Rutenberg J et al. (2002), Early embryonic expression of ion channels and ...

XB-ART-6126

Dev Dyn 2002 Dec 01;2254:469-84. doi: 10.1002/dvdy.10180.

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Early embryonic expression of ion channels and pumps in chick and Xenopus development.

Rutenberg J , Cheng SM , Levin M .

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An extensive body of literature implicates endogenous ion currents and standing voltage potential differences in the control of events during embryonic morphogenesis. Although the expression of ion channel and pump genes, which are responsible for ion flux, has been investigated in detail in nervous tissues, little data are available on the distribution and function of specific channels and pumps in early embryogenesis. To provide a necessary basis for the molecular understanding of the role of ion flux in development, we surveyed the expression of ion channel and pump mRNAs, as well as other genes that help to regulate membrane potential. Analysis in two species, chick and Xenopus, shows that several ion channel and pump mRNAs are present in specific and dynamic expression patterns in early embryos, well before the appearance of neurons. Examination of the distribution of maternal mRNAs reveals complex spatiotemporal subcellular localization patterns of transcripts in early blastomeres in Xenopus. Taken together, these data are consistent with an important role for ion flux in early embryonic morphogenesis; this survey characterizes candidate genes and provides information on likely embryonic contexts for their function, setting the stage for functional studies of the morphogenetic roles of ion transport.

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Species referenced: Xenopus
Genes referenced: aqp4 arfgap1 atp1a1 atp6ap1 atp6v0c atp6v0d2 dmbt1 gja4 gja4.2 gja5 gjb3 kcna1 kcnb2 kcnip2 kcnip4 kcnj3 kcnj5 kcnj8 magainins vegt

