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 orthologs (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.

1. Introduction

Embryogenesis is a complex process that requires constant feedback between the extracellular and intracellular environments to provide positional cues relative to the dorsal-ventral (D-V), anterior-posterior (A-P), and left-right (L-R) axes ^1–3. Many of these positional cues result from morphogen gradients emanating from signaling centers throughout the embryo, such as the node in mice and chick, the ‘shield’ in zebrafish, and the Spemann organizer in the frog, Xenopus ⁴. These organizing centers provide a complex suite of signaling cues to the surrounding cells to induce the three initial germ layers⁵. In Xenopus, the Spemann organizer determines the dorsal aspect of the embryo and functions to pattern the surrounding tissue using both instructive and antagonistic signaling, the latter through the expression of proteins such as chordin and noggin (BMP inhibitors), and Frzb-1 (Wnt inhibitor) ⁶. TGF-β, Wnt, and FGF signaling pathways are all required during the blastula stage embryo to pattern the three axes and the combination of all signals creates a cartesian map, allowing three axes and three germ layers to be simultaneously specified².

Xenopus mesoderm induction is a perfect example of the complexity involved in tissue specification. The mesodermal tissue is initially induced by nodal/activin signals of the TGF-β family originating from the vegetal hemisphere, while the dorsal-ventral axis of this tissue is specified through expression of BMP inhibitors (also of the TGF-β family) along the equator of the embryo⁷. The dorsal mesoderm is exposed to high levels of BMP antagonists, while the ventral side is exposed to little to no antagonist levels ⁸. Dorsal mesoderm further develops into the notochord and somites, while the ventral mesoderm gives rise to multiple tissues, including renal, heart, and blood⁹. The Fibroblast Growth Factor (FGF) pathway also plays a critical role in both the specification and the maintenance of mesoderm, by inducing a suite of mesodermal specific genes, as well as regulating cell migration and convergent extension ^10–14. FGF signaling is additionally required in the induction and patterning of neural tissue and endoderm ^15–19. The current working model of FGF signaling is for FGFs to bind to FGF receptors (FGFR), which in turn activates the Mitogen activated protein kinase (MapK) pathway. Upon ligand binding, the activated FGFR stimulates the monomeric GTPase Ras, which activates a phosphorylation cascade of signaling through Raf, (a MapK kinase kinase, Map3K), to MEK, (Map2K), to ERK, (MapK) ²⁰. Map kinases phosphorylate downstream target proteins, which in turn regulate transcription of target genes and other cellular responses. In addition to ERK, there are two other well-known Map kinases: p38 and c-JUN N-terminal kinase (JNK) ²¹, which are often activated by cellular and environmental stress signals, rather than canonical FGF signaling, and similarly affect target gene transcription.

p38 exists in four isoforms: p38ɑ/MAPK14, p38β/MAPK11, p38γ/MAPK12, and p38δ/MAPK13. The p38ɑ and p38β isoforms tend to be grouped together, sharing 75% identity, and the p38γ and p38δ isoforms grouped sharing 70% identity²². Among the four isoforms, there are differences in tissue expression and substrate specificity, with some substrates being better phosphorylated by p38ɑ/β than p38γ/δ and vice versa ²². In general, p38 functions downstream of a canonical MAP kinase cascade, activated by cytokines, cellular stresses, and G-protein couple receptor signaling, and has over 100 known potential phosphorylation targets ²³. In Xenopus laevis, p38 activates CREB and regulate the expression of the morphogen chordin, thus serving to help pattern the mesoderm ²⁴. Additionally, p38 was found to aid in anterior development through its activation of Nemo-Like Kinase ²⁵ and in myogenic development through its regulation of XMyf5 ²⁴. There are currently three known JNK genes in vertebrates, JNK1, JNK2, and JNK3, each of which also has multiple isoforms, creating a further complexity to MAPK signaling ²⁶. JNK proteins typically works through activation of c-JUN and FOS transcription factors to elicit downstream effects, including cell migration and polarity, as well as cell cycle regulation and apoptosis ^26,27. In developmental processes, JNK is often activated through non-canonical Wnt signaling, where it will facilitate cytoskeletal rearrangements and tissue morphogenesis, including convergent-extension movements, gut tube elongation, and pronephros development ^28–31.

