Treimer E et al. (2021), Galloway-Mowat syndrome: New insights from bioi...

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Gene Expr Patterns 2021 Dec 01;42:119215. doi: 10.1016/j.gep.2021.119215.

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Galloway-Mowat syndrome: New insights from bioinformatics and expression during Xenopus embryogenesis.

Treimer E , Niedermayer K , Schumann S , Zenker M , Schmeisser MJ , Kühl SJ .

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Galloway-Mowat syndrome (GAMOS) is a rare developmental disease. Patients suffer from congenital brain anomalies combined with renal abnormalities often resulting in an early-onset steroid-resistant nephrotic syndrome. The etiology of GAMOS has a heterogeneous genetic contribution. Mutations in more than 10 different genes have been reported in GAMOS patients. Among these are mutations in four genes encoding members of the human KEOPS (kinase, endopeptidase and other proteins of small size) complex, including OSGEP, TP53RK, TPRKB and LAGE3. Until now, these components have been functionally mainly investigated in bacteria, eukarya and archaea and in humans in the context of the discovery of its role in GAMOS, but the KEOPS complex members' expression and function during embryogenesis in vertebrates is still unknown. In this study, in silico analysis showed that both gene localization and the protein sequences of the three core KEOPS complex members Osgep, Tp53rk and Tprkb are highly conserved across different species including Xenopus laevis. In addition, we examined the spatio-temporal expression pattern of osgep, tp53rk and tprkb using RT-PCR and whole mount in situ hybridization approaches during early Xenopus development. We observed that all three genes were expressed during early embryogenesis and enriched in tissues and organs affected in GAMOS. More precisely, KEOPS complex genes are expressed in the pronephros, but also in neural tissue such as the developing brain, eye and cranial cartilage. These findings suggest that the KEOPS complex plays an important role during vertebrate embryonic development.

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Species referenced: Xenopus laevis
Genes referenced: apex1 dusp11 eya2 gon7 lage3 ocstamp osgep parp2 pnp prdm15 slc2a10 stambp tep1 tp53rk tprkb zmynd8

???displayArticle.disOnts??? Galloway-Mowat syndrome 1
???displayArticle.omims??? GALLOWAY-MOWAT SYNDROME 1; GAMOS1

