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Mech Dev
2015 Nov 01;138 Pt 3:256-67. doi: 10.1016/j.mod.2015.10.003.
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Gremlin1 induces anterior-posterior limb bifurcations in developing Xenopus limbs but does not enhance limb regeneration.
Wang YH
,
Keenan SR
,
Lynn J
,
McEwan JC
,
Beck CW
.
Abstract
Gremlin1 (grem1) has been previously identified as being significantly up-regulated during regeneration of Xenopus laevis limbs. Grem1 is an antagonist of bone morphogenetic proteins (BMPs) with a known role in limb development in amniotes. It forms part of a self-regulating feedback loop linking epithelial (FGF) and mesenchymal (shh) signalling centres, thereby controlling outgrowth, anteriorposterior and proximaldistal patterning. Spatiotemporal regulation of the same genes in developing and regenerating Xenopus limb buds supports conservation of this mechanism. Using a heat shock inducible grem1 (G) transgene to created temperature regulated stable lines, we have shown that despite being upregulated in regeneration, grem1 overexpression does not enhance regeneration of tadpole hindlimbs. However, both the regenerating and contralateral, developing limb of G transgenics developed skeletal defects, suggesting that overexpressing grem1 negatively affects limb patterning. When grem1 expression was targeted earlier in limb bud development, we saw dramatic bifurcations of the limbs resulting in duplication of anteriorposterior (AP) pattern, forming a phenotypic continuum ranging from duplications arising at the level of the femoral head to digit bifurcations, but never involving the pelvis. Intriguingly, the original limbs have AP pattern inversion due to de-restricted Shh signalling. We discuss a possible role for Grem1 regulation of limb BMPs in regulation of branching pattern in the limbs.
Fig. 1. Grem1 expression, relative to shh and fgf8 supports the two phase limb reciprocal signalling model of Verheyden and Sun (2008) in Xenopus. A–W in situ hybridisation (dark purple staining) of developing (A–L) and regenerating Xenopus laevis limb buds. Note that the posteriorlimb bud is uppermost as this reflects the posture in which the embryo develops; the tadpole itself is anterior to the left and dorsal uppermost, lying on its side. This orients the limbproximal to the left. Some embryos were pigmented and show black melanophores. A–G) Shh in developing limbs is localised to the ZPA, the posteriordistalmesenchyme, as previously shown. Expression appeared strongest at stage 52 and then declined up to stage 55, where it was found in digit V mesenchyme. H–L) Grem1 expression was first seen in distalmesenchyme at stage 50 (H), before becoming cleared from the mesenchyme directly under the AER by stage 51 (I). This trend continued at stage 52 (J) before gradual loss of expression in the autopod so that no transcripts were detectable by stage 54 (K, L). Black arrowheads in C and J indicate amputation site used for regenerating limb experiments and roman numerals indicate digit identity. M–Q) Expression of shh in regenerating limbs amputated at stage 52, midway through the limb bud. Shh was first detected at 2 dpa and was always localised to the posteriordistalmesenchyme under the AEC (N–Q). By 6 dpa it was starting to reduce, as the autopod re-differentiated. R–W) Grem1 expression in regenerating limb buds appeared at 1 dpa and from 2 to 4 dpa was localised in distalanteriormesenchyme, reciprocal to shh (R–U). Expression cleared from the autopod at 5–6 dpa (V, W). X) Model of Verheyden and Sun (2008) based on genetic manipulations in mice. Y) Xenopus model showing alignment with the mouse model, FGF data from Wang and Beck (2014) for fgf8. Note limb drawings have been “inverted” to put anterior uppermost as is the convention for amniotes, and black arrowheads indicate amputation level. Grem1 is initially directly in contact with the AER and a positive feedback loop is established between Grem1 and Shh in the mesenchyme and the AER FGFs. This is phase I and corresponds to stage 50 of Xenopus limb development. At stage 51, grem1 is cleared from the distalmesenchyme because fgf levels in the AER rise above the threshold required to switch on an inhibitory loop (phase II). Shh levels continue to rise but grem1 is now distant from the AER Fgfs and may fail to maintain expression (stage 52). This leads to termination of the loop starting at stage 53 and loss of first grem1 and then shh and fgf8 from the autopod. Regenerating limbs are similar although since grem1 is not cleared from the distalmesenchyme until 5 dpa, phase I may last longer, enabling extra growth required to regenerate lost structures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Inducible expression of grem1 in a stable transgenic line. A) Schematic of the transgene used. At ambient temperatures, grem1 transcription is not expected, but when the temperature is increased, triggering the heat shock proteins to bind to the Hsp70 promoter, grem1 is transcribed in all cells. RFP driven by the lens specific γ-crystallin promoter is always present acting as a visible marker for transgene presence. B) In situ hybridisation for grem1 transcripts in heterozygote and wild type offspring of the G6 male founder. Heat shock was applied for 30 min at stage 30 and the tadpoles fixed after 1 or 3 h, or at stage 15 and fixed after 24 h. Grem1 is overexpressed strongly in G6 F1 tadpoles, so staining reactions for all tadpoles were terminated at the same time, when endogenous grem1 is only just detectable in the WT siblings ( Hsu et al., 1998 and Pearl et al., 2008). Note that a phenotype, similar to that seen with Noggin overexpression, develops in embryos heat shocked at stage 15, and presumably results from BMP inhibition.