While there are multiple potential inputs for p38 and JNK activation during embryonic development, they both function as downstream effectors of the MAP kinase pathway. Within the MAPK signaling cascade, a group of Ste20-like MAP3Ks known as the Thousand And One amino acid kinases (Taok) have been described as effectors of both the p38 and the JNK pathways ³². Interestingly, they have additional roles as effectors of the Hippo pathway and regulators of both actin and microtubule dynamics ³³. Three Tao kinases, Taok1, Taok2, and Taok3, have been identified in vertebrates ^34–36, with a single homolog in invertebrates (tao in Drosophila and KIN-18 in C. elegans) ^32,37. The current data demonstrates that Taok1, Taok2, and Taok3 activate the p38 pathway through phosphorylation of MEK3/6, while Taok1 and Taok2 additionally activate the JNK/SAPK pathway through phosphorylation of MEK4/7 ³³. Conversely, while Taok3 was originally identified at ‘JNK Inhibitory Kinase’(JIK)³⁶, there is evidence that it can either activate^38–40 or inhibit^36,41 the JNK pathway, pointing to cell and/or tissue specific functions. The effects of the Tao kinases on the Hippo pathway come from studies investigating the Hippo ‘interactome’ and identified that Taok1 and Taok3 can directly phosphorylate both MST2/Hippo and Lats1/2 in parallel pathways ⁴². In a non-signaling context, Taok1 and Taok2 play opposing roles in microtubule stability, where Taok1 functions as a destabilizer, through phosphorylation of tau, and Taok2 can directly bind and stabilize microtubules ^43,44. Taok1 and Taok2 have also been shown to affect actin dynamics, although this regulation is poorly understood ^45,46. No cytoskeletal role as yet been attributed to Taok3. As a result of their ability to bind to and/or phosphorylate multiple targets, the Tao kinases have been linked to numerous cellular responses, such as proliferation, differentiation, DNA damage, apoptosis, cytokine induction, heat and osmotic shock, and cytoskeletal regulation ³³. Physiologically, these responses contribute to the regulation of inflammation and immunity ^47,48, neural development (described below) , and cancerous phenotypes ⁴⁹.

Despite our understanding of Tao kinases at the cellular level, there are limited investigations into their role in embryonic development. It is currently known that Taok1 and Taok2 are involved with neuronal development and synapse formation, and have been genetically linked to multiple neurodevelopmental disorders (NDDs) ^50,51. Mice defective for Taok1 in neural progenitor cells have impaired neural migration and mutations in Taok1 have been linked to NDDs characterized by similar traits, including distinct facial features, intellectual disability/ developmental delay, muscular hypotonia, and others ⁵². Similarly, Taok2 knockout mice exhibit abnormal brain development and decreases in dendrite morphology ⁵³. The mechanism of Taok2 function has been described in studies using Rat neurons, where a splice variant of Taok2, Taok2β, induces N-cadherin endocytosis in a p38 MAPK feedback loop to establish and maintain the neural network ⁵⁴. Conversely, Drosophila Tao mutants develop an increase in dendritic complexity, demonstrating a conserved role in brain development with potential species-specific differences in Taok regulation ⁵⁵. The earliest developmental role for the Tao kinases can be observed in C. elegans, where KIN-18 is required for localizing RHO-1 to facilitate cell contractility in the single-celled embryo, as well as establishing an anterior-posterior boundary for the PAR polarity proteins ⁵⁶. Unlike Taok1 and Taok2, a role for Taok3 in development has yet to be supported with the current data.

