Itpka depletion implicates defects in anterior neural development of Xenopus laevis

Abstract

Inositol 1,4,5-trisphosphate 3-kinase A (Itpka) is a neuronal isoform of the ITPK family that regulates both actin dynamics and calcium signaling. While itpka deficiency in adult mice mainly results in central nervous system phenotypes, its contribution to early development remains unclear. To study the role of Itpka in embryogenesis, we used the South African clawed frog, Xenopus laevis, as vertebrate model organism. Our analysis revealed that itpka is specifically expressed in distinct regions of the developing anterior neural tissue. To investigate Itpka function during early anterior neural development, we generated a morpholino oligonucleotide (MO)-mediated itpka knockdown approach. The depletion of Itpka leads to defects in head, brain, and eye development which can be rescued by Xenopus itpka RNA co-injection. An analysis of the underlying molecular basis revealed a reduced expression of key genes associated with head, brain and eye development in Itpka MO-injected embryos. These findings highlight a crucial role of Itpka during anterior neural development in Xenopus laevis and indicate that the function of Itpka needs to be further investigated.

Keywords: ITPKA; Xenopus laevis; anterior neural development; disease modeling; embryogenesis.

FIGURE 1

In silico analysis of itpka reveals a strong conservation across species. (A) Synteny analysis of itpka genes’ location and its neighboring genes among different species such as Homo sapiens, Mus musculus, Xenopus laevis, Xenopus tropicalis and Danio rerio. Conserved genes are depicted by boxes with identical colors, itpka is shown in red. Non-conserved neighboring genes are not shown. The orientations of the open reading frames are indicated by arrows. Gene lengths and 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. X. laevis L or S chromosome is specified next to the gene name. (B) Schematic representation of human ITPKA. Region required for cytoskeleton location (green), IPK region (blue) and calmodulin binding region (pink) are shown. (C) Human ITPKA protein sequence. Amino acid (aa) sequence is shown. The region required for cytoskeleton location (green), IPK region (blue) and calmodulin binding region (pink) are marked. (D) Homology of the aa sequences of full-length ITPKA among different species. Aa length is given in numbers. Percentages represent identical residues (percent identity) of the indicated species compared to Homo sapiens. Abbreviation: aa, amino acid; IPK, inositol polyphosphate kinases; itpka, inositol 1,4,5-trisphosphate 3-kinase A; LTK, leukocyte receptor tyrosine kinase; NDUFAF1, NADH:ubiquinone oxidoreductase complex assembly factor 1; NUSAP1, nucleolar and spindle associated protein 1; RPAP1, RNA polymerase II associated protein 1; RTF1, RTF1 homolog, Paf1/RNA polymerase II complex component; TYRO3, TYRO3 protein tyrosine kinase.

FIGURE 2

Itpka is expressed during X. laevis anterior neural development. (A–J) Spatio-temporal expression pattern of itpka visualized by whole mount in situ hybridization (WMISH) during X. laevis development. Embryonic stages and scale bars are indicated in each panel. Black dashed lines represent section planes. (A) Animal view of X. laevis at stage 10. (B) Anterior and (C) dorsal view of embryos at stage 13 and 15. (D) Transversal section of stage 15. (E) Anterior view of a X. laevis embryo at stage 20. (F) Transversal section at stage 23. (G) Lateral view. (H,I) Transversal sections and (J) horizontal section at stage 36. (K) Illustration of the isolated tissue (marked with asterisk) of the anterior (head) structures and the eye in different Nieuwkoop-Faber stages (st. 13, 23, 32, 42). (L) Temporal expression pattern of itpka during X. laevis anterior neural development analyzed by semi-quantitative reverse transcriptase (RT)-PCR with X. laevis cDNA templates of the indicated stages and isolated tissues. All three genes were detected in all investigated stages. Gapdh was used as loading control, and as negative control -RT which reaction lacks reverse transcriptase. Abbreviations: anp, anterior neural plate; b, brain; ba, branchial arch; bp, blastopore; cDNA, copyDNA; e, eye; epi, epidermis; ev, eye vesicle; gapdh, glyceraldehyde 3-phosphate dehydrogenase; ha, hyoid arch; le, lens; ma, mandibular arch; me, mesencephalon; µm, micrometer; nc, notochord; ncc, neural crest cells; itpka, inositol 1,4,5-trisphosphate 3-kinase A; PCR, polymerase chain reaction; re, retina; RPE, retinal pigmented epithelium; RT, reverse transcriptase; st., stage; WMISH, whole mount in situ hybridization.