Fig. 3. Gremlin overexpression does not enhance regeneration of stage 54 limbs: A) Bar chart comparing the percentage of limbs undergoing regeneration to any degree in WT vs. hindlimbs (G6 F1 or F2) overexpressing grem1. Following amputation at future knee level, fewer G6 limbs attempted regeneration than their wild type siblings. Numbers on top of the bars indicate sample size (N). B) Boxplots of the number of digits regenerated in WT vs. grem1 (G6 F1 or F2) overexpressing hindlimbs. Centre lines show medians, box limits are 25th to 75th percentiles, whiskers extend to maximum and minimum values, and crosses indicate the sample means. Significant differences in the means are indicated as * (p < 0.05) and ** (p < 0.01) and were determined using 2-tailed unpaired t-tests. Rather than enhancing regeneration, grem1 overexpression appears to significantly reduce the ability of stage 54 limbs to regenerate.
Fig. 4. Effects of grem1 overexpression on stage 52 regenerating and developing limbs. Stage 52 WT hindlimb amputation at knee level normally results in perfect or near perfect regeneration. A, B) Boxplots of the number of digits (A) or metatarsals (mtt) and phalanges (ph) (B) regenerated in WT ( = 19) vs. grem1 (G6) overexpressing hindlimbs. Centre lines show medians, box limits are 25th to 75th percentiles, whiskers extend to maximum and minimum values, and crosses indicate the sample means. Significant differences (p < 0.05) are indicated by * and determined using 2-tailed unpaired t-tests. Grem1 overexpression had little impact on regeneration in terms of the number of digits regenerated (A), or in terms of the number of mtt and ph in the autopod (B). Grem1 overexpression did have a significant effect on the developing contralateral limb (B). C) Bar graph of categorised autopodal defects. Anterior defects are more common in G6 regenerating limbs whereas the contralateral limb is more likely to show a defect in the posterior digits. D–G, skeletal preparations of representative WT and G1 hindlimbs. D) WT limbs amputated at knee level at stage 52 most commonly regenerate 5 toes, although defects can still occur: in this case a missing phalanx on digit IV (*). E) G1 amputated also most commonly regenerated 4–5 toes, in this example the black arrowhead indicates absent digit I. F) Wild type control limbautopod showing normal patterning. G) Example of grem1 overexpressing autopod with developmental defects including missing a phalange on digit V (*). Abbreviations: tf, tibia–fibula; ta, tarsus; mt, metatarsal; and p, phalanges; roman numerals indicate digit identity.
Fig. 5. Phenotypes of G6 tadpoles overexpressing grem1 at different limb bud stages. A, B) Bar charts indicating the frequency of limb duplications by proximodistal level (stylopod, zeugopod or autopod) and of pattern loss resulting in fewer than expected digits (oligodactyly), when the G6 transgene is activated by heat shock at different limb developmental stages. Numbers in brackets indicate sample size/number of individual tadpoles. A) Hindlimbs and B) forelimbs. C, D) box plots showing the highest median digit number and variance occurs when grem1 is overexpressed at stage 49. Centre lines show medians, box limits are 25th to 75th percentiles, whiskers extend to maximum and minimum values, and crosses indicate the sample means for hindlimbs (C), and forelimbs (D). Statistical analysis was not attempted due to the extremely unequal variance.
