Pleiotropic functions of embryonic sonic hedgehog expression link jaw and taste bud amplification with eye loss during cavefish evolution

Abstract

This study addresses the role of sonic hedgehog (shh) in increasing oral-pharyngeal constructive traits (jaws and taste buds) at the expense of eyes in the blind cavefish Astyanax mexicanus. In cavefish embryos, eye primordia degenerate under the influence of hyperactive Shh signaling. In concert, cavefish show amplified jaw size and taste bud numbers as part of a change in feeding behavior. To determine whether pleiotropic effects of hyperactive Shh signaling link these regressive and constructive traits, shh expression was compared during late development of the surface-dwelling (surface fish) and cave-dwelling (cavefish) forms of Astyanax. After an initial expansion along the midline of early embryos, shh was elevated in the oral-pharyngeal region in cavefish and later was confined to taste buds. The results of shh inhibition and overexpression experiments indicate that Shh signaling has an important role in oral and taste bud development. Conditional overexpression of an injected shh transgene at specific times in development showed that taste bud amplification and eye degeneration are sensitive to shh overexpression during the same early developmental period, although taste buds are not formed until much later. Genetic crosses between cavefish and surface fish revealed an inverse relationship between eye size and jaw size/taste bud number, supporting a link between oral-pharyngeal constructive traits and eye degeneration. The results suggest that hyperactive Shh signaling increases oral and taste bud amplification in cavefish at the expense of eyes. Therefore, selection for constructive oral-pharyngeal traits may be responsible for eye loss during cavefish evolution via pleiotropic function of the Shh signaling pathway.

Figure 1

Amplified shh expression in the cavefish oral pharyngeal region. A– D. Dorsal anterior (A, B) and lateral (C, D) views of 1 dpf surface fish (A, C) and cavefish (B, D) embryos showing expanded shh expression in the cavefish anterior midline (A, B arrowheads). C–F. Lateral (C, D) and rostral (E, F) views of 2 dpf surface fish (C, E) and cavefish (D, F) embryos showing expanded shh expression in the cavefish oral epithelium (o). O: oral area. OC-P: Oral-pharyngeal cavity. G–J. Ventral views of 3 dpf surface fish (G, I) and cavefish (H, J) embryos showing shh expression in taste buds (upward pointing arrowheads in I) and primary tooth germs (oblique pointing arrowheads in I) on the lips. I, J are two- fold magnifications of G, H showing the ring-like shh expression pattern in taste buds. K–N. Sections through 3 dpf surface fish (K) and cavefish (L, N) comparing the patterns of shh expression (K, L, N) and calretinin staining (M) in taste buds. MC: marginal cells. Scale bars: A (100 μm), E (50 μm), K (20 μm); M (4 μm); magnification is the same in A–D, E–H, I and J, K and L, M and N. O. Quantification by qRT-PCR showing increased levels of shh, vax1, and pax2a mRNA and decreased levels of pax6 mRNA relative to β-actin and α-actin mRNA in 3 dpf cavefish larvae. Asterisk: p<0.05 in one-way ANOVAs comparing cave and surface fish mRNA levels (n=4).

Figure 2

Constructive oral-pharyngeal features in cavefish. A–D. Dorsal (A, B) and ventral (C–F) views 6 dpf surface fish (A, C, E) and cavefish (B, D, F) showing wider jaw span (A, B; double-headed arrows), larger Alcian Blue stained mandibles (C, D), more calretinin-stained taste buds (E, F; upward pointing arrowheads), and wider oral palates (E, F; doubled headed arrows) in cavefish. Scale bars in A is 100μm; magnification is the same in A–D and E, F. G–I. Surface fish (top frames) and cavefish (bottom frames) show differences in jaw width (G, red bars) and taste bud numbers on the upper (H, blue bars) and lower (I, black bars) lips. Jaw width is indicated in units of 20 μm with unit 1 as 371–390 μm, unit 2 as 391–410 μm, and so forth.

