Identification of differentially expressed genes in early inner ear development

Abstract

To understand the etiology of congenital hearing loss, a comprehensive understanding of the molecular genetic mechanisms underlying normal ear development is required. We are identifying genes involved in otogenesis, with the longer term goal of studying their mechanisms of action, leading to inner ear induction and patterning. Using Agilent microarrays, we compared the differential expression of a test domain (which consisted of the pre-otic placodal ectoderm with the adjacent hindbrain ectoderm and the underlying mesendodermal tissues) with a rostral control domain (which included tissue that is competent, but not specified, to express inner ear markers in explant assays). We identified 1261 transcripts differentially expressed between the two domains at a 2-fold or greater change: 463 were upregulated and 798 were downregulated in the test domain. We validated the differential expression of several signaling molecules and transcription factors identified in this array using in situ hybridization. Furthermore, the expression patterns of the validated group of genes from the test domain were explored in detail to determine how the timing of their expression relates to specific events of otic induction and development. In conclusion, we identified a number of novel candidate genes for otic placode induction.

Fig. 1

Design strategy for microarray experiments. (A) Cartoon of a stage 7 chick embryo showing the test domain (a) and the more rostral control domain (b). The test domain is marked on its caudal boundary by the first somite (pink ovals) and includes all-three germ layers in the mediolateral plane from the pre-placodal non-neural ectoderm (blue circles) to the midline of the neural plate of the future caudal hindbrain. The neural plate is colored red, the floor plate orange, and the primitive streak green. (B) In situ hybridization (ISH) showing Fgf19 expression in an embryo in which the test and control domains were removed prior to fixation and processing for ISH. The loss of most tissue expressing Fgf19 in the extirpated area confirms that the test domain was correctly removed for the microarray study.

Fig. 2

FGF-signaling pathway. Members of the FGF-signaling pathway identified in our microarray study as differentially expressed were visualized by in situ hybridization at induction (A–L) and placodal (M–P) stages of development. Fgf3, 4, and 19 are all expressed in bilateral patches of the test domain (E–G). In contrast, Fgfr3 expression is reduced in the test domain as compared to the control domain (H and L, arrows). Stage 6–7 embryos (E–H) were sectioned through the test domain as indicated (black lines). The sections (E’–H’) provide details on the germ layer in which each gene is expressed.

Fig. 3

WNT-signaling pathway. Members of the WNT-signaling pathway identified in our microarray study as differentially expressed were visualized by in situ hybridization at induction (A–O) and placodal stages of development (P–T). At stages 4–5, Wnt5b, Ndp, and Fzd10 are all expressed in the primitive streak, whereas Fzd4 is not expressed at all, and Fzd8 is restricted to the most rostral portion of the embryo, rostral to the node (A–E). Wnt5b and Fzd10 are expressed throughout the entire length of the primitive streak (A, D) and Ndp is expressed only in the caudal half of the streak (B). By stages 6–7, Wnt5b and Fzd10 have extended their streak-associated expression rostrolaterally into the test domain (F, I). At this time, Ndp and Fz4 are also expressed in the test domain (G, H): Ndp is expressed robustly in bilateral patches within the test domain, with additional expression extending caudally into the developing somites, but its primitive streak expression is fading; and Fzd4 is also expressed in bilateral patches in the test domain, but faintly, as well as in the cardiac crescent. Stage 6–7 embryos (F–J) were sectioned through the test domain as indicated (black lines). The sections provide details on the germ layer in which each gene is expressed (F’–J’). By stages 8–9 (K–O), all 5 of these WNT-family genes are expressed to some degree in bilateral patches in the test domain (arrows). Expression of Wnt5b extends caudally from the test domain (K, arrows) throughout the developing somite region and persists in the caudal portion of the streak, Ndp expression (L) extends into the area of the developing somites, Fzd4 expression (M) persists in the test domain and in the developing heart tubes, and Fzd10 (N) expression continues robustly in the primitive streak region. At this stage, Fzd10 is faintly expressed in distinct patches in the test domain (N, arrows). Like Fzd10, Fzd8 is only faintly expressed bilaterally in the test domain (O, arrows), but in contrast to the other WNT-family members, Fzd8 is also expressed robustly in the most rostral portion of the embryo throughout the placode induction stages. At placodal stages (P–T), the expression of Wnt5b (P) is restricted to the tail bud and flanking mesoderm, Ndp is expressed throughout the spinal cord level of the neural tube (Q) and adjacent to the otic placode in rhombomere 4 of the hindbrain (Q, arrowheads), Fzd4 is expressed throughout the heart and optic vesicles (R), Fzd10 persists in the tail bud and surrounding mesoderm, extending rostrally throughout the spinal cord level of the neural tube and hindbrain (S), and Fzd8 is expressed weakly throughout the brain level of the neural tube (T).

Fig. 4

Expression of Ndp (A–C) and Fzd10 (D–E) at otocyst stages. At stages 15–19, Ndp is expressed (A, B) throughout most of the length of the neural tube from the spinal cord to the mid-diencephalon region. By stage 22, Ndp expression in the neural tube is faint and restricted to specific patches (C, C’, arrows); staining observed in the otic vesicle and other parts of the head in whole mounts was due to trapping. Ndp is also expressed at the dorsal region of the developing mesonephric kidneys (C”, arrowheads). Fzd10 was also expressed along the rostrocaudal extent of the neural tube in a pattern very similar to Ndp at stages 14 (D) and 19 (E). However in sections, Fzd10 expression within the neural tube was broader and restricted to its dorsal two-thirds (E’). ov, otic vesicle; nt, neural tube; lb, limb bud.

