Maternal thyroid hormones are essential for neural development in zebrafish

Abstract

Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.

Figure 1.

Lateral views of zebrafish embryos at 12 (A–D), 24 (E–H), 31 (I–L), 48 (M–P), and 72 (Q–T) hpf showing results of WISH for expression of mct8 (A, E, I, M, and Q), thraa (B, F, J, N, and R), thrab (C, G, K, O, and S), and thrb (D, H, L, P, and T). In A, black arrowheads denote rhombomeres 3 and 5. In E, F, and G, white arrows indicate the otic vesicle. Scale bars, 100 μm.

Figure 2.

MCT8MO titration. A, Bright-field images of 48-hpf wild-type zebrafish embryos noninjected or microinjected with 0.6 pmol CTRMO or 0.4, 0.6, or 0.8 pmol MCT8MO. Scale bars, 500 μm. B, To further understand the phenotype and penetrance of the MCT8MO, WISH for pax8 was carried out in 48-hpf wild-type zebrafish embryos noninjected or microinjected with 0.6 pmol CTRMO or 0.4, 0.6, or 0.8 pmol MCT8MO. Scale bars, 100 μm.

Figure 3.

Increased apoptosis in mct8 morphants is not rescued by suppression of p53 signaling. A, Lateral (first row) and dorsal (second row) bright-field images of wild-type 25-hpf zebrafish noninjected embryos or embryos microinjected (1 nL) with CTRMO (0.6 pmol), MCT8MO (0.8 pmol), or MCT8+p53MO (0.8 + 0.5 pmol). The black box denotes the otic vesicle presented in a higher magnification in the insets. Scale bars, 100 μm. B, Total fluorescence (570 nm) measurement of noninjected and CTRMO, MCT8MO, or MCT8+p53MO microinjected wild-type 25-hpf zebrafish embryos after TUNEL using TMR-red. In the graph in B, bars denote SE, and experimental groups that do not differ significantly bear the same letter (P > .05) and those with different letters are significantly different (P < .0001) after Bonferroni multiple-comparison test.

Figure 4.

MCT8MO phenotype is rescued by MCT8 mRNA coinjection. A, Lateral (upper row) and dorsal (lower row) bright-field images of Tg(fli1:EGFP) 48-hpf zebrafish embryos noninjected and CTRMO (0.6 pmol), mutated mct8 mRNA (100 g), MCT8MO (0.8 pmol), or mutated mct8 mRNA+MCT8MO (100 pg + 0.8 pmol) microinjected (1 nL). Scale bars, 100 μm. B, Distribution of normal, morphant, and rescued animals in the different experimental groups according to axial trunk bending phenotype. C, Fluorescent images of central arteries invading the hindbrain of Tg(fli1:EGFP) 48-hpf zebrafish embryos noninjected and CTRMO, mutated mct8 mRNA, MCT8MO, or mutated mct8 mRNA+MCT8MO microinjected. The red chevron denotes midcerebral vein, red arrowheads denote central arteries invading the hindbrain, and cyan asterisk denotes otic capsule. Scale bars, 50 μm.

Figure 5.

MCT8 knockdown affects brain and spinal cord development. A, Fluorescent images of 25-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO. Red and green represent, respectively, Pax7 and acetylated β-tubulin immunostaining. The white and cyan boxed area denotes, respectively, the high-magnification images presented in B and C. Scale bars, 100 μm. B, Higher magnification of the most anterior spinal cord section of 25-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO (white boxed area in A) after immunostaining for Pax7. The red brackets denote Pax7-positive spinal cord cells along the dorsal region of the developing spinal cord. In the MCT8 morphant, only the most anterior dorsal spinal cord has a few Pax7-positive cells. Scale bars, 50 μm. C, Higher magnification of sections 4 to 10 of the spinal cord in 25-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO (white boxed area in A) after immunostaining for acetylated β-tubulin. Dorsal (red arrowheads), medial (cyan arrowheads), and ventral (green arrowheads) positioned spinal cord neurons are highlighted. Scale bars, 50 μm.

Figure 6.

pax2a is a target of maternal THs in a context-related fashion. shha (A), fgf8a (B), and pax2a (C, D and, E) WISH expression in 25-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO. Arrowheads in B denote the otic vesicle expression field of fgf8. Arrowheads in C denote otic vesicle expression of pax2a, and arrows denote the optic stalk expression field. Boxed areas in D represent the high-magnification images shown in E. Scale bars, 50 μm (A, B, and E) and 100 μm (C and D).

Figure 7.

At 31 (A–D) and 48 (E and F) hpf, mct8 morphants have fewer hindbrain and spinal cord neurons. A, Immunodetection of Zn8 in the hindbrain of 31-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO. Arrowheads in A denote hindbrain interneurons, and red asterisks indicate trigeminal neurons. Scale bars, 50 μm. B, Expression of pax2a in 31-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO. Black arrowheads indicate otic vesicle expression of pax2a. Scale bars, 100 μm. C, Immunodetection of acetylated β-tubulin in segments 4 to 10 of the spinal cord in 31-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO. Scale bars, 50 μm. D, Expression of pax8 in 31-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO. Arrows indicate first pax8-positive hindbrain interneurons. Scale bars, 50 μm. (e) Spinal cord expression of pax8 in 48-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO. Scale bars, 200 μm. The boxed area in E corresponds to a higher-magnification image in F. Scale bars, 50 μm (F).

Figure 8.

mct8 morphants have impaired mobility at 72 hpf. A, Typical image depicting the total distance covered in 2 minutes by 72-hpf wild-type zebrafish embryos or embryos microinjected with either CTRMO or MCT8MO. Scale bar, 1 cm. B, Box and whisker graphic (minimum and maximum) of the distance covered in 1 minute after touch of 72-hpf wild-type zebrafish embryos microinjected with either CTRMO or MCT8MO. ***, Significant statistical differences (P < .0001) between the experimental groups (Dunnet's multiple-comparison test).