Kratzer MC et al. (2019), Evolution of the Rho guanine nucleotide exchang...

XB-ART-55758

Gene Expr Patterns 2019 Jun 01;32:18-27. doi: 10.1016/j.gep.2019.02.004.

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Evolution of the Rho guanine nucleotide exchange factors Kalirin and Trio and their gene expression in Xenopus development.

Kratzer MC , England L , Apel D , Hassel M , Borchers A .

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Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by accelerating their GDP/GTP exchange. Trio and its paralog Kalirin (Kalrn) are unique members of the Rho-GEFs that harbor three catalytic domains: two functional GEF domains and a serine/threonine kinase domain. The N-terminal GEF domain activates Rac1 and RhoG GTPases, while the C-terminal GEF domain acts specifically on RhoA. Trio and Kalrn have an evolutionary conserved function in morphogenetic processes including neuronal development. De novo mutations in TRIO have lately been identified in patients with intellectual disability, suggesting that this protein family plays an important role in development and disease. Phylogenetic and domain analysis revealed that a Kalrn/Trio ancestor originated in Prebilateria and duplicated in Urbilateria to yield Kalrn and Trio. Only few taxa outside the vertebrates retained both of these highly conserved proteins. To obtain first insights into their redundant or distinct functions in a vertebrate model system, we show for the first time a detailed comparative analysis of trio and kalrn expression in Xenopus laevis development. The mRNAs are maternally transcribed and expression increases starting with neurula stages. Trio and kalrn are detected in mesoderm/somites and different neuronal populations in the neural plate/tube and later also in the brain. However, only trio is expressed in migrating neural crest cells, while kalrn expression is detected in the cranial nerves, suggesting distinct functions. Thus, our expression analysis provides a good basis for further functional studies.

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Species referenced: Xenopus laevis
Genes referenced: kalrn npb rac1 rho rho.2 rhoa rhog trio tub tubb2b twist1
GO keywords: Rho guanyl-nucleotide exchange factor activity [+]

