Gene expression analysis of the Tao kinase family of Ste20p-like map kinase kinase kinases during early embryonic development in Xenopus laevis

Abstract

Development of the vertebrate embryo requires strict coordination of a highly complex series of signaling cascades, that drive cell proliferation, differentiation, migration, and the general morphogenetic program. Members of the Map kinase signaling pathway are repeatedly required throughout development to activate the downstream effectors, ERK, p38, and JNK. Regulation of these pathways occurs at many levels in the signaling cascade, with the Map3Ks playing an essential role in target selection. The thousand and one amino acid kinases (Taoks) are Map3Ks that have been shown to activate both p38 and JNK and are linked to neurodevelopment in both invertebrate and vertebrate organisms. In vertebrates, there are three Taok paralogs (Taok1, Taok2, and Taok3) which have not yet been ascribed a role in early development. Here we describe the spatiotemporal expression of Taok1, Taok2, and Taok3 in the model organism Xenopus laevis. The X. laevis Tao kinases share roughly 80% identity to each other, with the bulk of the conservation in the kinase domain. Taok1 and Taok3 are highly expressed in pre-gastrula and gastrula stage embryos, with initial expression localized to the animal pole and later expression in the ectoderm and mesoderm. All three Taoks are expressed in the neural and tailbud stages, with overlapping expression in the neural tube, notochord, and many anterior structures (including branchial arches, brain, otic vesicles, and eye). The expression patterns described here provide evidence that the Tao kinases may play a central role in early development, in addition to their function during neural development, and establish a framework to better understand the developmental roles of Tao kinase signaling.

Keywords: Embryonic development; Gene expression; In situ hybridization; Map kinase; Tao kinase; Xenopus laevis.

Figure 1:

Xenopus laevis Tao kinases are orthologous to the Tao kinases in other species. A. Diagram representing the Tao kinases, including a conserved N-terminal kinase domain (KD, shaded box) with the conserved serine in the activation loop (red line and red box in C), and a conserved serine-rich domain (SR, lined box). Unshaded regions represent non-conserved regions of the protein. B. Phylogenetic analysis of maximum likelihood of vertebrate tao kinases from X. laevis (Xl), D. rerio (Dr), and H. sapiens (Hs), as well as the single tao kinase from the basal chordate C. intestinalis (Ci) and arthropod D. melanogaster (Dm). Saccharomyces cerevisiae (Sc) Ste20p was used as the outgroup. Tree was assembled using PhyML 3.0 - ATGC-Montpellier (bootstrapping was set at 100). All alignments were created using Clustal Omega (EMBL-EBI). C. Alignment of the metazoan kinase domains. Conserved kinase subdomains underlined and denoted with a roman numeral. The activation loop and conserved serine residue highlighted in yellow and red box, respectively. D. Comparison of X. laevis tao kinases to each other, shown as % identity for the entire protein, the kinase domain (as in C), and the C-terminal domain (representing all sequence C-terminal to the kinase domain).

Figure 2.

Tao kinase 1, 2, and 3 are differentially expressed during X. laevis development. mRNA was extracted from X. laevis embryos at the indicated developmental stages and converted to cDNA for qPCR analysis. Relative expression levels of taok1 (shaded), taok2 (open), and taok3 (crosshatch) are reported as the average from three technical replicates performed on three biological replicates for each stage. Expression levels were normalized using the housekeeping gene slc35b1 and calculated using the DC_q method. qPCR was performed on a Bio-Rad CFX96 machine. Standard curves were performed for each primer set used to calculate efficiency at 100.5%, 99.7%, 98.8%, and 99.3% for Taok1, Taok2, Taok3, and slc35b1, respectively. qPCR was performed on a Bio-Rad CFX96 machine.

Figure 3.

Tao kinase 1 and 3 are expressed in the pre-gastrula embryo. Whole mount in situ hybridization using antisense riboprobes to tao kinase 1 (A, A’, D, D’), tao kinase 2 (B, B’, E, E’), and tao kinase 3 (C, C’, F, F’). A-C. Animal and lateral (A’-C’) views of NF stage 2 embryos. Browning in A-C is residual pigment from incomplete bleaching. D-F. Animal and bisected lateral (D’-F’) of late blastula (NF stage 9) embryos. N = ≥50 embryos per probe.

Figure 4.

Tao kinase 1 and 3 are expressed throughout gastrulation. Whole mount in situ hybridization using antisense riboprobes to tao kinase 1 (A, A’, D, D’), tao kinase 2 (B, B’, E, E’), and tao kinase 3 (C, C’, F, F’). A-C. Animal and bisected lateral (A’-C’) views of NF stage 10 embryos. Dorsal is left in A’-C’. D-F. Vegetal and bisected lateral (D’-F’) of late gastrula (NF stage 12) embryos. Blastopore (marked with a ‘bp’) visible en face in D-F. Asterisk in D’-F’ represents the dorsal lip. N = ≥ 50 embryos per probe. Scale bars = 0.5mm

Figure 5.

Tao kinases 1, 2, and 3 are expressed during neurulation and the early tailbud. Whole mount in situ hybridization using antisense riboprobes to tao kinase 1 (A, A’, D), tao kinase 2 (B, B’, E), and tao kinase 3 (C, C’, F). A-C. Dorsal and transverse (A’-C’) views of NF stage 18 embryos. Dotted line represents the plane of transverse section in A’-C’. Anterior is up in A-C, dorsal is up in A’-C’ (posterior view). D-F. Lateral view of tailbud (NF stage 24) embryos. Dorsal to the left. Structures are labeled as follows neural plate (np), notochord (nc), somites (s), neural tube (nt), branchial arches (ba), eye (e), and cement gland (cg). N = ≥ 50 embryos per probe. Scale bars = 0.5mm

Figure 6.

Tao kinases 1, 2, and 3 have overlapping expression in the late tailbud. Whole mount in situ hybridization using antisense riboprobes to tao kinase 1 (A, A’), tao kinase 2 (B, B’), and tao kinase 3 (C, C’). A-C. Lateral and transverse (A’-C’) views of NF stage 30 embryos. Dotted line represents the plane of transverse section in A’-C’. Anterior is to the right in A-C, and dorsal is up in all images. Structures are labeled as follows notochord (nc), neural tube (nt), eye (e), and cement gland (cg), branchial arches (ba), otic vesicle (ov), brain (b), pronephros (pn). N = ≥ 50 embryos per probe. Scale bars in A-C = 1 mm, and A’-C’ = 0.25mm