FIGURE 3

Itpka MO knockdown results in defects in anterior neural development in a dose-dependent manner. (A) The dorsal and lateral views of the embryo show the head and eye phenotype at stage 42/43. Representative embryos are shown. (B) Statistical evaluation of smaller and deformed heads and eyes as indicated in (A). Abbreviations: CoMO, control morpholino oligonucleotide; inj., injected; itpka, inositol 1,4,5-trisphosphate 3-kinase A; µm, micrometer; MO, morpholino oligonucleotide; n, number of independent experiments; N, number of analyzed embryos in total; ng, nanogram; st., stage; uninj., uninjected; WT, wildtype. Error bars indicate standard errors of the means: ****, p ≤ 0.0001.

FIGURE 4

Itpka MO injection leads to a severe head phenotype that is rescued by Xitpka RNA. (A) The dorsal views of representative embryos at stage 42/43 are shown. Red lines indicate the measured head width and area. (B) Statistical evaluation of smaller and/or deformed heads between wildtype/CoMO-injected embryos compared to Itpka MO-injected embryos. (C) Statistical evaluation of the head width and area as indicated in (A). (D) Co-injection of Xenopus Itpka-RNA. The dorsal views of representative stage 43 embryos are shown. Red lines indicate the measured width and area. (E) Statistical evaluation of smaller and/or deformed heads between Itpka MO-injected embryos compared to Itpka MO-injected embryos with Xitpka co-injection. (F) Statistical evaluation of the head width and area as illustrated in (D). (G) Ventral view of Alcian blue-stained and dissected cranial cartilages from control and Itpka morphants. Deformed cartilage structures are shown by black arrowheads, especially at the Meckel’s cartilage (MC) and branchial arch (ba). Abbreviations: Ba, Branchial arch; CoMO, control morpholino oligonucleotide; GFP, green fluorescent protein; inj., injected; itpka, inositol 1,4,5-trisphosphate 3-kinase A; MC, Meckel’s cartilage; µm, micrometer; MO, morpholino oligonucleotide; n, number of independent experiments; N, number of analyzed embryos in total; Ps, Parasphenoid; st., stage; uninj., uninjected; WT, wildtype, Xitpka, Xenopus itpka. Error bars indicate standard errors of the means. **, p ≤ 0.01; ****, p ≤ 0.0001.

FIGURE 5

Itpka knockdown results in malformed cranial nerves. (A) Dorsal and ventral views of CoMO- and Itpka MO-injected embryos show the branching of cranial nerves upon Itpka MO KD visualized by 3A10 antibody staining. (B) Statistical evaluation of embryos with shortened or absent branching of cranial nerves as illustrated in (A). (C) Statistical analysis of cranial nerve length of the Nervus opticus (N. opticus) and the Nervus glossopharyngeus (N. glossop.) illustrated in (A). (D,E) Confocal images of an Itpka MO-injected embryo are shown. An overview (D) of the stained Nervus opticus (N. opticus), Nervus trigeminus (N. trigeminus) and Nervus mandibularis (N. mandibularis) and a detailed view (E) of the stained Nervus opticus (N. opticus). Abbreviations: CoMO, control morpholino oligonucleotide; inj., injected; itpka, inositol 1,4,5-trisphosphate 3-kinase A; KD, knockdown; µm, micrometer; MO, morpholino oligonucleotide; st., stage; uninj., uninjected. Error bars indicate standard errors of the means. *, p ≤ 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001.

FIGURE 6

Itpka MO injection impairs neural crest cell induction and migration at stage 15. (A) Anterior view of CoMO- and Itpka MO-injected embryos at stage 15 after whole mount in situ hybridization (WMISH) with NCC-specific genes twist1 and snai2 during NCC induction and migration. Reduced gene expression on the Itpka MO-injected side is indicated by black arrowheads. (B) Statistical evaluation of NCC-specific gene expression as illustrated in (A). (C) Anterior view of CoMO- and Itpka MO-injected embryos at stage 15. The analyzed gene expression area and the area of the measured mean intensity of gene expression of twist1 and snai2 is marked with a dashed line. Reduced gene expression on the Itpka MO-injected side is indicated by black arrowheads. (D) Statistical evaluation of the gene expression area of twist1 and snai2 as illustrated in (C). (E) Statistical evaluation of the mean intensity of gene expression of twist1 and snai2 as illustrated in (C). Abbreviations: CoMO, control morpholino oligonucleotide; inj., injected; MO, morpholino oligonucleotide; n, number of independent experiments; N, number of analyzed embryos in total; itpka, inositol 1,4,5-trisphosphate 3-kinase A; snai2, snail family transcriptional repressor 2; st., stage; twist1, twist family bHLH transcription factor 1; uninj., uninjected; WMISH, whole mount in situ hybridization. Error bars indicate standard errors of the means: *, p ≤ 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001.