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	Graphical abstract
	Fig. 1. In silico analysis of osgep (A), tp53rk (B), and tprkb (C) revealed high conservation across species. Synteny analysis of osgep (A, upper part), tp53rk (B, upper part) and tprkb (C, upper part) and its neighboring genes in Homo sapiens, Mus musculus, Xenopus laevis, Xenopus tropicalis and Danio rerio. Schematic overview of the genes' location and its surrounded genes among different species. Conserved genes are depicted by boxes with identical colors, osgep (A), tp53rk (B) and tprkb (C) are shown in red. Non-conserved neighboring genes are not shown. The orientations of the open reading frames are indicated by arrows. Gene lengths as well as the distances are not proportional to their actual size. More distanced genes on the same chromosome are emphasized by a vertical dashed line. Chromosomal location is listed below the species name. Xenopus laevis S or L chromosome is specified next to the gene name. Osgep, o-sialoglycoprotein endopeptidase; parp2, poly (ADP-ribose) polymerase 2; tep1, telomerase associated protein 1; klhl33, kelch like family member 33; apex1, apurinic/apyrimidinic endodeoxyribonuclease 1; pip4p1, phosphatidylinositol 4,5-bisphosphate 4-phosphatase 1; pnp, purine nucleoside phosphorylase. tp53rk, TP53 regulating kinase; znf334, zinc finger protein 334; ocstamp, osteoclast stimulatory transmembrane protein; slc13a3, solute carrier family 13 member 3; slc2a10, solute carrier family 2 member 10; eya2, EYA transcriptional coactivator and phosphatase 2; zmynd8, zinc finger MYND-type containing 8. tprkb, TP53RK binding protein; egr4, early growth response 4; alms1, ALMS1 centrosome and basal body associated protein; nat8, N-acetyltransferase 8 (putative); dusp11, dual specificity phosphatase 11; c2orf78, chromosome 2 open reading frame 78; stambp, STAM binding protein. Homology of the amino acid sequences of full-length Osgep (A, lower left part), Tp53rk (B, lower left part) and Tprkb (C, lower left part) among different species. Amino acid length is given in numbers. Percentages represent identical residues (percent identity) of the indicated species compared to Homo sapiens. Phylogenetic trees (Phylogram) of Osgep (A, lower right part), Tp53rk (B, lower right part) and Tprkb (C, lower right part) on the basis of its alignment across indicated species show an evolutionary relationship between human, mouse, frog and fish. Genetic distances between the nodes of the phylogenetic tree were indicated by numbers above the branching lines which were generated by Clustal Omega and iTOL.
	Fig. 2. Osgep, tp53rk and tprkb are temporally expressed during Xenopus embryogenesis. Temporal expression pattern of osgep, tp53rk and tprkb during Xenopus laevis embryogenesis analyzed by semi-quantitative reverse transcriptase (RT)-PCR with Xenopus cDNA templates of the indicated stages. All three genes were detected in all investigated stages. Glyceraldehyde 3-phosphate dehydrogenase (gapdh) was used as loading control, and as negative control -RT which reaction lacks reverse transcriptase.
	Fig. 3. Osgep expression during Xenopus development. Spatio-temporal expression pattern of osgep visualized by whole mount in situ hybridization (WMISH). Embryonic stages and scale bars are indicated in each panel. Upper part: WMISH with exterior view. Scale bars are equivalent to 500 m. Black dashed lines represent section planes. Lower part: WMISH following sections. Scale bars are equivalent to 100 m. (A) Animal view, (B) lateral view and (C) vegetal view of Xenopus embryos in very early stages showed an osgep expression in the animal pole (arrowhead) and in the developing neural plate (arrow) beside the blastopore (bp). During Xenopus embryogenesis osgep is strongly expressed in neural tissue which is revealed by dorsal view (D, G) in the dorsal neural tissue and neural tube (nt) and by anterior view (E, F) in the anterior neural plate (anp), neural tube (nt) and the developing eye, more precisely in the eye vesicle (ev). (H, I, J, K) Lateral view, osgep expression is detected in the somites (s), the eye vesicle (ev) or eye (e), the embryonic kidney (pronephric anlage (pa), pronephros (p)), the otic vesicle (ov), the brain (b) and the mandibular (ma), hyoid (ha) and branchial arch (ba) which will develop into the cranial cartilage. (L) Parasagittal view showed an expression of osgep in the neuroectoderm (ne). The transversal view (M, P) revealed osgep transcripts in the mesencephalon (m), neural crest cells (ncc), eye vesicle (ev). Horizontal sections (N, O, Q, R) showed an expression in the somites (s), spinal cord (sc), the mandibular (ma), hyoid (ha) and branchial arches (ba), embryonic kidney (pronephric tubule (pt)) as well as in the eye, more detailed in the lens (le) and ciliary marginal zone (cmz).