Fig. 6. Phenotypes arising from overexpression of grem1 in G6 F1 tadpoles that have been heat shocked to activate transcription of grem1 at stage 49. A–D) G6 tadpoles at stage 58, after hatching of the forelimbs. White arrows indicate examples of apparent limb bifurcation at the level of the soft tissue in G6 tadpoles where all four limbs are duplicated or bifurcated. A) Ventral view with anterior uppermost, showing complete forelimb duplications and bifurcated hindlimbs. B) A tadpole from the same cohort showing bifurcations at elbow/knee level. C) Example of bifurcated hindlimb at ankle level, side view with anterior to left. D) Example of hindlimb polydactyly, side view with anterior to left. F–H) Skeletal preparations stained with Alcian blue (cartilage) and Alizarin red (bone). Red (ventral most) and black (dorsal most) labels indicate the bones of each pair of limbs. Roman numerals indicate digit identity. F) Individual shown in B, showing complete duplication of hindlimbs articulating with a single pelvis (black arrowheads), soft tissue is joined to the level of the knee (white arrows). Note that the ventral limbs have a normal anteriorposterior polarity (V–I, I–V) while the dorsal pair have opposing polarity (I–V, V–I). The polarity of the zeugopod is also inverted in dorsal limbs. G) Individual shown in C, righthindlimb demonstrating the duplication arises at the zeugopod level (black arrowheads). H) Individual shown in D, exhibiting polydactyly and a malformed zeugopod (*). Digits red II and black V have additional bifurcations of the phalanges (II′, V′) and “?” indicates a thickened digit where red digit IV would be expected, which may have formed by incomplete separation. Finally, digit red V is vestigial. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. Expression key marker genes for limb developmental patterning in G6 tadpole limbs. In situ hybridisation with limb patterning markers confirms that PD and DV axes are normal in branched limbs. A, B) fgf8b expression in approx. stage 51 limb buds of G6 tadpoles showing that a new AER forms in addition to the original (black arrowheads) as the limb bud bifurcates. The dorsal limb appears to be the original. C, D) Lmx1b marks the dorsal mesenchyme of the zeugopod and autopod. The bifurcated limb establishes or retains DV identity. In C, the new axis is perpendicular to the original and in D it is parallel. Mirror imaged dorsal ventral polarity was not observed. Limb bud is stage approx. 52, side view in C and stage 53, ventral view in D. E, F) sox9 expression in the forming digitcartilage of autopod duplications, stage 54. E) Side view. F) Distal (palm) view. Digit IV is indicated by roman numerals showing the mirror imaging of the AP axis V–II–II–V. Digit I is still forming but appears duplicated as well (*).
Fig. 8. Expression of hindlimbshh following overexpression of grem1 at stage 49. Shh expression (left column) at stage 51 to 52 is normally restricted to the ZPA but was very variable in grem1 overexpressed hindlimbs. Despite this, we always observed distinct anteriorposterior polarity of digits. Here, we attempt to link these patterns via predicted intermediate stages (middle two columns, side and distal view of limb stage 54) to the observed outcome phenotypes (right two columns, side view and digit identity as viewed distally). A) Zeugopod (or stylopod) duplications may arise from early dorsal budding of the limb, and expanded shh expression leading to inversion of the original limbanterior-posterior axis. B) Double autopods (side to side duplications) may arise from splitting of the ZPA, resulting in two autopods that have the same anteriorposterior orientation. C) Later branching results in polydactyly or autopod duplication through expansion of the autopod and loss of shh polarity. This also results in an inversion of polarity and an I–V–I autopod which is opposite to the phenotype of a ZPA graft to the anterior. D) Budding from the side of the original limb bud may result in a second ZPA forming perpendicular to the original, leading to partial mirroring of the autopod. Keys: Side view and distal view, purple in stage 54 predicted intermediate represents predicted shh expression intensity is represented by darker shading. Observed outcome (in terms of skeleton) for limb at metamorphosis, digit order from I (most anterior) to V (most posterior). Open circles indicate digits without a claw (posterior digits V and IV) and orange circles indicate digits with claws (anterior digits I and II and digit III). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