While much of the current data describes a role for the Tao kinases in brain/neural development, previously published RNAseq data in X. laevis shows that all three Tao kinases are expressed throughout early development, long before neural induction occurs ⁵⁷. However, an early developmental role for these critical regulators of the MAPK pathway has yet to be elucidated. In the present study, we describe the spatiotemporal expression of Taok1, Taok2, and Taok3 throughout the development of the Xenopus laevis embryo. This is a first critical step in understanding how these Map3Ks contribute to the vertebrate body plan.

2. Results

2.1. Sequence analysis and comparison of X. laevis Tao kinases

Tao kinases share structural similarity to the Ste20p family of serine kinases and are known members of the GCK sub-family³². The protein sequences of Taok1 (1001 AA), Taok2 (1025 AA), and Taok3 (896 AA) from X. laevis cluster together with paralogous proteins from other vertebrates, including human (H. sapiens) and zebrafish (D. rerio), but not with the basal chordate C. intestinalis (Fig 1A). For each of the Tao kinase orthologs, the X. laevis paralog shares a node with human, when compared with zebrafish. The conserved N-terminal kinase domain of X. laevis Taok1, Taok2, and Taok3 contains the 11 kinase subdomains associated with Ste20 kinases (Fig 1 B, underlined), as well as the conserved activation loop (Fig 1B, yellow shading) and critical serine residue (Fig 1B, red box) ^63,64. Overall, the X. laevis Tao kinases share 82–84% sequence identity with each other, at the amino acid level, with the highest degree of identity in the N-terminal kinase domain with much lower conservation outside of this domain. (Fig. 1C). For example, the Taok1 kinase domain is roughly 90% identical to TaoK2, while only 69% identical outside of this region. Comparatively, Taok2 and Taok3 are 85.5% identical in their kinase domains and 61% identical across the rest of the protein sequence.

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).

2.2. Tao kinase expression level analysis

In order to establish the spatiotemporal patterns for the Tao kinases during Xenopus development, qPCR and in situ hybridization were used to determine the relative amounts and location of gene expression during early embryogenesis. Prior to gastrulation, taok3 is expressed at greater than 6-fold higher levels than taok1 and taok2, when normalized to slc35b1.L, and continues to be expressed at higher levels throughout all stages tested (Fig 2). taok3 levels decrease at the onset of gastrulation and continue to decrease to mid-gastrulation, where it reaches a steady level of expression through the late tailbud stage (Stage 30). taok1 exhibits a slight increase in expression at the onset of gastrulation and then steadily declines through the late tailbud stage. taok2 is expressed at the lowest levels of all the Tao kinases through neurulation (Stage 18), with expression decreasing at the onset of gastrulation and then slightly increasing through the neurula and tailbud stages. From the neurula stage through the late tailbud, taok2 is expressed at the second highest levels, just below taok3. The qPCR expression trends are similar to those found through the published RNAseq data on Xenbase⁶⁵, but do exhibit minor differences (Fig 1S). Notably, our data show that taok3 is expressed at higher levels than taok1 and taok2 throughout development, whereas the RNA-Seq data show higher taok1 expression in the pre-gastrula stages and higher taok2 levels in the post-gastrula stages.

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.

2.3. Localization of the Tao kinases during early development of X. laevis

Expression of taok1 and taok3 is uniformly distributed across the animal half of the embryo in the 2-cell stage embryo (Fig 3, A and C) while taok2 is not detectable through in situ hybridization (Fig 3, B). This pattern persists in the late blastula (St. 9) with taok1 and taok3 also migrating with the edge of the animal cap towards the vegetal pole, as epiboly commences (Fig 3, D, D’, F, F’). taok2 remains undetectable at stage 9. At the onset of gastrulation (St. 10), taok1 and taok3 are ‘cleared’ from the animal pole (Fig 4, A and C). taok1 is still uniformly distributed around the equatorial region (Fig 4 A’), while the limits of taok3 expression are more asymmetrically aligned (Fig 4, C’), with the dorsal side expression extending further towards the vegetal aspect of the embryo. By the late gastrula state (St 12.5), both taok1 and taok3 are expressed in the vegetal half of the embryo (Fig 4, D and F), with higher expression in the dorsal ectoderm and involuting mesoderm (Fig 4, asterisk in D’ and F’), and lower levels in the presumptive ventral ectoderm and mesoderm. taok2 remains undetectable throughout the gastrula stages (Fig 4, B, B’, E, E’).

