Wang YH and Beck CW (2014), Distal expression of sprouty (spry) genes durin...

XB-ART-49068

Gene Expr Patterns 2014 May 01;151:61-6. doi: 10.1016/j.gep.2014.04.004.

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Distal expression of sprouty (spry) genes during Xenopus laevis limb development and regeneration.

Wang YH , Beck CW .

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The eukaryotic Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling pathways, which in turn provide readout for morphogens such as fibroblast growth factors (Fgfs) that are essential for many developmental processes, including limb development. In a transcriptome analysis of early proximo-distal patterning of the Xenopus laevis limb bud, spry1a, 2 and 4 were predicted to be expressed predominantly in the distal third. Expression of all three in the distal limb initially corresponded to the progress zone mesenchyme, adjacent to the fgf8b-positive cryptic apical ectodermal ridge (AER). Spry2 transcripts were also expressed ectodermally, and later localized to the AER. During formation of the autopod, spry1a and spry4 became restricted to the anterior distal mesenchyme. All three spry genes were re-expressed in the mesenchyme cells of the blastema during hindlimb regeneration, with spry2 also overlapping with the region of fgf8b re-expression. However, the anterior bias seen in developing limbs was not recapitulated. We conclude that Spry1a, 2 and 4 have partially overlapping expression in developing and regenerating Xenopus limbs, which correspond with known areas of Fgf signaling. Sprys may therefore be involved in refining of Fgf signal transduction from the AER during development and regeneration.

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Species referenced: Xenopus laevis
Genes referenced: fgf10 fgf8 spry1 spry2 spry3 spry4

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	Fig. 1. Identification of three spry genes in a stage 51 limb bud transcriptome. (A) stacked column graph showing percentage of reads originating from each third of the limb bud: proximal, middle or distal. fgf8 and -8b are included for comparison. (B) Bar graph to show relative numbers of reads for each transcript across the whole limb, normalised for transcript size. Higher numbers of reads indicate higher levels of expression. (C–F) In situ hybridisation to stage 32 X. laevis embryos for spry1a, 2, 3 and 4. Abbreviations: ba: branchial arches, mhb: mid/hindbrain boundary, op: olfactory placode, ov: otic vesicle, sc: spinal cord, tb: tail bud.
	spry1 (sprouty homolog 1, antagonist of FGF signaling ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	spry2 (sprouty homolog 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	spry3 (sprouty homolog 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	spry4 (sprouty homolog 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	Fig. 2. In situ hybridisation of spry1a, spry2, spry4 and fgf8b in Xenopus laevis developing hindlimbs from stage 49 to 55. Fgf8b is included to show the location of the AER at developmental stages 50–54. Specific staining is dark purple. Black spots in some panels are melanophores in pigmented specimens. Black arrows indicate distal expression, red arrows localised expression in more proximal mesenchyme of older limbs, and thin white arrows indicate possible forming blood vessels. Roman numerals show detectable staining in some digits. The AER is indicated in some panels to show absence of spry1a and spry4 and presence of spry2 and fgf8b transcripts. All limbs are oriented proximal to the left and posterior uppermost, with the tadpole on its right side, except for panels B, I and V where both hind limbs are viewed from the anterior with the tadpole placed on its back. For stage 54 and 55 limbs only the autopod is shown for spry1a, spry4 and fgf8b, as by this stage there is no proximal staining. Developmental stage is in the bottom right of each panel and limbs are photographed to the same scale, except for panel T which shows histological section through a stage 53 limb bud to show expression of spry2 in the AER around digit IV. Scale bar in T is 50 um.
	spry1 (sprouty homolog 1, antagonist of FGF signaling ) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 50, proximal to the left and posterior uppermost, with the tadpole on right side.
	spry1 (sprouty homolog 1, antagonist of FGF signaling ) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 52, proximal to the left and posterior uppermost, with the tadpole on right side.
	spry1 (sprouty homolog 1, antagonist of FGF signaling ) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 53, proximal to the left and posterior uppermost, with the tadpole on right side.
	spry1 (sprouty homolog 1, antagonist of FGF signaling ) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 55, proximal to the left and posterior uppermost, with the tadpole on right side.
	spry4 (sprouty homolog 4) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 50, proximal to the left and posterior uppermost, with the tadpole on right.
	spry4 (sprouty homolog 4) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 51, proximal to the left and posterior uppermost, with the tadpole on right.
	spry4 (sprouty homolog 4) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 52, proximal to the left and posterior uppermost, with the tadpole on right.
	spry4 (sprouty homolog 4) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 53, proximal to the left and posterior uppermost, with the tadpole on right.
	spry4 (sprouty homolog 4) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 54, proximal to the left and posterior uppermost, with the tadpole on right.
	spry4 (sprouty homolog 4) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 55, proximal to the left and posterior uppermost, with the tadpole on right.
	spry2 (sprouty homolog 2) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 50, proximal to the left and posterior uppermost, with the tadpole on right side.
	spry2 (sprouty homolog 2) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 51, proximal to the left and posterior uppermost, with the tadpole on right side.
	spry2 (sprouty homolog 2) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 52, proximal to the left and posterior uppermost, with the tadpole on right side.
	spry2 (sprouty homolog 2) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 53, proximal to the left and posterior uppermost, with the tadpole on right side.
	spry2 (sprouty homolog 2) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 54, proximal to the left and posterior uppermost, with the tadpole on right side.
	fgf8 (fibroblast growth factor 8 (androgen-induced)) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 50, proximal to the left and posterior uppermost, with the tadpole on right side.
	fgf8 (fibroblast growth factor 8 (androgen-induced)) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 51, proximal to the left and posterior uppermost, with the tadpole on right side.
	fgf8 (fibroblast growth factor 8) gene expression in Xenopus laevis embryonic hindlimb, assayed via in situ hybridization, NF stage 52, proximal to the left and posterior uppermost, with the tadpole on right side. Key: aer= apical ectodermal ridge
	Fig. 3. In situ hybridisation of spry1a, spry4, spry2 and fgf8b in Xenopus laevis regenerating stage 52 hindlimbs from 1 to 6 days post amputation (dpa). Panels A–F; whole- mount spry1a, whole-mount panels H–M; spry4; whole-mount panels N–S, spry2. Panels U–Z show whole-mount fgf8b, which marks the functional apical ectodermal cap (AEC), for comparison. Scale bar in U is 250 lm. Specific staining is dark purple, and the distal limits of the regeneration bud AEC, which are hard to see, are outlined by red dashes. White dashed lines indicate approximate plane of the original amputation. Black spots in some panels are melanophores in pigmented specimens. Black arrows indicate de novo distal expression. Dpa in the bottom right of each panel indicates the time after amputation in days. Panels G and T are histological sections through 3 dpa regenerating hindlimbs with scale bars representing 50 lm. G) expression of spry1a is restricted to the regeneration bud (b). T) expression of spry2 is seen in the mesenchymal regeneration bud (b), the functional AEC (fa), a row of columnar epithelial cells adjacent to the mesenchyme and in the inner layers of the AEC. All limbs are oriented proximal to the left and posterior uppermost, with the tadpole on its right side.