Cosse-Etchepare C et al. (2018), Pou3f transcription factor expression during em...

XB-ART-55002

Int J Dev Biol 2018 Jan 01;624-5:325-333. doi: 10.1387/ijdb.170260RL.

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Pou3f transcription factor expression during embryonic development highlights distinct pou3f3 and pou3f4 localization in the Xenopus laevis kidney.

Cosse-Etchepare C , Gervi I , Buisson I , Formery L , Schubert M , Riou JF , Umbhauer M , Le Bouffant R .

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The POU (Pit-Oct-Unc) genes encode a large transcription factor family comprising 6 classes (pou1f to pou6f ) involved in many developmental processes, such as cell commitment and differentiation. The pou3f class contains four members (pou3f1, pou3f2, pou3f3, pou3f4) characterized by expression in ectodermal tissue derivatives, such as nervous system and otic vesicle, during mammalian development. In order to obtain insights into the potential conservation of this class of transcription factors in vertebrates, we carried out a phylogenetic analysis and a comprehensive comparative study of pou3f expression in the frog Xenopus laevis. All vertebrates examined possessed members of the four pou3f subfamilies, excepting the zebrafish, which lacked a pou3f4 gene. Whole mount in situ hybridization and real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that Xenopus pou3f genes were expressed in the forming neural tube and their expression was maintained in the brain, mostly in the dorsal part, at tailbud stages. The pou3f2, pou3f3, and pou3f4 genes were also expressed in the developing otic vesicle, and pou3f1 in some cells of the epidermis. Besides ectodermal derivatives, pou3f3 and pou3f4 were expressed in the developing kidney. Their expression started at the early tailbud stage in the pronephric anlage and partly overlapped. In the mature pronephric tubule, pou3f3 was restricted to the intermediate tubule, while pou3f4 was also expressed in the distal and connecting tubule. Together, our results highlight a significant conservation of pou3f gene expression in vertebrates and indicate that they may have distinct but also redundant functions during neural and renal development.

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Species referenced: Xenopus laevis
Genes referenced: gars1 pax8 pou3f1 pou3f2 pou3f3 pou3f4
GO keywords: kidney development [+]

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	Fig. 1. Sequence analysis of vertebrate Pou3f proteins. (A) Sequence alignments of the Xenopus laevis (Xl) Pou3f1, Pou3f2, Pou3f3, and Pou3f4 proteins. Conserved residues are shown in bold, the POU-specific domain and the POU-homeodomain are highlighted in yellow, and the linker region is indicated in cyan. (B) The percentage of sequence identity for each vertebrate Pou3f subfamily (Pou3f1, Pou3f2, Pou3f3, and Pou3f4) relative to the Xenopus laevis sequence is indicated for the POU-specific domain, the linker, the POU-homedomain as well as for the whole (i.e. full-length) protein. The percentages are the average sequence identities of the human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), and spotted gar (Lepisosteus oculatus) sequences relative to the African clawed frog (Xenopus laevis) sequence.
	Fig. 2. Phylogenetic relationship of vertebrate Pou3f proteins. Phylogenetic tree of the Pou3f family based on sequences from human (Homo sapiens, Hs), mouse (Mus musculus, Mm), chicken (Gallus gallus, Gg), African clawed frog (Xenopus laevis, Xl), zebrafish (Danio rerio, Dr), medaka (Oryzias latipes, Ol), Japanese puffer (Takifugu rubripes, Tr), spotted gar (Lepisosteus oculatus, Lo), Florida amphioxus (Branchiostoma floridae, Bf), and the purple sea urchin (Strongylocentrotus purpuratus, Sp). The Pou3f1, Pou3f2, Pou3f3, and Pou3f4 subfamilies are respectively highlighted in blue, green, yellow, and orange. The tree was rooted with members of the Pou4f1 subfamily. Although trees were calculated using both the Maximum Likelihood (ML) and Bayesian Inference (BI) methods, only the ML tree is shown, with branch lengths being representative of sequence substitution rates. Branch support is indicated as bootstrap percentages from the ML analysis (ranging from 0 to 100) / posterior probabilities (ranging from 0 to 1) from the BI analysis. “--” indicates that the branching patterns of the ML and BI analyses diverged at this node and that one of the scores is thus unavailable.
	Fig. 3. Genomic organization of vertebrate pou3f loci. Genomic organization and gene synteny comparisons of vertebrate pou3f1, pou3f2, pou3f3, and pou3f4 genes. Dotted lines represent chromosome loci in the genomes of human (Homo sapiens, Hs), chicken (Gallus gallus, Gg), Western clawed frogs (Xenopus tropicalus, Xt), zebrafish (Danio rerio, Dr), medaka (Oryzias latipes, Ol), Japanese puffers (Takifugu rubripes, Tr), and spotted gars (Lepisosteus oculatus, Lo). Genes are shown as colored pentagon arrows, with the transcriptional orientation of a given gene locus being indicated by the direction of the arrow. When a pou3f paralog is not found in a given species, the pentagon is white. Gene loci are named according to the nomenclature used in human and orthologs in the different species are shown in identical colors. Gene paralogs are indicated by letters. Bold black lines between polygons mark changes in genetic linkage and hence in gene synteny relative to the situation in the human genome.
	Fig. 4 (left). Spatio-temporal expression of pou3f1 during Xenopus laevis development. (A) Expression of pou3f1 analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) at indicated stages of development. Average values from three independent experiments. (B) Whole mount in situ hybridization for pou3f1 at indicated stages of development. Early gastrula, vegetal view (a). Neurula, dorsal view (b,c). Tailbud, dorsal (d) and lateral (f) view. Tadpole, lateral view (g,i). Transverse sections at the level of the developing brain at tailbud stage (e) and at the level of the developing eye at tadpole stage (h). Abbreviations: e, eye; ep, epidermis; fb, forebrain; hb, hindbrain; mb, midbrain; nf, neural fold; np, neural plate; nt, neural tube; op, optic placode. (C) Expression of pou3f1 analyzed by RT-qPCR on dissected explants from tailbud-stage embryos, stage 25. Embryos were dissected either into ventral and dorsal halves or into tailbud, pronephros and head. Average values from three independent experiments. : P < 0,05; *: P < 0,005, relative to the whole embryo.
	pou3f1 (POU class 3 homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5 , blastoporal view, dorsal up.
	pou3f1 (POU class 3 homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anterior right.
	pou3f1 (POU class 3 homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, (d) lateral view, anterior right, and (e) transverse sections at the level of the developing brain, dorsal up.
	pou3f1 (POU class 3 homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.