FIGURE 7

Itpka MO knockdown influences migration of neural crest cells at stage 20. (A) Anterior view of CoMO- and Itpka MO-injected embryos at stage 20 after whole mount in situ hybridization (WMISH) with NCC-specific genes during NCC migration such as twist1 and snai2. Reduced gene expression on the Itpka MO-injected side is indicated by black arrowheads. (B) Statistical evaluation of NCC-specific gene expression as illustrated in (A). (C) Anterior view of CoMO- and Itpka MO-injected embryos at stage 20. The analyzed gene expression area and the area of the measured mean intensity of gene expression of twist1 and snai2 is marked with a dashed line. Reduced gene expression on the Itpka MO-injected side is indicated by black arrowheads. (D) Statistical evaluation of the gene expression area of twist1 and snai2 as illustrated in (C). (E) Statistical evaluation of the mean intensity of gene expression of twist1 and snai2 as illustrated in (C). Abbreviations: CoMO, control morpholino oligonucleotide; inj., injected; MO, morpholino oligonucleotide; n, number of independent experiments; N, number of analyzed embryos in total; ns, non-significant; itpka, inositol 1,4,5-trisphosphate 3-kinase A; snai2, snail family transcriptional repressor 2; st., stage; twist1, twist family bHLH transcription factor 1; uninj., uninjected; WMISH, whole mount in situ hybridization. Error bars indicate standard errors of the means: ns, p > 0.05; *, p ≤ 0.05; ****, p ≤ 0.0001.

FIGURE 8

Itpka MO injection hinders proper migration of neural crest cells at stage 23. (A) Lateral view of CoMO- and Itpka MO-injected embryos at stage 23 after whole mount in situ hybridization (WMISH) with NCC-specific genes during NCC migration twist1, foxd3 and egr2. Reduced gene expression on Itpka MO-injected side is indicated by black arrowheads. (B) Statistical evaluation of NCC-specific gene expression as illustrated in (A). (C) Lateral view of CoMO- and Itpka MO-injected embryos at stage 23. The analyzed gene expression area and the area of the measured mean intensity of gene expression of twist1, foxd3 and egr2 is marked with a dashed line. Reduced gene expression on Itpka MO-injected side is indicated by black arrowheads. (D) Statistical evaluation of the gene expression area of twist1, foxd3 and egr2 as illustrated in (C). (E) Statistical evaluation of the mean intensity of gene expression of twist1, foxd3 and egr2 as illustrated in (C). (F) Lateral view of CoMO- and Itpka MO-injected embryos at stage 23 showing the analyzed length (1,2,3) of twist1 gene expression and statistical evaluation of the length of twist expression (1,2,3). (G) Lateral view of CoMO- and Itpka MO-injected embryos at stage 23 showing the analyzed gene expression length (1,2,3) of foxd3 and the statistical evaluation of NCC-specific genes’ expression length. (H) Lateral view of CoMO- and Itpka MO-injected embryos at stage 23, showing the analyzed egr2 gene expression length (1,2) and the statistical evaluation of egr2 expression length (1,2) in detail. Abbreviations: CoMO, control morpholino oligonucleotide; egr2, early growth response 2; foxd3, forkhead box D3; inj., injected; MO, morpholino oligonucleotide; n, number of independent experiments; N, number of analyzed embryos in total; ns, non-significant; itpka, inositol 1,4,5-trisphosphate 3-kinase A; st., stage; twist1, twist family bHLH transcription factor 1; WMISH, whole mount in situ hybridization. Error bars indicate standard errors of the means: ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