Figure 3

Effect of MO-mediated shh inhibition on oral and taste bud development. A–H. Cavefish were injected with control (A, C, E, G) or shh (B, D, F, H) MOs and analyzed at the tailbud stage (A, B) or 6 dpf (C–H). A, B. In situ hybridization showing downregulation of nkx2.1a but not pax2a (asterisks) expression in shh MO injected embryos at the neural plate stage. C–F. Reduced jaw span (C, D; double headed arrows) and oral pharyngeal region (F, arrowhead) in shh MO injected larvae at 6 dpf. C, D: Ventral views. E, F: Lateral views. Double headed arrows: jaw span. G, H. Reduced numbers of calretinin-stained taste buds are formed in 6 dpf cavefish larvae injected with shh MO. G: Ventral view. H. Anterior view. Downward and upward pointing arrowheads indicate upper and lower jaws respectively. Scale bars: A (250 μm), C, E, and G (200 μm); same magnification in A and B, C and D, E and F, G and H. I. Reduced jaw span (μm; red bars) and taste bud numbers on the upper (blue bars) and lower (black bars) lips in 6 dpf cavefish injected with shh MO (middle three bars) compared to control MO (left three bars). Injection of a mixture of shh MO and zebrafish shh mRNA decreases the effects on jaw width and taste bud number (right three bars). JW: jaw width. UJ: Upper jaw. LJ: Lower jaw. Error bars indicate SE of the mean.

Figure 4

Effect of shh overexpression on oral and taste bud development. A–H. Surface fish (A–F) or cavefish (G, H) embryos were injected with shh (B, E, F–H) or GFP (A, C, D) mRNAs and analyzed at the tailbud stage (A, B) or 6 dpf (C–H). A, B. In situ hybridization showing expansion of nkx2.1a but not pax2a expression in the neural plate of cavefish embryos injected with shh MO (B). C–H. Increase in the oral pharyngeal region and calretinin-stained oral taste bud numbers (arrowheads) in shh mRNA injected surface fish (C–F) and cavefish (G, H) embryos. Lateral (C, E, G), ventral (D, F), and anterior ventral (H) views at 6 dpf. (C–F). 20 pg shh mRNA. (G, H). 800 pg shh mRNA (E, F). DE: pigmented remnant of degenerate eye. Arrowheads: calretinin stained taste buds. Doubled headed arrows: mouth opening. Scale bars: A (250 μm), C (200 μm); magnification is the same in A and B, C–F. I. Increased jaw span (red bars) and taste bud numbers on the upper (blue bars) and lower (black bars) lips in 6 dpf larvae that developed from embryos 20 pg shh mRNA (middle) or 800 pg shh mRNA (right) relative to controls injected with 20 pg GFP mRNA (left). Error bars indicate SE of the mean. JW: jaw width. UJ: Upper jaw. LJ: Lower jaw.

Figure 5

The effects of shh overexpression on oral-pharyngeal development and eye degeneration in surface fish embryos injected with an hsp70:shh:GFP transgene and heat shocked at various stages of development. A, B. A transgene injected embryo (6 dpf) heat shocked at the tailbud (TB) stage showing a gaping mouth, enlarged forebrain (FB), and small degenerate eyes. C, D. A transgene injected embryo (6 dpf) heat shocked beginning at 1 dpf showing a normal mouth and eye. A and C: dorsal views B and D: lateral views. Scale bar in D is 200 μm.; magnification is the same in A–D. E. The shh sensitivity periods for increased taste bud development and eye degeneration in transgene injected surface fish embryos determined by heat shocking at different developmental stages. Red bars. oral width (μm). Blue bars: taste bud number on upper lips. Black bars: taste bud number on lower lips. Error bars indicate SE of the mean. Blue dots: Percentage of embryos with normal eye development. Error bars indicate SE of the mean. JW: jaw width. UJ: Upper jaw. LJ: Lower jaw. Asterisk indicates that the pooled differences between taste buds on the upper and lower jaws at the tail bud and 1-somite stage are significantly different from those at 1, 2, 2.5, and 3 dpf (p = 0.003).

Figure 6

Relationship between oral pharyngeal traits and eye size in the F3 hybrid progeny of a surface fish X cavefish cross. A. Examples of small- (A) and large- (B) eyed hybrid. The eye(s) of small-eyed hybrids are sunken into the orbit and unpigmented, resembling those of cavefish, whereas the eyes of large-eyed hybrids are exposed and pigmented, resembling those of surface fish. C–E. Differences in jaw width (red bars) and taste bud numbers on the upper (blue bars) and lower (gray bars) lips of small- and large-eyed F3 hybrids. Jaw width is indicated in units of 20 μm with unit 1 as 331–350 μm, unit 2 as 351–370 μm, and so forth.

Figure 7

Diagram illustrating the relationship between Shh signaling, oral pharyngeal constructive traits, and eye development along the Astyanax embryonic midline. A. Surface fish. B. Cavefish indicating the effects on oral pharyngeal, lens, and optic cup development of enhanced Shh signaling. Letter size indicates relative increase or decrease in cavefish compared to surface fish. See text for other details.