Lukas P and Olsson L (2018), Bapx1 upregulation is associated with ectopic m...

XB-ART-55067

Zoological Lett 2018 Jan 01;4:16. doi: 10.1186/s40851-018-0101-3.

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Bapx1 upregulation is associated with ectopic mandibular cartilage development in amphibians.

Lukas P , Olsson L .

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BACKGROUND: The emergence of novel structures during evolution is crucial for creating variation among organisms, but the underlying processes which lead to the emergence of evolutionary novelties are poorly understood. The gnathostome jaw joint is such a novelty, and the incorporation of bapx1 expression into the intermediate first pharyngeal arch may have played a major role in the evolution of this joint. Knockdown experiments revealed that loss of bapx1 function leads to the loss of the jaw joint, because Meckel's cartilage and the palatoquadrate fuse during development. We used Xenopus laevis and Ambystoma mexicanum to further investigate the function of bapx1 in amphibians. Bapx1 expression levels were upregulated through the use of Ly-294,002 and we investigated the potential consequences of the enhanced bapx1 expression in amphibians to test the hypothesized joint inducing function of bapx1. RESULTS: We show that Ly-294,002 upregulates bapx1 expression in vivo. Additionally, ectopic mandibular arch derived cartilages develop after Ly-294,002 treatment. These ectopic cartilages are dorsoventrally oriented rods situated lateral to the palatoquadrate. The development of these additional cartilages did not change the muscular arrangement of mandibular arch-derived muscles. CONCLUSIONS: Development of additional mandibular cartilages is not unusual in larval anurans. Therefore, changes in the bapx1 expression during evolution may have been the reason for the development of several additional cartilages in the larval anuran jaw. Furthermore, our observations imply a joint-promoting function of bapx1, which further substantiates its hypothetical role in the evolution of the gnathostome jaw joint.

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Species referenced: Xenopus laevis
Genes referenced: barx1 col2a1 fubp1 nkx3-2 pik3cb
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	Fig. 1 Quantitative analysis of X. laevis tadpole survival after Ly-294,002 treatment. X. laevis larvae were treated with different amounts of Ly-294,002 at different developmental stages until NF 45. As controls, X. laevis larvae were raised either in 0,1 μM DMSO or in 0,1× MBS. Larvae treated with DMSO and with 0,1× MBS show no significant differences in survival rates. The survival rate in Ly-294,002 treated larvae declines with increasing Ly-294,002 concentration and early start of the incubation
	Fig. 2 Effects of Ly-294,002 treatment on external head morphology and bapx1 expression in X. laevis. Lateral view of larvae at NF 44 after rearing in a 0,1× MBS as control, b 0,1 μM DMSO and c 20 μM Ly-294,002 from NF 29 onwards. No significant differences in the external morphology can be observed. In situ hybridisation of NF 37 X. laevis larvae raised in d 0,1xMBS as control and e 20 μM Ly-294,002 from NF 29 onwards in lateral view reveal differences in the expression of bapx1 in the mandibular arch. After bapx1 upregulation the mandibular arch expression domain is broader and extends postero-dorsally. Scale bar 500 μm
	Fig. 3 3D reconstructions based on confocal laser scanning microscopy showing the effects of Ly-294,002 treatment on cranial cartilage morphology in Xenopus laevis larvae. Lateral view of ZO 10 (a, b) and ZO 17 (c, d) larvae with normal bapx1 expression (left column) and elevated bapx1 expression (right column). In Ly-294,002-treated specimen with increased bapx1 expression an ectopic cartilage develops within the mandibular arch lateral to the palatoquadrate. Depictions of X. laevis larvae in lateral view show the main differences between e normal and f perturbed development. In short, the processus muscularis is reduced in size and an ectopic cartilage arises lateral to the palatoquadrate after upregulation of bapx1. ch, ceratohyal; cm, Meckel’s cartilage; ec, ectopic cartilage; ir, infrarostral cartilage; pq, palatoquadrate; sp., suprarostral plate
	Fig. 4 3D-reconstructions based on confocal laser scanning microscopy of specimen stained with antibodies showing the effects of bapx1 upregulation on cranial muscle morphology in Xenopus laevis larvae. Lateral view of the jaw articulation and selected muscles of ZO 17 larvae. a Muscle morphology in a control specimen. b Muscle morphology in a specimen after bapx1 upregulation. The m. levator mandibulae articularis originates more anteriorly on the dorsal surface of the processus muscularis than in control specimens. The m. quadratohyoangularis surrounds the ectopic cartilage without originating from or inserting onto this cartilage. cm, Meckel’s cartilage; ec, ectopic cartilage; lma, M. levator mandibulae articularis; qha, M. quadratohyoangularis
	Fig. 5 Effect of Ly-294,002 treatment on bapx1 expression in Xenopus laevis at NF 45. Expression levels were determined through relative quantification (Livak method). Error bars indicate standard deviation of PCR runs (n = 3). DMSO reared specimens show no significant reduction in expression compared to control specimens. Ly-294,002 treated specimens show a ~ 5 times higher expression level of bapx1 in comparison to controls
	Fig. 6 Effect of Ly-294,002 injection into mandibular arch precursors on cartilaginous cranial morphology in X. laevis larvae. 3D-reconstructions based on confocal laser scanning microscopy of a control and b injected specimens at ZO 17 in lateral view. Maximum intensity projections of c control and d injected specimens at ZO 20 stained with monoclonal II6B3-collagen II antibody and Alexa 568 in dorsal view. The reduction of the processus muscularis and the presence of an ectopic cartilage can be observed in injected specimens. ch, ceratohyal; cm, Meckel’s cartilage; ec, ectopic cartilage; pmpq, processus muscularis of the palatoquadrate; pq, palatoquadrate. Scale bar 200 μm
	Fig. 7 Effect of Ly-294,002 treatment on the external and internal morphology of A. mexicanum. Dorsal view of a control and b Ly-294,002 treated specimens reveals no differences in external morphology. Lateral view of 3D-reconstructions based on confocal laser scanning microscopy of c control and d Ly-294,002 treated specimen shows the development of an ectopic cartilage lateral to the jaw articulation in treated specimens. The musculature of e control and f treated specimens are similar and no changes in origination or insertion of muscles can be observed after bapx1 upregulation. cm, Meckel’s cartilage; dm, M. depressor mandibulae; ec, ectopic cartilage; lma, M. levator mandibulae articularis; lme, M. levator mandibulae externus; lml, M. levator mandibulae longus; pq, palatoquadrate. Scale bar 500 μm
	Fig. 8 Overview of the diversity of adrostral cartilages among anurans. Ventral view of the jaw region of selected anuran larvae. a Bombina orientalis shows a typical anuran condition of the jaw region consisting of paired infrarostral cartilages, a plate-like suprarostral cartilage and horizontally oriented Meckel’s cartilages. b In Pelobates fuscus (drawn after Nikitin 1986) two so-called adrostral cartilages can be observed lateral to the paired suprarostral cartilages. c Heliophryne orientalis possesses two additional cartilages in the lower jaw. The submeckelian cartilages lie beneath Meckel’s cartilages and the adrostral cartilages are situated lateral to the suprarostral cartilage. In Ly-294,002-treated Xenopus laevis larvae (d) the ectopic cartilages arise lateral to the palatoquadrate and are free from any muscle insertion or origination similar to P. fuscus and H. orientalis

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