos.Download figure to PowerPoint
	Figure 6. Effects of knockdown of Klf factors on embryonic development. A: Western blotting analysis on the efficiency of control MO (ctrlMO) and Klf MOs to inhibit protein translation in Xenopus embryos. Blots for Actin were similar for all the samples and only one of them was shown as control. A total of 500 pg of each mRNA and each 40 ng of ctrlMO, K2MO, K5MO, K6MO, K7MO, K15MO, 15 ng of K8MO, and 50 ng of K11MO were injected. B,H: Embryos injected with ctrlMO at tail bud (B) and tadpole (H) stages, respectively. C–G,I–J: Developmental defects after injection of different Klf MOs, as indicated in each panel. Lateral view is shown for all embryos, with the anterior being orientated to the left. ctrlMO, K8MO, K11MO, and K15MO were injected at 50 ng each, K2MO was injected at 30 ng, K5MO was 80 ng, K6MO was 40 ng, and K7MO was injected at 15 ng. K: Percent of normal or defect embryos among the total numbers (n) of each morpholino injected embryos.Download figure to PowerPoint
	Figure 7. Effects of knockdown of Klf factors on germ layer formation. A: Germ layer marker gene expression in embryos injected with different MOs. The doses of each injected MO were the same as those in Figure 6B–J. All embryos for t expression are shown as lateral view with animal pole being placed up. All embryos for sox17a expression are placed in vegetal view. All embryos for sox2 expression are shown as dorsal view with the anterior being placed to the left. B: Ratios of embryos with normal or altered gene expression among the total numbers (n) of injected embryos.Download figure to PowerPoint
	Figure 8. Analysis on the expression of genes responsible for germ layer formation in response to knockdown of Klf factors. A: Effects of knockdown of Klf factors on gene expression. The doses of each injected MO were the same as those in Figure 6B–J. All embryos are shown as vegetal view with the dorsal being placed up or as dorsal view with the animal pole being orientated up. B: Ratios of embryos with normal or disturbed transcription among the total numbers (n) of injected embryos.Download figure to PowerPoint
	Figure 9. Effect of double knockdown on gene expression. A: Effect of single knockdown and double knockdown of Klf2 and Klf7 on the expression of sox17a. B: Effect of single knockdown and double knockdown of Klf7 and Klf8 on the expression of gsc. C: Ratios of embryos with normal or disturbed transcription among the total numbers (n) of injected embryos. ctrlMO, K2MO, and K7MO were injected at 30 ng per embryo, and K8MO was injected at 50 ng per embryo. l, lateral view with animal pole to the top of each panel; v, vegetal view with dorsal side to the top of each panel.Download figure to PowerPoint
	Fig. 2. A: Spatial expression patterns klf2, during Xenopus embryogenesis detected with whole mount in situ hybridization. a, animal view; aa, aortic arches; ad, adenohypophysial placode; an, anterior view, dorsal is up; ba, branchial arches; bl, blastopore lip; cg, cement gland; d, dorsal view, the anterior is up; da, dorsal anterior view; dv, dorsal vegetal view; ey, eye; fb, forebrain; fep, facial epibranchial placode; hb, hindbrain; he, heart anlage; hg, hatching gland; hm, hypaxial muscles; is, intersomitic ves- sels; l, lateral view, the animal pole is up; la, lateral view, the anterior is up and the ventral is to the left; ld, lateral dorsal view, the animal pole is up; le, lens; li, liver primordium; lu, lung primordium; mb, midbrain; nc, neural crest; np, neural plate; op, olfactory placode; ov, otic vesicle; pcv, posterior cardinal vein; pl, plexus; pn, pronephros; pop, presumptive olfactory placode; ppe, preplacodal ectoderm; prp, preplacodal region; pt, proctodeum; sd, primordia of stomach/duodenum; sm, somite; tg, trigeminal placode; v, ventral view, the anterior is up; vbi, ventral blood island; vg, vegetal view, the dorsal is up.
