Expression of the zic1, zic2, zic3, and zic4 genes in early chick embryos

Abstract

Background: The zic genes encode a family of transcription factors with important roles during early development. Since little is known about zic gene expression in chick embryos, we have characterized the expression patterns of the zic1, zic2, zic3, and zic4 (zic1-4) genes during neurulation and somitogenesis.

Findings: We used in situ hybridization to analyze the expression patterns of the zic1-4 genes during early chick development (HH stages 7-19). The zic1-3 genes showed both overlapping and gene-specific expression patterns along the length of the dorsal neural tube and in the dorsal parts of the somites. In addition, unique expression domains of zic genes included: zic2 in the neural plate, periotic mesoderm and limb buds; zic3 in the paraxial mesoderm surrounding the neural plate, in presomitic mesoderm and in the most recently formed epithelial somites; zic2 and zic3 in developing eyes. zic4 expression was limited to dorsal fore- and midbrain regions and, unlike the expression of the zic1-3 genes, zic4 expression was not detected in the hindbrain and trunk. This was in contrast to more extensive zic4 expression in other vertebrates.

Conclusions: The zic1-3 genes were expressed in both overlapping and unique domains within the neural tube, somites and other ectoderm and mesoderm-derived structures in the future head and trunk. zic4 expression, however, was limited to dorso-anterior regions of the future brain. This is the first comprehensive study of zic1-4 gene expression in chick embryos during neurulation and somitogenesis.

Figure 1

Expression of the zic1 gene in whole mount chick embryos at HH stages 7-11 determined by in situ hybridization. A: In stage 7 embryos, zic1 is expressed at very low levels in the most anterior portion of the head fold (white arrow). B: zic1 is expressed in the neural folds of the future forebrain (white arrow) and hindbrain (white arrowheads) at stage 8. It is not expressed in the future spinal cord or in somites (black arrows). C: By stage 9, zic1 is expressed in the anterior region of the future forebrain and in a domain of the future hindbrain (white arrowheads). D: Very weak zic1 expression in anterior somites is first observed at stage 10 (black arrowheads). E (dorsal view), F (side view): zic1 expression in anterior somites becomes more robust by stage 11 (black arrowheads), as does zic1 expression in the anterior portion of the trunk neural tube (E). In the brain, zic1 continues to be expressed in the anterior prosencephalon (best seen in F, white arrow) and in the limited hindbrain domain (best seen in E, white arrowheads). zic1 is not expressed in the optic vesicles. G: At stage 11, anterior somites express zic1, while it is not expressed in posterior somites. zic1 expression in the trunk neural tube diminishes towards posterior regions. H: zic1 is not expressed in the neural tube, neural folds, or somites of the posterior trunk in stage 11 (and older) embryos. Overall, expression levels of zic1 are very low in these early stage embryos. FB: future forebrain; MB: future midbrain; HB: future hindbrain; HN: Hensen's Node; PS: primitive streak; NF: neural folds; Pro: prosencephalon; Mes: mesencephalon; Rhom: rhombencephalon; white arrowheads: hindbrain expression domain of zic1; white arrow: forebrain expression domain of zic1; black arrows: somites not expressing zic1; black arrowheads flank somites expressing zic1; the anterior of the embryos in all figures is to the left.

Figure 2

Expression of the zic2 gene in chick embryos at HH stages 6+ to 12. A: zic2 is expressed in all of the head fold and neural plate region of stage 6+ embryos. B: At stage 8, zic2 expression continues in the neural folds and throughout the neural plate along the entire anterior-posterior axis of the embryo. C: zic2 expression is downregulated in much of the forebrain and midbrain regions of stage 9 embryos. zic2 expression persists in a small domain of the forebrain and in a large region of the hindbrain. Contiguous with its expression in the hindbrain, zic2 is expressed in the trunk neural tube. D: zic2 is not yet expressed in somites of stage 10 embryos. E, F: dorsal view (E) and side view (F) of stage 11 embryo; although zic2 expression appears weaker in the mesencephalon region (E), the side view shows that zic2 expression is present along the entire dorsal brain and trunk neural tube (F). The optic vesicles express zic2 at stage 11. G: Weak zic2 expression begins in the anterior somites of stage 11 embryos. H: zic2 is expressed in the most posterior neural tube and neural folds. I: in stage 12+ embryos, zic2 expression in anterior somites is more robust and zic2 is expressed in the periotic mesoderm (bracket). Overall, expression levels of zic2 are very low in early stage embryos. Abbreviations as in legend for Figure 1; NP: neural plate; black arrows: somites not expressing zic2; black arrowheads flank somites expressing zic2; bracket: periotic mesoderm.

