Neff AW et al. (2005), Expression of Xenopus XlSALL4 during limb devel...

XB-ART-34616

Dev Dyn 2005 Jun 01;2332:356-67. doi: 10.1002/dvdy.20363.

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Expression of Xenopus XlSALL4 during limb development and regeneration.

Neff AW , King MW , Harty MW , Nguyen T , Calley J , Smith RC , Mescher AL .

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The multi-C2H2 zinc-finger domain containing transcriptional regulators of the spalt (SAL) family plays important developmental regulatory roles. In a competitive subtractive hybridization screen of genes expressed in Xenopus laevis hindlimb regeneration blastemas, we identified a SAL family member that, by phylogenetic analysis, falls in the same clade as human SALL4 and have designated it as XlSALL4. Mutations of human SALL4 have been linked to Okihiro syndrome, which includes preaxial (anterior) limb defects. The expression pattern of XlSALL4 transcripts during normal forelimb and hindlimb development and during hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages is temporally and regionally dynamic. We show for the first time that a SAL family member (XlSALL4) is expressed at the right place and time to play a role regulating both digit identity along the anterior/posterior axis and epimorphic limb regeneration.

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Species referenced: Xenopus laevis
Genes referenced: odc1 sall1 sall2 sall4 tbx2

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	Figure 3. XlSALL4 expression in a stage 17 neurula (anterior and posterior views of two neurulae) and stage 28 tail bud stage embryos. Antisense XlSALL4 probe showed similar whole-mount in situ hybridization signals as Xsal-3 and chick cxal4 (Onuma et al., 1999; Barembaum and Bonner-Fraser, 2004). At the neurula stage, expression is localized to the developing central nervous system (CNS) and neural crest. At the tail bud stage, expression is prominent in the in the CNS, branchial arches, and tail bud/tail tip. Sense XlSALL4 probe gave no signal (data not shown).
	Figure 4. Expression of XlSALL4 during normal Xenopus limb development. A–I,K,L: Whole-mount in situ hybridization with the anti-sense XlSALL4 probe of stage 48 (A), 50 (B), 51 (C), 52 (D), 52+ (E), 53 (F), 54 (G), 55 (H), 57 (I) hindlimbs and stage 51+ (K), stage 53+ (L) forelimbs. J: A stage 53 hindlimb sense XlSALL4 probe control is shown. Dorsal views with the posterior side (pigmented) up and proximal to the left. Melanocytes are colored brown, while positive XlSALL4 signals are blue/purple. Magnifications are variable to fit different size limbs into constant boxes.
	Figure 5. Expression of XlSALL4 during Xenopus hindlimb development and in response to wounding. A,B: Longitudinal hindlimb sections of whole-mount in situ hybridization (WISH) limbs through the anterior–posterior plane of a stage 47/48 limb and a stage 50 limb. Note the absence of XlSALL4 expression in the epidermis and in the distal subepidermal zone (labeled in A with thin arrows). Also note the asymmetry of XlSALL4 mesenchymal expression along the anterior–posterior axis. Magnifications are variable to fit different size limbs into constant boxes. C: WISH of a stage 55 hindlimb wounded on the anterior side at the base of the autopod (thick arrow) 3 days after wounding, showing no expression at the wound site, while also showing the typical stage 55 interdigital XlSALL4 expression pattern. Posterior is up.
	Figure 6. Expression of XlSALL4 during Xenopus hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages. A: Whole-mount in situ hybridization (WISH) using anti-sense XlSALL4 probe at the stages (stage 53, 55, 57) and at the times after amputation (t = 0 control, 1-day, 3-days, 5-days, and 7-days) as indicated. Amputations were through the zeugopodia just distal to the knee joint. Dorsal views with the posterior side (pigmented) of the limb up and proximal to the left. Magnifications are variable to fit different size limbs into constant boxes. B: Expression of XlSALL4 in stage 53 amputated hindlimb stumps 1 day after amputation. End-on WISH images showing polar expression in distal amputated stump. The upper row shows three examples of the distal limb stump 1 day after amputation, while the bottom shows three controls fixed for WISH immediately after amputation (t = 0). Note intense XlSALL4 expression primarily in the posterior side of the distal limb stump 1 day after amputation compared with the t = 0 controls. By 3 days after amputation XlSALL4 expression is uniform throughout the mesenchyme of the blastema (data not shown). Anterior–posterior and ventral–dorsal limb axes are shown. All magnifications are the same.
	sall4 (sal-like 4) gene expression in Xenopus laevis embryos, NF stage 17, as assayed by in situ hybridization, anterior and posterior views, dorsal up.
	sall4 ( sal-like 4 ) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	sall4 ( sal-like 4 ) gene expression in Xenopus laevis, developing hindlimb, NF stage 48, as assayed by in situ hybridization, proximal is to the left.
	sall4 ( sal-like 4 ) gene expression in Xenopus laevis developeing hindlimb, NF stage 55, as assayed by in situ hybridization, proximal is to the left.
	Figure 2. Open in figure viewerDownload Powerpoint slide Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), analysis of XlSALL4 expression in developing hindlimbs and blastemas. A: Semiquantitive RT-PCR, showing levels of XlSALL4 product relative to ornithine decarboxylase (ODC) internal control. B: Relative quantitative real-time polymerase chain reaction (QPCR) data normalized to expression in stage 57 three-day pseudoblastema (3DB) corrected for ODC levels (see Experimental Procedures for more details). 53L, stage 53 hindlimb; 57L, stage 57 hindlimb; 1–5DB on the left are 1-, 3-, and 5-day-old stage 53 blastemas; 1–5DB on right are stage 57 1-, 3-, and 5-day-old pseudoblastemas.