	Fig. 2. B: Spatial expression patterns klf5, during Xenopus embryogenesis detected with whole mount in situ hybridization. a, animal view; aa, aortic arches; ad, adenohypophysial placode; an, anterior view, dorsal is up; ba, branchial arches; bl, blastopore lip; cg, cement gland; d, dorsal view, the anterior is up; da, dorsal anterior view; dv, dorsal vegetal view; ey, eye; fb, forebrain; fep, facial epibranchial placode; hb, hindbrain; he, heart anlage; hg, hatching gland; hm, hypaxial muscles; is, intersomitic ves- sels; l, lateral view, the animal pole is up; la, lateral view, the anterior is up and the ventral is to the left; ld, lateral dorsal view, the animal pole is up; le, lens; li, liver primordium; lu, lung primordium; mb, midbrain; nc, neural crest; np, neural plate; op, olfactory placode; ov, otic vesicle; pcv, posterior cardinal vein; pl, plexus; pn, pronephros; pop, presumptive olfactory placode; ppe, preplacodal ectoderm; prp, preplacodal region; pt, proctodeum; sd, primordia of stomach/duodenum; sm, somite; tg, trigeminal placode; v, ventral view, the anterior is up; vbi, ventral blood island; vg, vegetal view, the dorsal is up.
	Fig. 2. C: Spatial expression patterns klf6, during Xenopus embryogenesis detected with whole mount in situ hybridization. a, animal view; aa, aortic arches; ad, adenohypophysial placode; an, anterior view, dorsal is up; ba, branchial arches; bl, blastopore lip; cg, cement gland; d, dorsal view, the anterior is up; da, dorsal anterior view; dv, dorsal vegetal view; ey, eye; fb, forebrain; fep, facial epibranchial placode; hb, hindbrain; he, heart anlage; hg, hatching gland; hm, hypaxial muscles; is, intersomitic ves- sels; l, lateral view, the animal pole is up; la, lateral view, the anterior is up and the ventral is to the left; ld, lateral dorsal view, the animal pole is up; le, lens; li, liver primordium; lu, lung primordium; mb, midbrain; nc, neural crest; np, neural plate; op, olfactory placode; ov, otic vesicle; pcv, posterior cardinal vein; pl, plexus; pn, pronephros; pop, presumptive olfactory placode; ppe, preplacodal ectoderm; prp, preplacodal region; pt, proctodeum; sd, primordia of stomach/duodenum; sm, somite; tg, trigeminal placode; v, ventral view, the anterior is up; vbi, ventral blood island; vg, vegetal view, the dorsal is up.
	Fig. 2. D: Spatial expression patterns klf7, during Xenopus embryogenesis detected with whole mount in situ hybridization. a, animal view; aa, aortic arches; ad, adenohypophysial placode; an, anterior view, dorsal is up; ba, branchial arches; bl, blastopore lip; cg, cement gland; d, dorsal view, the anterior is up; da, dorsal anterior view; dv, dorsal vegetal view; ey, eye; fb, forebrain; fep, facial epibranchial placode; hb, hindbrain; he, heart anlage; hg, hatching gland; hm, hypaxial muscles; is, intersomitic ves- sels; l, lateral view, the animal pole is up; la, lateral view, the anterior is up and the ventral is to the left; ld, lateral dorsal view, the animal pole is up; le, lens; li, liver primordium; lu, lung primordium; mb, midbrain; nc, neural crest; np, neural plate; op, olfactory placode; ov, otic vesicle; pcv, posterior cardinal vein; pl, plexus; pn, pronephros; pop, presumptive olfactory placode; ppe, preplacodal ectoderm; prp, preplacodal region; pt, proctodeum; sd, primordia of stomach/duodenum; sm, somite; tg, trigeminal placode; v, ventral view, the anterior is up; vbi, ventral blood island; vg, vegetal view, the dorsal is up.
	Fig. 2. E: Spatial expression patterns klf8, during Xenopus embryogenesis detected with whole mount in situ hybridization. a, animal view; aa, aortic arches; ad, adenohypophysial placode; an, anterior view, dorsal is up; ba, branchial arches; bl, blastopore lip; cg, cement gland; d, dorsal view, the anterior is up; da, dorsal anterior view; dv, dorsal vegetal view; ey, eye; fb, forebrain; fep, facial epibranchial placode; hb, hindbrain; he, heart anlage; hg, hatching gland; hm, hypaxial muscles; is, intersomitic ves- sels; l, lateral view, the animal pole is up; la, lateral view, the anterior is up and the ventral is to the left; ld, lateral dorsal view, the animal pole is up; le, lens; li, liver primordium; lu, lung primordium; mb, midbrain; nc, neural crest; np, neural plate; op, olfactory placode; ov, otic vesicle; pcv, posterior cardinal vein; pl, plexus; pn, pronephros; pop, presumptive olfactory placode; ppe, preplacodal ectoderm; prp, preplacodal region; pt, proctodeum; sd, primordia of stomach/duodenum; sm, somite; tg, trigeminal placode; v, ventral view, the anterior is up; vbi, ventral blood island; vg, vegetal view, the dorsal is up.