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	Fig. 1. Graphical summary of the protein domain structures of Kalrn and Trio using an idealized animal tree of life. The tree reflects the currently accepted phylogeny of animals. Different protein domains are indicated by color. (The domain architecture of different Kalrn/Trio proteins in respect to protein length (in amino acids) is depicted in Supplementary Fig. 2). Species abbreviation Ap: Acanthaster planci; Aq: Amphimedon queenslandica; Bb: Branchiostoma belcheri; Ce: Caenorhabditis elegans; Ct: Capitella teleta; Dm: Drosophila melanogaster; Fo: Frankliniella occidentalis; Hv: Hydra vulgaris; Lg: Lottia gigantea; Lg: Sk: Saccoglossus kowalevskii; Ta: Trichoplax adhaerens; Tc: Tribolium castaneum; Xl: Xenopus laevis; Xt: Xenopus tropicalis.//indicate a gap in the protein domain structure, as two shorter fragments were assembled for graphical representation.
	Fig. 2. Bayesian phylogenetic tree of vertebrate and invertebrate Kalrn and Trio. Species abbreviation as in Fig. 1. Accession numbers are given in Supplementary Table 1.
	Fig. 3. Temporal gene expression pattern of Xenopus laevis trio and kalrn. Expression levels were determined by RT-PCR at different stages of Xenopus laevis development. Amplification of Histone H4 (H4) was used as an internal control. Reactions performed in the absence of reverse transcriptase (-RT) served as negative control. The temporal expression pattern of trio (upper lane), kalrn.L (middle lane) and kalrn.S (lower lane) are shown.
	Fig. 4. Spatial gene expression pattern of trio. Trio gene expression was analyzed by whole mount in situ hybridization. Embryonic stages are indicated. The detailed expression pattern is described in the text. A Embryo at cleavage stage, lateral view. B Blastula stage embryo, lateral view. C Gastrula stage embryo, blastopore lip is indicated. D Transverse section of embryo in C; section plane is indicated in C. E Control in situ hybridization using a sense trio probe; gastrula stage embryo. F,G Neurula stage embryos, dorsal view. H Tailbud stage embryo, dorsal view. I,J Transverse sections of embryo shown in H. Section planes are indicated in H. Arrowhead in I marks staining between the meso- and endoderm likely representing migrating NC cells. K-M Tailbud stage embryos, lateral view. Sense control at stage 28 is indicated in the insert in L. N Higher magnification of the head of the embryo shown in L. O Double in situ hybridization showing trio (blue) and twist (red) co-expression in cranial NC cells. P, Q Transverse sections of tadpole embryos analyzed by trio/twist double in situ hybridization. Section planes are indicated in O. Abbreviations: a: animal, b: brain; ba: branchial arches; bl: blastopore lip; cg: cement gland; e: eye; ec: ectoderm; ha: hyoidal arch; ma: mandibular arch; me: mesoderm; nc: neural crest; no: notochord; np: neural plate; npb: neural plate border; nt: neural tube; ov: otic vesicle; s: somites; pom: periocular mesenchyme; sc: sclerotome; tg: trigeminal ganglion; v: vegetal. Scale bar: 500 μm (I,J: 200 μm).
	Fig. 5. Spatial gene expression pattern of kalrn. Kalrn gene expression was analyzed by whole mount in situ hybridization. Embryonic stages are indicated. The detailed expression pattern is described in the text. A Embryo at cleavage stage, lateral view. B Blastula stage embryo, lateral view. C Gastrula stage embryo, blastopore lip is indicated. D Transverse section of embryo in C; section plane is indicated in C. E Sense control of gastrula stage embryo. F-H,K Neurula stage embryos, dorsal view. I,J Transverse sections of embryo shown in H. Section planes are indicated in H. K Anterior view of embryo shown in H. M-N Tailbud stage embryos, lateral view. O,P Higher magnification of the embryo heads shown in M,N, respectively; the mandibular branch (Vmd) and the ophthalmic branch (Vop) of the trigeminal nerve (V), the facial VII, the glossopharyngeal (IX), and the vagus (X) nerve are indicated in P. Q Control in situ hybridization using a sense trio probe; tailbud stage embryo. Abbreviations: a: animal; bl: blastopore lip; cn: cranial nerves; fb: forebrain; hb: hindbrain; i: intermediate domain of primary neurons; in: interneuron; l: lateral domain of primary neurons; le: lens; m: medial domain of primary neurons; mb: midbrain; me: mesoderm; mn: motor neuron; no: notochord; np: neural plate; npb: neural plate border; nt: neural tube; op: olfactory placode; pg: pineal gland; rb: Rohon-Beard neuron; s: somites; sc: sclerotome; tg: trigeminal ganglion; tn: trigeminal nerve; tp: trigeminal placode; v: vegetal; vn: vestibulo cochlear nerve. Scale bar: 500 μm (I,J: 200 μm).
	Fig. 6. Kalrn is co-expressed with n-tubulin in different neurons. Kalrn (blue) and n-tubulin (red) co-expression was analyzed by double whole mount in situ hybridization. A Neurula embryo (stage 13) analyzed by in situ hybridization using a kalrn antisense probe. B Stage 13 embryo analyzed by kalrn (blue) and n-tubulin (red) double in situ hybridization. C Transverse section of the embryo seen in A. D Transverse section of the embryo seen in B shows that the purple areas expressing kalrn and n-tubulin significantly overlap with the kalrn expression in C. Abbreviations: i: intermediate domain of primary neurons; l: lateral domain of primary neurons; m: medial domain of primary neurons; n-tub: n-tubulin; no: notochord; np: neural plate. Scale bar: A,B: 500 μm; C,D: 200 μm.
	FigS2. A Vertebrate Trio proteins. B Vertebrate Kalrn proteins. C Invertebrate Trio/Kalrn orthologs. Different protein domains are indicated by color. Protein length is depicted representing number of amino acids (indicated on the right).
	SuppTable1. Summary of the species, given names of sequences and accession numbers of all vertebrate and invertebrate sequences used for the domain analysis (Fig. 1) and for the phylogenetic tree (Fig. 2).
	trio (trio Rho guanine nucleotide exchange factor) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 9, lateral view, animal side up.
	trio (trio Rho guanine nucleotide exchange factor) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior up.
	trio (trio Rho guanine nucleotide exchange factor) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	kalrn (kalirin RhoGEF kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 9, lateral view, animal side up.
	kalrn (kalirin RhoGEF kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anterior up.
	kalrn (kalirin RhoGEF kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	kalrn (kalirin RhoGEF kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 38, lateral view, anterior left, dorsal up.