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

In post-gastrula stage embryos, taok1, taok2, and taok3 exhibit a high degree of overlapping expression (Figs 5 and 6). In late neurula stage embryos (St. 18), all three Tao kinases are expressed in the neural plate along the entire anterior-posterior axis (Fig 5 A–C). In embryos transversely bisected, taok1, taok2, and taok3 are all expressed in the somitic tissue lateral to the midline, while taok1 and taok2 are additionally expressed in the notochord at equal levels to their somitic expression and taok3 notochord expression is reduced compared to the corresponding somitic expression (Fig 5, A’-C’). In the early tailbud stage embryo (St. 24), all three Tao kinases are still present along the anterior-posterior axis in the dorsal neural and mesodermal tissues. Additional anterior tissues, including the branchial arches and eye, also express taok1, taok2, and taok3 (Fig 5 D–F). Compared to taok1 and taok2, taok3 exhibits weaker expression in the branchial arches. Expression of taok1, taok2, and taok3 persists in the late tailbud (St. 30), with a significant overlap within the dorsal and anterior tissues (Fig 6 AC, A’–C’). Compared to taok1 and taok2, the dorsal aspect of taok3 expression extends further posteriorly, but stops just short of the tailbud. In addition to the neural tube, notochord, eye, and branchial arches, taok1, taok2, and taok3 are present in the otic vesicle and brain. taok2 appears to also be expressed at low levels in the pronephros, based on location and developmental timing (Fig 6 B, pn). Within the neural tube, taok1 is uniformly expressed along the dorsal-ventral axis, while both taok2 and taok3 have a stronger expression in the ventral region compared to the dorsal aspect (Compare Fig 6 A’ to B’ and C’).

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

3. Discussion

Map kinase signaling is a critical regulator of cell function, ranging from apoptosis and cell division to cell migration and differentiation. Due to the vast array of outcomes resulting from Map kinase signaling, a high degree of regulation is required in order to prevent aberrant activation or repression of downstream targets. Map kinase effector proteins, such as ERK, p38, and JNK are activated by upstream kinases, any of which can serve as critical regulation points of the overall pathway. For example, in the early Zebrafish embryo, p38α is asymmetrically activated in the cleavage stage, where it likely functions to control cell division on the future dorsal side of the embryo ⁶⁶. However, the upstream activator(s) in this case has not yet been identified. In contrast, MEKK1 (a Map3K) is specifically expressed in the developing eyelid epithelium and has a specific role in the differential activation of JNK 1 and JNK2 during eyelid closure, where disturbances in this pathway lead to the EOB (eyes open at birth) phenotype ^67,68. Because of the redundancy in these signaling pathways, differential expression as well as dose-dependency play central roles in how these pathways are interpreted.

The Tao kinases are a highly conserved group of Map3ks with homologs across the metazoans. Invertebrates only have a single Tao kinase, while vertebrates have three identified orthologs (Taok1, Taok2, and Taok3). The vertebrate orthologs share a high degree of similarity at the protein level and cluster together in paralogous groups with other vertebrates (Fig 1A). The conservation of the paralogs indicates that they have defined and unique functions from one another, and while they all function in Map kinase signaling, their spatiotemporal regulation during development may provide a mechanism to separate potential redundancies. In Xenopus laevis embryos, taok1 and taok3 exhibit some distinct, but mostly overlapping expression throughout development (Figs 3–6). The high expression levels of taok1 and taok3 in the early embryo supports their potential roles in the regulation of Map kinase signaling, with each potentially targeting the downstream effectors, p38 and JNK, in a tissue specific manner. Both p38 and JNK are necessary for the early patterning events in the Xenopus embryo and their synergistic effects are likely required for tissue specific signaling ⁶⁹. Due to their distinct, yet overlapping functions in the Map kinase pathway, a deeper investigation is necessary to determine their specific roles in early embryonic development. The high levels of taok3 expression in the pre-gastrula supports the requirement of JNK signaling for axis formation and apoptotic decision making in the pre-gastrula embryo ^70,71. Conversely, taok2 is expressed at much lower levels than taok1 and taok3 and is not detectable via in situ hybridization in the early embryo, supporting a role in neural development as described in other organisms. The ectodermal expression of taok1 and taok3 in the gastrula could also provide a mechanism for the early specification of neural tissue. Taok1 and Taok2 have both been previously described as activating p38, so their overlapping expression patterns would likely have some redundancy in signaling, which could affect axis formation and tissue specification in the early embryo ^24,25,72.

