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. 2022 Apr 1;10:797352. doi: 10.3389/fcell.2022.797352

FIGURE 2.

FIGURE 2

Axolotl Shh crispants completely lack forelimbs. (A,B) Phenotypes like body axis, position of the eye, and Alcian blue/Alizarin red staining of craniofacial structures of CRISPR control and Shh crispant larvae (n = 23 for CRISPR control and n = 67 for Shh crispant larvae for body axis and eye position analysis, n = 10 each for CRISPR control and Shh crispant larvae for Alcian blue/Alizarin red staining of craniofacial structures). Green dotted line, tail length; red dotted line, distance between the eyes; red asterisk, loss of anterior cranio-facial structures. Scale bar = 1 mm (for body axis) and 500 µm (for eye position and cranio-facial structures). (B,C) Alcian blue/Alizarin red staining for stage 48 limbs of CRISPR control and Shh crispant larvae (n = 10). Scale bar = 500 µm. (D) In situ hybridization for genes Ptch1, Grem1, and Fgf8 in CRISPR control and Shh crispant larvae at stage 46 limbs (n = 3 or 4 per treatments). Red arrows, expression domains. Scale bar = 100 µm. m, meckel; hh, hypohyale; bb1, basibranchial 1; hb1, hypobranchial 1; bb2, basibranchial 2; hb2, hypobranchial 2; ch, ceratohyal; and 4 cb1-4, ceratobranchials.