	Fig. 2. F: Spatial expression patterns klf11 [KLF transcription factor 11], during Xenopus embryogenesis detected with whole mount in situ hybridization. a, animal view; aa, aortic arches; ad, adenohypophysial placode; an, anterior view, dorsal is up; ba, branchial arches; bl, blastopore lip; cg, cement gland; d, dorsal view, the anterior is up; da, dorsal anterior view; dv, dorsal vegetal view; ey, eye; fb, forebrain; fep, facial epibranchial placode; hb, hindbrain; he, heart anlage; hg, hatching gland; hm, hypaxial muscles; is, intersomitic ves- sels; l, lateral view, the animal pole is up; la, lateral view, the anterior is up and the ventral is to the left; ld, lateral dorsal view, the animal pole is up; le, lens; li, liver primordium; lu, lung primordium; mb, midbrain; nc, neural crest; np, neural plate; op, olfactory placode; ov, otic vesicle; pcv, posterior cardinal vein; pl, plexus; pn, pronephros; pop, presumptive olfactory placode; ppe, preplacodal ectoderm; prp, preplacodal region; pt, proctodeum; sd, primordia of stomach/duodenum; sm, somite; tg, trigeminal placode; v, ventral view, the anterior is up; vbi, ventral blood island; vg, vegetal view, the dorsal is up.
	Fig. 2. G: Spatial expression patterns klf15, during Xenopus embryogenesis detected with whole mount in situ hybridization. a, animal view; aa, aortic arches; ad, adenohypophysial placode; an, anterior view, dorsal is up; ba, branchial arches; bl, blastopore lip; cg, cement gland; d, dorsal view, the anterior is up; da, dorsal anterior view; dv, dorsal vegetal view; ey, eye; fb, forebrain; fep, facial epibranchial placode; hb, hindbrain; he, heart anlage; hg, hatching gland; hm, hypaxial muscles; is, intersomitic ves- sels; l, lateral view, the animal pole is up; la, lateral view, the anterior is up and the ventral is to the left; ld, lateral dorsal view, the animal pole is up; le, lens; li, liver primordium; lu, lung primordium; mb, midbrain; nc, neural crest; np, neural plate; op, olfactory placode; ov, otic vesicle; pcv, posterior cardinal vein; pl, plexus; pn, pronephros; pop, presumptive olfactory placode; ppe, preplacodal ectoderm; prp, preplacodal region; pt, proctodeum; sd, primordia of stomach/duodenum; sm, somite; tg, trigeminal placode; v, ventral view, the anterior is up; vbi, ventral blood island; vg, vegetal view, the dorsal is up.
	Fig. 2. H: Spatial expression patterns klf17 [ KLF transcription factor 17], during Xenopus embryogenesis detected with whole mount in situ hybridization. a, animal view; aa, aortic arches; ad, adenohypophysial placode; an, anterior view, dorsal is up; ba, branchial arches; bl, blastopore lip; cg, cement gland; d, dorsal view, the anterior is up; da, dorsal anterior view; dv, dorsal vegetal view; ey, eye; fb, forebrain; fep, facial epibranchial placode; hb, hindbrain; he, heart anlage; hg, hatching gland; hm, hypaxial muscles; is, intersomitic ves- sels; l, lateral view, the animal pole is up; la, lateral view, the anterior is up and the ventral is to the left; ld, lateral dorsal view, the animal pole is up; le, lens; li, liver primordium; lu, lung primordium; mb, midbrain; nc, neural crest; np, neural plate; op, olfactory placode; ov, otic vesicle; pcv, posterior cardinal vein; pl, plexus; pn, pronephros; pop, presumptive olfactory placode; ppe, preplacodal ectoderm; prp, preplacodal region; pt, proctodeum; sd, primordia of stomach/duodenum; sm, somite; tg, trigeminal placode; v, ventral view, the anterior is up; vbi, ventral blood island; vg, vegetal view, the dorsal is up.
	nodal 5 (nodal homolog 5) gene expression in X. laevis embryo, assayed via in situ hybridization, NF stage 10, blastoporal view, dorsal up.