Fig. 5

Notch-signaling pathway. Members of the Notch-signaling pathway identified in our microarray study as differentially expressed were visualized by in situ hybridization at induction (A–I), placodal (J–L), and post-placodal (M–N) stages of development. All three genes from the Notch-signaling pathway identified in our microarray are expressed in bilateral patches in the test domain beginning at stages 6–7 (D–F). Hes5 (E) and Hes6 (F) are also expressed in the node, whereas Jag1 (D) is also expressed in the developing heart field. Stage 6–7 embryos (D–F) were sectioned through the test domain as indicated (black lines). The sections provide details on the germ layer in which each gene is expressed (D’–F’). At placodal stages, Hes5 (K) and Hes6 (L) are expressed in the hindbrain neural tube. In addition, Hes5 is expressed near the otic placodes in the pharyngeal pouch endoderm (K: arrowheads, K’). Jag1 (J) expression at this time is concentrated in the developing heart. Jag1 expression is more prominent in the post-placodal stages of otic development, with persistent expression in the heart and new areas of expression in the lens, mesonephric kidneys, and otic vesicles at both stage 15 (M) and stage 19 (N). Dorsal view (N’) and sections at both stages 15 (M’) and 19 (N”) show Jag1 expression in the otic vesicle. op, otic placode; ov, otic vesicle; nt, neural tube; nc, notochord; mn, mesenephros; ht, heart.

Fig. 6

Hox genes. Members of the Hox gene family of transcription factors identified in our microarray study as differentially expressed were visualized by in situ hybridization at induction (A–L) and placodal (M–P) stages of development. Hoxa1–4 (Hoxa1–3 shown here) and Hoxb1 genes are all differentially upregulated in the test domain. Each of the Hox genes is expressed in the primitive streak at stage 4 (A–D). Expression in the primitive streak persists throughout placode induction stages, overlapping the test domain (E–P). The expression of Hoxb1 is unique among the Hox genes identified in our microarray in that at stages 6–7 it occurs in bilateral patches in the test domain, separated from the broader expression domain along the caudal half of the embryo (H, arrow). Stage 6–7 embryos (E–H) were sectioned through the test domain as indicated (black lines). The sections provide details on the germ layer in which each gene is expressed (E’–H’).

Fig. 7

Other transcription factors. A subset of other transcription factors identified in our microarray study as differentially expressed was visualized by in situ hybridization at induction (A–R) and placodal (S–Y) stages of development. Five other transcription factors chosen from our microarray data for further study are upregulated in the test domain, and all five are expressed in the bilateral patches in the test domain at stages 6–7 (G–L). Stage 6–7 embryos (G–L) were sectioned through the test domain as indicated (black lines). The sections provide details on the germ layer in which each gene is expressed (G’–L’). Gbx2 expression spans the test domain patches and extends into the developing cardiac crescent (G). Meox1 expression extends caudally from the test domain patches into the area of the developing somites(H, N, T), whereas Nkx6.2 extends slightly rostrally from the test domain into the hindbrain (I, O, U). Tbx22 is initially expressed in two small patches within the test domain (J) and later expression extends rostrally into the mid- and hindbrain and caudally into the forming somites (P). At placodal stages, Tbx22 expression persists in the somites and in undefined head mesenchyme (V, inset; inset sectioned at level indicated by the black line in V). Tshz3 is first expressed at stage 6 throughout the test domain, and in the regressing streak (K). Expression extends caudally throughout the middle portion of the embryo in the developing neural tube (Q). In addition to persistent expression of Tshz3 through the mid-level of the neural tube, Tshz3 is also expressed in rhombomere 4, adjacent to the otic placode (W, arrowheads). Dlx5 is expressed in the pan-placodal region from stage 4 (F) through stages 6–7 (L). By stage 9 Dlx5 is expressed in the heart tube and prosencephalon (R). At the placodal stages, Dlx5 is expressed within the otic placode (Y, arrowheads).

Fig. 8

Expression of selected transcription factors at otocyst stages. Nkx6.2 expression (A–B). At stage 15, Nkx6.2 is expressed in the neural tube from the spinal cord to the mid- and hindbrain regions (A). Sections revealed that Nkx6.2 expression is restricted to the ventral midline of the neural tube, extending up into the isthmus at the juncture between the metencephalon and mesencephalon (A’, A”). Expression persists along the neural tube and in the mid- and hindbrain regions through stage 18 (B). Tbx22 expression (C–D). During otocyst stages Tbx22 is expressed in the anteroventral portion of the developing somites (C–D). At stage 14, Tbx22-expressing mesenchymal cells flank the floor of the developing midbrain as two columns of cells (C, C’). By stage 16, these columns of mesenchymal cells expand to surround the developing pituitary gland (D, inset). Tshz3 expression (E–F). At stage 17, Tshz3 is expressed in a striped pattern along the anterior-posterior axis of the body. Tshz3 is expressed throughout the neural tube extending rostrally from the spinal cord into the posterior region of the hindbrain, adjacent to the otic vesicles (E, inset). Laterally and parallel to the neural tube, Tshz3 is expressed in two bands on either side of the embryo flanking the developing gut tube (E’). By stage 21, Tshz3 is expressed throughout much of the embryo, including the limb buds and neural tube (F). Sections through the otic vesicles revealed that Tshz3 is expressed in its anterior-most portion (F’), as well as in the marginal zone of the neural tube. e, eye; ov, otic vesicle; nt, neural tube; st, stomach.