	Fig. 4. Tp53rk expression during Xenopus development. Spatio-temporal expression pattern of tp53rk visualized by whole mount in situ hybridization (WMISH). Embryonic stages and scale bars are indicated in each panel. Upper part: WMISH with exterior view. Scale bars are equivalent to 500 m. Black dashed lines represent section planes. Lower part: WMISH following sections. Scale bars are equivalent to 100 m. (A) Animal view, (B) lateral view and (C) vegetal view of Xenopus embryos in very early stages showed a tp53rk expression in the animal pole (arrowhead) and in the developing neural plate (arrow) beside the blastopore (bp). During Xenopus embryogenesis tp53rk is strongly expressed in neural tissue which is revealed by dorsal view (D, G) in the dorsal neural tissue and neural tube (nt) and by anterior view (E, F) in the anterior neural plate (anp), neural tube (nt) and the developing eye, more precisely at the eye vesicle (ev). (H, I, J, K) Lateral view, tp53rk expression is detected in the somites (s), the eye vesicle (ev) or eye (e), the embryonic kidney (pronephric anlage (pa), pronephros (p)), the otic vesicle (ov), the brain (b) and the mandibular (ma), hyoid (ha) and branchial arch (ba) which will develop into the cranial cartilage. (L) Parasagittal view showed an expression of tp53rk in the neuroectoderm (ne). The transversal view (M, P) revealed tp53rk transcripts in the mesencephalon (m), neural crest cells (ncc) and eye vesicle (ev). Horizontal sections (N, O, Q, R) showed a strong expression in the neural crest cells (ncc), mesencephalon (m), the mandibular (ma), hyoid (ha) and branchial arches (ba), embryonic kidney (pronephric tubules (pt)) as well as in the eye, more detailed in the lens (le) and ciliary marginal zone (cmz).
	Fig. 5. Tprkb expression during Xenopus development. Spatio-temporal expression pattern of tprkb visualized by whole mount in situ hybridization (WMISH). Embryonic stages and scale bars are indicated in each panel. Upper part: WMISH with exterior view. Scale bars are equivalent to 500 m. Black dashed lines represent section planes. Lower part: WMISH following sections. Scale bars are equivalent to 100 m. (A) Animal view, (B) lateral view and (C) vegetal view of Xenopus embryos in very early stages showed a very mild tprkb expression in the animal pole (arrowhead). During Xenopus embryogenesis tprkb is slightly expressed in neural tissue which is revealed by anterior view (E, F) in the anterior neural plate (anp), neural tube (nt) and the developing eye, more precisely in the eye vesicle (ev) and by dorsal view (G) in the dorsal neural tissue and neural tube (nt). (H, I, J, K) Lateral view, tprkb expression is detected in the eye vesicle (ev) or eye (e), the embryonic kidney (pronephros (p)), the otic vesicle (ov), the brain (b) and the mandibular (ma), hyoid (ha) and branchial arch (ba) which will develop into the cranial cartilage. (L) In the parasagittal view tprkb is detected in the neuroectoderm (ne). The transversal view (M, P, R) showed an expression in the mesencephalon (m), neural crest cells (ncc) and eye vesicle (ev) as well as in the eye, more detailed in the lens (le) and ciliary marginal zone (cmz). Horizontal sections (N, O, Q) revealed tprkb transcripts in the neural crest cells (ncc), mesencephalon (m), the mandibular (ma), hyoid (ha) and branchial arches (ba) and a few transcripts in the embryonic kidney (pronephric tubules (pt)).
	Supplemental Figure 1. Multiple sequence alignment from Osgep shows high amino acid conservation, especially at the patients’ mutation amino acids (black shadowed). Multiple sequence alignment illustrated by QIAGEN CLC Genomics Workbench. Varied amino acids are shown in red and conserved amino acids in black. The known patients’ mutations amino acids are black shadowed.
	Supplemental Figure 2. Multiple sequence alignment from Tp53rk shows high amino acid conservation, especially at the patients’ mutation amino acids (black shadowed). Multiple sequence alignment illustrated by QIAGEN CLC Genomics Workbench. Varied amino acids are shown in red and conserved amino acids in black. The known patients’ mutations amino acids are black shadowed.
	Supplemental Figure 3. Multiple sequence alignment from Tprkb shows high amino acid conservation. Multiple sequence alignment illustrated by QIAGEN CLC Genomics Workbench. Varied amino acids are shown in red and conserved amino
	Supplemental Figure 4. Gene specific RNA-antisense probes from osgep, tp53rk and tprkb bind specifically to the complementary antisense mRNA using WMISH. (A-C) Antisense probes from osgep (A), tp53rk (B) and tprkb (C) showed a specific expression whereas sense probes reveal no distinct expression. Staining conditions for sense and antisense probes were identical. Embryonic stages and scale bars are indicated in each panel. Scale bars are equivalent to 500 m. Stage 4, 13 and 20 dorsal views. Stage 36 lateral view. (D) Antisense probes from osgep (horizontal: 5/5, sagittal: 2/2), tp53rk (horizontal: 3/3, sagittal: 4/5) and tprkb (horizontal: 6/6, sagittal: 3/5) showed also in sectioned embryos a gene specific expression whereas sense probes from osgep (horizontal: 4/4, sagittal: 2/2), tp53rk (horizontal: 2/3, sagittal: 5/5) and tprkb (horizontal: 4/6, sagittal: 4/4) reveal no distinct and clear expression. Staining conditions for sense and antisense probes were identical and representative sections were shown. Embryos are shown at stage 36 and scale bars are indicated. Scale bars are equivalent to 100 m.
	Supplemental Figure 5. RNA-antisense probes bind specifically to the complementary mRNA. Dotblot revealed no cross binding of the different antisense probes