	pou3f1 (POU class 3 homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior right, dorsal up.
	Fig. 5 (right). Spatio-temporal expression of pou3f2 during Xenopus laevis development. (A) Whole mount in situ hybridization for pou3f2 at indicated stages of development. Neurula, dorsal view (a,b). Tailbud, dorsal (c) and lateral (e) view. Tadpole, lateral view (f,i). Transverse sections at the level of the developing brain (d,g,h), and otic vesicle (h). Abbreviations: (fb) forebrain, (hb) hindbrain, (mb) midbrain, (nt) neural tube, (om) otic mesenchyme, (ov) otic vesicle. (B) Expression of pou3f2 analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) on dissected explants from tailbud embryos (stage 25). Embryos were dissected either into ventral and dorsal halves or into tailbud, pronephros and head. : P < 0,05; **: P < 0,001, relative to the whole embryo.
	pou3f2 (POU class 3 homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior right.
	pou3f2 (POU class 3 homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anterior right.
	pou3f2 (POU class 3 homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, dorsal view, anterior right.



	pou3f2 (POU class 3 homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior right, dorsal up.
	Fig. 6 (left). Spatio-temporal expression of pou3f3 during Xenopus laevis development. (A) Expression of pou3f3 analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) at indicated stages of development. (B) Whole mount in situ hybridization for pou3f3 at indicated stages of development. Neurula, dorsal view (a,b). Tailbud, dorsal (c) and lateral (d,f) view. Tadpole, lateral view (g,i). Transverse sections at the level of the developing pronephros (e,h). Abbreviations: (fb) forebrain, (hb) hindbrain, (mb) midbrain, (nt) neural tube, (ov) otic vesicle, (p) pronephros. (C) Expression of pou3f3 analyzed by RT-qPCR on dissected explants from tailbud-stage embryos (stage 25). Embryos were dissected either into ventral and dorsal halves or into tail bud, pronephros and head. : P < 0,005; *: P < 0,001, relative to the ventral half.
	pou3f3 (POU class 3 homeobox 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior right.
	pou3f3 (POU class 3 homeobox 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anterior right.
	pou3f3 (POU class 3 homeobox 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25,(d) lateral view, anterior right, dorsal up, ( e) in transverse section at the level of pronephros, dorsal up.



	pou3f3 (POU class 3 homeobox 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior right, dorsal up.
	Fig. 7 (right). Spatio-temporal expression of pou3f4 during Xenopus laevis development. (A) Expression of pou3f4 analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) at indicated stages of development. Average values from three independent experiments. (B) Whole mount in situ hybridization for pou3f4 at indicated stages of development. Neurula, dorsal view (a). Tailbud, dorsal (b) and lateral view (c,e). Tadpole, lateral (f,i) view. Transverse sections at the level of the developing pronephros (d,h) and otic vesicle (g). Abbreviations: (fb) forebrain, (hb) hindbrain, (mb) midbrain, (nt) neural tube, (om) otic mesenchyme, (ov) otic vesicle, (p) pronephros. (C) Expression of pou3f4 analyzed by RT-qPCR on dissected explants from tailbud-stage embryos (stage 25). Embryos were dissected either into ventral and dorsal halves or into tail bud, pronephros and head. : P < 0,05; *: P < 0,005, relative to the whole embryo.
	pou3f4 (POU class 3 homeobox 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anterior right.
	pou3f4 (POU class 3 homeobox 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, (b) dorsal view, anterior right, and (c) lateral view, anterior right, dorsal up.
	pou3f4 (POU class 3 homeobox 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, traverse section at the level of the developing pronephric kidney.



	pou3f4 (POU class 3 homeobox 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior right, dorsal up.
	Fig. 8. Differential pronephric expression of pouf3 and pou3f4 in Xenopus laevis. Whole mount in situ hybridization analysis of pronephric expression of pou3f3 and pou3f4 (dark blue) compared to pax8 (light blue) in tailbud-stage embryos (stage 25) (A) and in the differentiated tubule in late tailbud-stage embryos (st 35) (B). Transverse sections in the anterior (A.f,j) and posterior part (A.g,k) of the anlage, and in the anterior part of the tubule (B.f,i). (C) Schematic representation of pou3f3 and pou3f4 expression in the pronephros: transverse section of a tailbud-stage embryo (left), lateral view of the pronephric anlage (middle) and of the differentiated tubule (right).