Later stage embryos (neurula and later) exhibit almost complete overlapping expression between taok1, taok2, and taok3, with a few notable differences. Expression of all three Tao kinases at these later stages suggests a coordinated effect on Map kinase signaling throughout development, with each possibly responding to a different set of upstream signals. Since both p38 and JNK signaling are required for organogenesis of multiple structures, the overlapping expression of the Tao kinases is not surprising ^29,73–75. The expression patterns of the Tao kinases also strongly support their role in neural development, with early ectodermal expression in the gastrula of taok1 and taok3 providing a possible mechanism for initial neural specification and the later stage expression of taok1, taok2, and taok3 functioning to direct more specific development of neuronal tissues. While their expression does overlap in many neural tissues and sensory organs, one notable difference is the strong ventral neural tube expression of taok2 and taok3, compared to the low expression levels of taok1. This could be due to gene specific responses to Sonic hedgehog (Shh) signaling emanating from the notochord, which is required for patterning the ventral neural tube ⁷⁶. It is possible that taok2 and taok3 are induced by the ventral Shh signal or, conversely, repressed by the dorsal BMP gradient. taok2 is also weakly expressed in the pronephros, indicating a potential role in embryonic kidney development, where it could function to regulate either p38 or JNK signaling ^30,77. While the Tao kinases most commonly function through Map kinase signaling, they have been implicated as effectors of the Hippo Pathway. Interestingly, Tao kinase expression overlaps significantly with the transcription factors Yap and Taz throughout all stages of development ⁷⁸. Future studies on Tao kinase function in developmental process will certainly need to include an in-depth focus on Hippo signaling, in addition to their role as Map kinase effectors.

Overall this expression data highlights the complexity of Map Kinase signaling throughout development, through the overlapping, co-expression of the Tao kinase family of Map3Ks. While each ortholog has been described as an effector of p38 and JNK, a true understanding of their developmental roles will require a detailed analysis of their upstream activators, downstream targets, and specific effects on morphogenesis. The data presented here provides a spatiotemporal framework for studies focused on Tao kinase signaling during development.

4. Experimental Procedures

4.1. Xenopus laevis care and husbandry

All experimental protocols that involved the use of X. laevis were approved by the Institutional Animal Care and Use Committee at the University of Central Arkansas. X. laevis eggs and embryos were obtained and handled by standard techniques ⁵⁸. Standard Nieuwkoop staging of embryos was used ⁵⁹.

4.2. Sequence and phylogenetic analysis

Orthologous X. laevis Tao kinase sequences were acquired through Xenbase and used for BLAST search to identify homologous sequences in other metazoan genomes (Table 1) ⁵⁷. The search verified the known sequences in H. sapiens and D. melanogaster and identified Tao kinases in D. rerio and C. intestinalis. Yeast Ste20p was used as an outgroup for phylogenetic analysis. Selected Tao kinase orthologs were aligned using Clustal Omega (EMBL- EMI) and the maximum likelihood phylogenetic tree was created with PhyML 3.0 (ATGC-Montpellier) ⁶⁰.

