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Kazald1 is expressed during skeletal and tooth development in tetrapods, and potentially in all jawed vertebrates (A) Quantification of Kazald1 expression in the developing whole limbs of embryonic mice, the intact, regenerating (R), and non-regenerating (NR) digit tips of amputated adult mice, the developing humerus of stage HH36 embryonic chickens, and whole embryos of different developmental stages of the axolotl. (B) Quantification of Kazald1 expression in adult axolotl during limb and mandible regeneration, and in the intact bone and cartilage of limbs. (C) Movat’s pentachrome staining of 15 dpa juvenile axolotl limb. cg = cartilaginous growth, Ul = ulna, Ra = radius. Scale bar, 500 μm. (D and D′) Maximum Z projection of whole-mount HCR for Kazald1 and negative control in the lower jaw of stage 46 larval axolotl. Dashed line indicates the location of Meckel’s cartilage (mc). Arrows indicate Kazald1 expression associated with the ossifying dentary bone. Arrowheads indicate Kazald1 expression associated with a tooth bud. Scale bars, 250 μm. (E and E′) HCR of Kazald1 and negative control in a sectioned tooth bud of adult axolotl. Scale bars, 100 μm. (F) Quantification of Kazald1 expression in the jaw and spine of the lungfish, bichir, and paddlefish, and in the cartilage of the bamboo shark. Kazald1 was duplicated in the lineage leading to paddlefish with no subsequent loss, creating Kazald1a and Kazald1b. (G) Whole-mount in situ for Kazald1 in the stage 29 catshark embryo head and upper trunk. Arrowheads indicate expression associated with the pharyngeal arches. Scale bar, 1 mm. (H) Tail tip of embryo in G. Scale bar, 500 μm. (I) Quantification of Kazald1 expression in the tooth of juvenile alligator, at different stages of tooth development in adult catshark, and teeth of adult bamboo shark. Non-tooth tissue is included as controls for expression in other areas of the jaw of the catshark. PRJ IDs indicate the publicly available RNA-Seq datasets that generated the raw data for the listed tissues. Dots represent biological replicates in examined datasets, and error bars represent standard deviation when calculable. CPM = counts per million, St. = stage, dpa = days post amputation, hpa = hours post amputation. Data are represented as mean ± SD.
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