Figure 3

Expression of the zic3 gene in chick embryos at HH stages 7 to 13. A: zic3 is expressed in the future forebrain in a limited and weak expression domain. It is broadly expressed in the future hindbrain. B: At stage 8+, weak zic3 expression continues in a limited region of the forebrain. It is expressed in the neural folds of the hindbrain and more weakly in the anterior trunk. C, D, D': In stage 9 embryos (7-9 somites), the first epithelial somites (somites I-III and sometimes somite IV) express zic3, while more anterior somites do not express zic3 (black arrows, C). zic3 is expressed at low levels in the presomitic mesoderm and at a slightly increased level in the area that will form the next somite (red arrowheads). D and D': posterior region of a stage 9 embryo. In D', the area that represents the neural plate and expresses zic2 (see Figure 2B) is outlined with red dots. The outer limit of the adjacent presomitic mesoderm, which expresses zic3, is marked by black dots. E: At stage 10, low levels of zic3 expression continue in presomitic mesoderm and in epithelial somites I-III. The neural folds do not express zic3. F: zic3 is first expressed along the entire brain in stage 11 embryos. Anterior somites do not express zic3 (black arrows). G, H: By stage 13, anterior somites (H, black arrowheads) and the optic vesicles first express low levels of zic3. The presomitic mesoderm expresses zic3, while there is very little or no expression of zic3 in epithelial somites (G). The brain and anterior neural tube express zic3 (H), while the posterior neural tube does not (G). Abbreviations as in legend for Figure 1; NP: neural plate; black arrows: somites not expressing zic3; black arrowheads flank somite showing weak zic3 expression; red arrowheads: increased zic3 expression in presomitic mesoderm.

Figure 4

Comparison of zic1-4 gene expression in chick embryos at stage HH 14/15. A-D: side views of whole embryos; E-H: dorsal views of midbrain, hindbrain, anterior trunk; I-L: mid-trunk; M-P: tail with epithelial somites marked (I - V). A, E, I, M: zic1 is expressed in the dorsal brain and in the neural tube, except in the most posterior neural tube (M). zic1 is expressed in most somites. B, F, J, N: zic2 is expressed along the entire dorsal brain and neural tube of the trunk with enhanced expression in the neural tube of the tail (N). zic2 is expressed in the developing eye (B), in the periotic mesoderm (F) and in anterior somites (F). C, G, K, O: zic3 is expressed in the dorsal brain, in the eye (C), and in the neural tube, except in the neural tube of the tail (O). zic3 is expressed in the presomitic mesoderm (O). D, H, L, P: zic4 is expressed in the anterior brain (D) and is not detectable in any other parts of stage 14/15 embryos. Pro: prosencephalon; Mes: mesencephalon; Rhom: rhombencephalon; ov: otic vesicle; NT: neural tube; PSM: presomitic mesoderm; arrowheads outline somites; arrow points to zic4 expression; bracket: periotic mesoderm. The stage of the embryos is HH 14 with the exception of D, H: HH 14-; M, F, N, P: HH 15.

Figure 5

Comparison of zic1-4 gene expression in chick embryos at stage HH 18/19. A-D: whole embryos; E-H: close-up side-views of the eye and hindbrain regions; I-L: dorsal views of midbrain, hindbrain, and anterior trunk; M-P: dorsal views of the mid-trunk; Q-T: dorsal views of the tail. A, E, I , M, Q: zic1 is expressed in the dorsal brain and in the trunk neural tube, except in the tail tip (Q). zic1 is strongly expressed in all somites, including caudal somites (Q). B, F, J, N, R: zic2 is expressed in the dorsal brain and trunk neural tube. zic2 is strongly expressed in the neural tube of the tail tip (B), but more weakly in caudal somites (R). zic2 is expressed in the developing eye and periotic mesoderm (B, F) and in the limb buds (B, N, R). C, G, K, O, S: zic3 is expressed in the dorsal brain and spinal cord and in the somites. zic3 is expressed in the developing eye (C, G) and in the mesoderm of the tail (C, S). D, H, L, P, T: zic4 is expressed in the forebrain (D) and midbrain (D, L). No significant expression is detected in the hindbrain or in the trunk. zic1-4 are expressed in the optic stalk (E, F, G, H). Tel: telencephalon; Di: diencephalon; Mes: mesencephalon; Met: metencephalon; Myelen: myelencephalon; ov: optic vesicle; white bracket indicates extent of periotic mesoderm; each pair of arrowheads outlines a somite. The stage of the embryos is HH 18 with the exception of A, E, N, R, S, T: HH 19 embryos.

Figure 6

Sections of trunk and hindbrain regions of stage HH 14/15 embryos. A-F: zic1 expression by in situ hybridization. zic1 is not expressed in the posterior neural tube (A, B). More rostrally, it is expressed in the dorsal neural tube and in the dorsal portion of recently formed somites (arrow) (C). zic1 expression continues in the dorsal neural tube, dermomyotome (arrow heads), and sclerotome (asterisks) (D, E) and in the dorsal hindbrain (F). G-L: zic2 expression. zic2 is expressed throughout the dorsal/ventral axis of the neural tube in the tail (G). Rostral to the tail region, zic2 expression is restricted to the dorsal portion of the trunk neural tube (H-K). zic2 is expressed in the dermomyotome and sclerotome of rostral somites (K; arrow head and asterisk, respectively). It is expressed in the dorsal hindbrain and in the periotic mesoderm (L). M-R: zic3 expression. zic3 is expressed in the presomitic mesoderm (M, N). It is not expressed in epithelial somites (O) and only further rostrally in more mature somites in the sclerotome (P, Q; asterisks) and in the dermomyotome (Q; arrow head). zic3 is expressed in the dorsal neural tube of the mid- and anterior trunk (P, Q) and in the dorsal hindbrain (R). S-X: zic4 is not expressed in the trunk or hindbrain of stage 14/15 embryos. PSM: presomitic mesoderm; ES: epithelial somite; DS: dissociating somite; POM: periotic mesoderm; arrowheads: dermomyotome; asterisks: sclerotome.