Table 1:

Accession numbers for all protein sequences used for maximum likelihood analysis

	Taok1	Taok2	Taok3	Taok	Ste20p
X. laevis (L)	NP 001084574.1	NP 001085661	NP 001086943	-	-
H. sapiens	AAI33040.1	AAI44345.1	AAH02756.1	-	-
D. rerio	XP_009293944.1	XP_009297645.1	XP_009299531.1	-	-
C.intestinalis	-	-	-	XP_009862271.1
D.melanogaster	-	-	-	BAF51959.1	-
S.cerevisiae	-	-	-	-	EWH18031.1

4.3. qPCR

mRNA was extracted from X. laevis embryos at stages 2, 9, 10, 12.5, 18, 24, and 30 using 20uL RNAzol (Sigma) per embryo (15–20 embryos per stage) and three biological replicates were used for each stage. cDNA was synthesized from 1μg of mRNA using ProtoScript^® II First Strand cDNA Synthesis (NEB). qPCR was performed using Luna^® Universal qPCR Master Mix (NEB) on a BioRad CFX96 Real Time PCR machine. Each primer set was designed to be specific to each paralog taok1, taok2, and taok3, but able to recognize both the L and S versions of each gene. All normalization was performed using the reference gene slc35b1.L ⁶¹. Primers used were: taok1, (5’-CCAACACGAACGAGAGATAC-3’) and (5’CTAGCCTCATGCTTTCAGAG-3’), taok2, (5’- CAGTTCTTACACTGCACAGG-3’) and (5’- CCATTTCTCCACTGTCATCG-3’), taok3, (5’- CAAGGCCCTAAAGAATCACC-3’) and (5’- GCTTGTGAGGCCATCATTTCG-3’), and slc36b1.L, (5’- CGCATTTCCAAACAGGCTCC-3’) and (5’- CAAGAAGTCCCAGAGCTCGC-3’). Cycling conditions were as follows: 95°C x 1 min, then 40 cycles of 95°C x 15s → 60°C x 30s with a plate read after every cycle. Three technical replicates were performed for each of the biological replicates. Efficiency was calculated from the slope of a standard curve generated from qPCR of serially diluted template DNA. Data was normalized to the expression of slc35b1.L using the Livak method (ΔΔC_T).

4.4. In situ hybridization

Whole mount in situ hybridization was performed as described in Jerry, et al. 2019⁶² and based on the protocol from Sive et al., 2000) ⁵⁸. For probe synthesis, plasmids containing cDNA for taok1(BC068781.1), taok2 (BC073108.1), and taok3 (BC077802.1) (HorizonDiscovery) were sub-cloned into pcDNA3, using fragments consisting of the non-conserved C-terminal sequences: Taok1 bases 1801–2957, Taok2 bases 1934–3082, Taok3 bases 1224–2692. DNA was linearized with BamHI for taok1, XhoI for taok2, and BamHI for taok3 and UTP-digoxygenin labeled probes were synthesized in vitro (Promega) and purified with RNAeasy columns (QIAGEN). Images were obtained with a Zeiss Axiocam eRC 5s camera and analyzed with Affinity Photo.

Supplementary Material

1

Figure 1S. Comparison RNAseq values to qPCR quantification. TPM values for the ‘L’ and ‘S’ alleles of taok1 (A), taok2 (B), and taok3 (C) from previously published data (Xenbase) were combined to account for the total expression values (left side Y axis). These data were plotted against their developmental stages (gray lines) and overlaid with the qPCR relative expression amounts from Figure 1 (blue lines, right side Y axis), which includes both the ‘L’ and ‘S’ alleles. qPCR values for taok1, taok2, and taok3 were normalized to slc35b1 expression. Embryos were staged according to Nieuwkoop Faber (NF).

Highlights.

• Taok1, Taok2, and Taok3 orthologs are conserved across vertebrates

• Taok1 and Taok3 are expressed from pre-gastrula through late-stage embryos

• Taok1 and Taok3 expression tracks with presumptive to differentiating neural tissue

• Taok1 and Taok3 are expressed in mesoderm and later in somitic mesoderm

• Taok2 expression is limited to neural and mesodermal tissues in late-stage embryos