FIGURE 9

Itpka knockdown affects the development of the brain. (A) Dorsal view of isolated brains from control and Itpka morphants (stage 42). Smaller brain area on the injected side (white arrowhead). (B) Statistical evaluation of the brain area as indicated in (A). (C) Anterior and dorsal view of control MO (CoMO)- and Itpka MO-injected embryos of egr2 and pax6 at stage 13 visualized by whole mount in situ hybridization (WMISH). Reduced expression is indicated by a black arrowhead. (D) Statistical evaluation of the brain-specific gene expression as illustrated in (C). (E) Anterior and dorsal view of CoMO- and Itpka MO-injected embryos at stage 15 and 23 after WMISH with brain-specific genes egr2, pax6 and otx2. Reduced gene expression on the Itpka MO-injected side in the mid- and hindbrain is indicated with black arrowheads. (F) Statistical evaluation of the brain-specific gene expression as illustrated in (E). Abbreviations: CoMO, control morpholino oligonucleotide; egr2, early growth response 2; inj., injected; itpka, inositol 1,4,5-trisphosphate 3-kinase A; µm, micrometer; MO, morpholino oligonucleotide; n, number of independent experiments; N, number of analyzed embryos in total; otx2, orthodenticle homeobox 2; pax6, paired box 6; st., stage; uninj., uninjected; WMISH, whole mount in situ hybridization. Error bars indicate standard errors of the means: *, p ≤ 0.05; ****, p ≤ 0.0001.

FIGURE 10

Itpka is required for eye development, while Xitpka rescues the Itpka MO-induced eye phenotype in X. laevis. (A) Lateral and detailed views of a representative wildtype, CoMO and Itpka MO-injected embryos at stage 42/43. Black arrowheads point to smaller eyes. (B) Statistical evaluation of smaller and deformed eyes as shown in (A). (C) Statistical analysis of the eye area (red dashed lines) as illustrated in (A). (D) Lateral and detailed views of Itpka MO-injected embryos in combination with Xitpka-RNA co-injection. Black arrowheads point to smaller eyes. Representative embryos at stage 43 are shown. (E) Statistical evaluation of smaller and deformed eyes as illustrated in (D). (F) Statistical analysis of the eye area (red dashed line) as illustrated in (D). (G) Transversal vibratome sections after whole mount in situ hybridization (WMISH) of Itpka MO-injected embryos at stage 43. Genes for specific cell populations of the retina are used as described in the main text. (H) Lens-specific genes celf1 and cryba1 are also affected. Abbreviations: celf1, CUGBP elav-like family member 1; CoMO, control morpholino oligonucleotide; cryba1, crystallin beta A1; GFP, green fluorescent protein; inj., injected; itpka, inositol 1,4,5-trisphosphate 3-kinase A; MO, morpholino oligonucleotide; n, number of independent experiments; N, number of analyzed embryos in total; pax6, paired box 6; pou4f1, POU class 4 homeobox 1; prox1, prospero homeobox 1; rho, rhodopsin; st., stage; vsx1, visual system homeobox 1; WT, wildtype; Xitpka, Xenopus itpka. Error bars indicate standard errors of the means: *, p ≤ 0.05; ****, p ≤ 0.0001.

FIGURE 11

Itpka is necessary for proper differentiation of eye-specific genes in X. laevis. (A) Anterior expression (black dashed line) of the eye-specific genes rax and pax6 as well as the pan-neural marker gene sox3 in control and Itpka morphants at stage 13, during eye field induction, visualized by whole mount in situ hybridization (WMISH). Reduced expression is shown by black arrowheads. (B) Statistical evaluation of embryos with reduced gene expression as described in (A). (C) Anterior view of CoMO- and Itpka MO-injected embryos illustrate eye-specific gene expression (black dashed line) of rax, pax6, and otx2 at stage 23. Black arrowheads show a reduced expression in the developing eye. (D) Statistical evaluation of embryos with reduced gene expression as described in (C). Abbreviations: CoMO, control morpholino oligonucleotide; inj., injected; itpka, inositol 1,4,5-trisphosphate 3-kinase A; MO, morpholino oligonucleotide; n, number of independent experiments; N, number of analyzed embryos in total; ns, non-significant; otx2, orthodenticle homeobox 2; pax6, paired box 6; rax, retina and anterior neural fold homeobox; sox3, SRY-box transcription factor 3; st., stage; uninj., uninjected; WMISH, whole mount in situ hybridization. Error bars indicate standard errors of the means. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01.