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	Figure 2. Ion pump genes expressed in Xenopus. A variety of known ion channel and pump genes are also expressed during early Xenopus embryogenesis. Clones with specific expression patterns are shown here at representative stages. A: Sense probe controls show no signal. B: A probe for the maternal gene Xombi shows that whole-mount in situ hybridization can detect vegetal mRNA localization when it is present (arrow). C: Sectioning perpendicular to the animalegetal axis of a four-cell embryo stained in whole-mount in situ hybridization with a probe for the Ac45 V-ATPase subunit EST and embedded in JB4 shows (arrowheads) nuclear mRNA in the center of cells, as well as cytoplasmic mRNA. D,E: The neural β3 subunit of the Na+/K+ ATPase is detected at st. 11 in cells around the ventral margin of the blastopore (arrows). F: At st. 32, it is detected in the neural tube and in the posterior gut (arrows). G: Maternal mRNA encoding a subunit of the H+ pump (V-ATPase) is present throughout the animal hemispheres of the four-cell embryo (arrows). H: It is later expressed throughout the neural tube and head of the tail bud stage embryo (arrows). I: mRNA for the H+/K+-ATPase (ion exchanger) is present in a more laterally restricted region of the two-cell embryo (arrow). J: mRNA for the 16-kDa proteolipid component of the H+ synthase is expressed in bilateral stripes of deep tissue ventral to the neural tube, demonstrating that signal along the length of the neural tube and in the head is not obligatory or artifactual at tail bud stages. Red arrows indicate expression; white arrows indicate regions of no detectable expression. G is a photograph of the internal surface of two blastomeres of a four-cell embryo manually separated after in situ hybridization. I is a view of the internal surface of one blastomere of a two-cell embryo.
	Figure 3. Ion channel genes expressed in Xenopus. The inward rectifier Girk4 (kir3.4) K+ channel is expressed in neural tissue in hatched Xenopus embryos (red arrowhead) and is specifically detected in a spot (green arrowheads) on the side of the posterior head (A, close-up in B). C,D: The inward rectifier Kir6.1 is detected as a maternal message in the animal half of vegetal cells during cleavage (arrowheads). E: It is expressed in the neural tissues at somite stages but is also detected in the posterior gut (arrowheads). Maternal mRNA for the inward rectifier channel Girk1 (Kir3.1) is localized in the middle of animal cells during cleavage (arrowheads, F,G), and around the blastopore lip during gastrulation (arrowhead, H). I: Magainin mRNA is detected in the cells of the animal cap of the blastula-stage embryo (arrowheads). The K+ channel Kv1.1 can be detected by radioactive in situ hybridization in animal cells at st. 5 (arrowhead, J) and in cells undergoing ingression at the blastopore at st. 10 (K). A are chromogenic in situ hybridization, whereas J and K are radioactive sections (signal is lighter-colored, background is darker).
	Figure 4. Comparison of ion channel and pump gene expression in chick and Xenopus. We compared the expression of the same ion channel clones from chick and Xenopus by in situ hybridization. A: The K+ channel xKv2.2 was expressed very strongly in the organizer during Xenopus gastrulation (arrow). B,C: Sectioning reveals staining deep in organizer cells (arrows). Similarly, in chick, cKv2.2 was expressed in the base of the nascent primitive streak at stage (st.) 1 (arrow, D) and in the streak itself as it elongates (arrows, E,F). The potassium channel K(v)LQT-1 is also expressed in the primitive streak in the chick (arrow, G) and then in most tissues at st. 8 (arrow, H). I: In contrast, in Xenopus, we detect no expression by in situ hybridization at gastrulation stages (data not shown), but at neurulation, it is expressed in a horse-shoe pattern very similar to the location of the neural crest (arrows). Maternal mRNA for K(v)LQT-1 is located in the animal halves of cells at early cleavage stages (J, showing the inside surface of half of a four-cell embryo manually split down the cleavage plane after in situ hybridization; Ja: section of 1-cell embryo, Jb: inside surface of wholemount 2-cell embryo split down the cleavage plane). K: In chick embryos, the 16-kDa proteolipid subunit of the vacuolar ATPase is expressed in the head-folds of the closing neural tube (red arrows); it can also be seen in the regressing primitive streak (yellow arrow). In Xenopus, maternal mRNA for the 16-kDa proteolipid subunit can be detected as maternal mRNA in animal cells during cleavage (arrows, L), and similarly to the chick, is localized to the neural tissues in the dorsal aspect of the embryo at somite stages (arrows, M). Red arrows indicate regions of expression.
	Figure 5. Expression of accessory genes in chick and Xenopus. We examined the expression of several genes that, although not strictly ion pumps or channels, are relevant to maintaining a cell's membrane potential. The 14-3-3 family member ϵ is weakly expressed in the primitive streak in chick embryos at stage (st.) 3 (arrow, A), but by st. 4, its expression is extremely strong in all embryonic tissues (red arrow, B), except for the posterior-most margin of the area opaca (yellow arrow, B). C: The 14-3-3 α/β is expressed in the primitive streak at st. 3 (red arrowhead) but is specifically excluded from Hensen's node (white arrowhead). D,E: Aquaporin 7 mRNA is present around the circumference of the embryo at st. 7 (red arrows) but is not detected in the yolky vegetal cells. D: The inside view of a st. 7 embryo split in half parallel to the animalegetal axis after in situ hybridization. E: At somite stages, Aquaporin 7 is detected (red arrowheads) in the brain, eye, branchial arches, and somites. F: Aquaporin 4 is detected (red arrowheads) in the cortex of the animal portion of the embryo at the two-cell stage. F: The interior view of half of a two-cell embryo; the blastomeres were separated manually after in situ hybridization. G: By the four-cell stage, the mRNA is detected only in the nucleus. H: By somite stages, Aquaporin 4 can be detected in the brain, somites, and tail bud. In early chick embryos, hensin is expressed in the base of the primitive streak at st. 3 (arrowhead, I) and then in stripes in the lateral plate of head-fold stage embryos (arrows, J). KCHIP4.2 is not detected in the early chick (arrow, K), but KCHIP2 is expressed in the primitive streak (arrow, L). Red arrows indicate regions of expression.
	Figure 6. Expression of connexin genes in frog and chick embryos. A: Cx31 is expressed in the posterior margin of the area pellucida in the chick embryo at stage (st.) 4+ (arrows). B: Cx47 is expressed within the posterior third of the primitive streak at st. 4 (arrow). Cx37 is expressed in the early primitive streak (arrow, C), and in a punctate pattern identifying a subset of the cells of the primitive ridges and neural plate at st. 7 (arrows, D). In Xenopus, maternal Cx40 mRNA is in the anterior pole of the two-cell embryo (arrow, E) and then in a band perpendicular to the animalegetal axis in the four-cell embryo (arrow, F). G: During blastula stages, it is expressed in the cells of the animal cap (arrowhead). H: Radioactive in situ hybridization on sections shows that Cx38 is present in the animal cap and in gastrulating cells (arrow, radioactive section). Cx41 mRNA is spread throughout the animal pole of the two-cell embryo (arrow, I) but is only detected in the nucleus by the four-cell stage (arrow, J). Red arrows indicate domain of expression.
	kcnj5 (potassium inwardly rectifying channel subfamily J member 5 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28/29, lateral view, anterior left, dorsal up.
	kcnj8 (potassium inwardly rectifying channel subfamily J member 8) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 5 (16 cell), lateral view, anterior left, dorsal up.
	kcnj8 (potassium inwardly rectifying channel subfamily J member 8 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 4 (8cell), dorsolateral view.
	kcnj8 (potassium inwardly rectifying channel subfamily J member 8 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
	kcnj3.1 ( potassium inwardly rectifying channel subfamily J member 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 5 (16-cell), dorsolateral view.
	kcnj3 ( potassium inwardly rectifying channel subfamily J member 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 4 (8-cell), animal view.
	kcnj3 (potassium inwardly rectifying channel subfamily J member 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, vegetal/blastoporal view.
	magainins (magainins) gene expression in bisected Xenopus laevis blastula embryo, NF stage 8, assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	kcna1 (potassium channel, voltage gated shaker related subfamily A, member 1) gene expression in Xenopus laevis embryo, detected by radioactive in situ hybridization, NF stage 5 (16-cell) inJ, left) and in bisected NF stage 10 embryo (K, right).