Connective tissue cells, but not muscle cells, are involved in establishing the proximo-distal outcome of limb regeneration in the axolotl

Limb development and regeneration involve the coordinated growth and patterning of several tissues, including bone, soft connective tissue, muscle, epidermis, peripheral nerve and blood vessels. During limb development, lateral plate mesoderm-derived cells can form recognisable limb segments in the absence of muscle formation and it has been proposed that they are a dominant cell type directing limb patterning (Christ et al., 1977; Grim and Wachtler, 1991; Kardon et al., 2002; Kardon et al., 2003; Hasson et al., 2010; Mathew et al., 2011). Molecular factors that influence the formation of the three distinct limb segments (upper arm, lower arm and hand), such as MEIS and HOX genes, are expressed in multiple limb progenitor cell types, including cells derived from lateral plate mesoderm, as well as myogenic cells derived from somitic mesoderm (Yamamoto et al., 1998; Hashimoto et al., 1999; Mercader et al., 1999; Cooper et al., 2011; Roselló-Díez et al., 2011). These factors probably exert their patterning influence via expression in the lateral plate mesoderm derivatives. Their expression in myogenic cells has been linked to myogenic differentiation, although an influence on muscle patterning has not been excluded (Knoepfler et al., 1999; Yamamoto and Kuroiwa, 2003; Heidt et al., 2007).

Salamander limb regeneration provides another context in which to study specification of proximo-distal limb identity. Upon limb amputation anywhere along the limb axis, only the missing portion of the limb distal to the amputation plane regenerates. Furthermore, when a hand blastema was transplanted to an upper arm stump, a normal limb regenerated. Examination of melanocytes or labelled cartilage nuclei indicated that the hand blastema cells contributed only to the hand, and the lower arm was formed from the upper arm stump cells through a phenomenon called intercalation (Stocum, 1975; Maden, 1980; Pescitelli and Stocum, 1980). This and other transplantation experiments showed that, as a whole, a limb blastema is autonomously specified to form the limb structures distal to its site of origin (for review, see Nacu and Tanaka, 2011). This is termed the rule of distal transformation.

The limb blastema is also composed of lineage-restricted progenitors (Kragl et al., 2009). Therefore, it is important to know which cells obey the rule of distal transformation and thus determine the proximo-distal outcome of regeneration, a crucial aspect of regenerating a properly patterned limb. Kragl and colleagues (Kragl et al., 2009) showed that upper limb blastema cells show heterogeneity in the nuclear expression of transcription factors associated with limb patterning. Cartilage-derived and muscle-derived blastema cells expressed MEIS, HOXA9 and HOXA13, as found for limb development, but Schwann cells did not. Interestingly, the cellular behaviour of cartilage-derived cells versus Schwann cells correlated with expression of these factors: cartilage-derived cells obeyed the rule of distal transformation, whereas Schwann cells did not. In addition to understanding which cells obey the rule of distal transformation, it is also important to determine the molecular mechanisms implemented in patterning during limb regeneration, and to what extent they are common to those used during development.

Here, we investigate the role of connective tissue (CT) cells, which derive from lateral plate mesoderm, and muscle cells in patterning during limb regeneration by asking whether they obey the rule of distal transformation. We find that CT-derived blastema cells display nuclear MEIS in upper arm, but not lower arm or hand blastemas. At a cellular level, CT-derived blastema cells obey the rule of distal transformation in the intercalation assay. By contrast, nuclear MEIS signal is not restricted to upper arm blastemas in myogenic blastema cells. At a cellular level, myogenic cells break the rule of distal transformation. These data indicate that, similarly to limb development, lateral plate mesoderm-derived cells probably play a dominant role in patterning of distal structures, whereas myogenic cells follow the patterning cues of lateral plate mesoderm-derived cells. We highlight an additional parallel to limb development by showing that β-catenin activity influences muscle patterning.

Ambystoma mexicanum (axolotl) expressing GFP in connective tissue cells (GFP-CT) or muscle cells (GFP-M) were generated by embryonic transplantation as previously described (Kragl et al., 2009; Nacu et al., 2009) (supplementary material Fig. S1).

SceI-CarAct:GFP plasmid (kind gift of Hajime Ogino, Nara Institute of Science and Technology, Japan) was injected into axolotl eggs as previously described (Khattak et al., 2009).

All animal procedures were carried out in accordance with the laws and regulations of the State of Saxony. Animals were anesthetised in 0.03% benzocaine (Sigma) prior to surgery. Intercalation assays were performed as previously described (Maden, 1980). Blastemas, 7 or 10 days post amputation (dpa), were transplanted on animals 2.5-3.0 cm and 3.0-4.0 cm in length (snout to cloaca), respectively. Blastemas were transplanted onto hosts amputated at mid-upper arm or shoulder. Animals were covered by benzocaine-soaked tissues for 1 hour and afterwards transferred into tap water. Only animals that regenerated distinct upper arm, lower arm and hand were analysed.

Samples were fixed in MEMFA, embedded in Tissue-tek (O.C.T. compound, Sakura) or 7.5% gelatine (Sigma), sectioned at 10 μm thickness and stained with antibodies (supplementary material Table S1) as previously described (Mchedlishvili et al., 2007; Mchedlishvili et al., 2012).

For MEIS/PAX7 double staining, the slides were first incubated with anti-MEIS, then with Fab goat anti-mouse, and finally with Alexa647-conjugated Fab rabbit anti-goat. Slides were then blocked with 20% goat serum in PBS and then incubated with anti-PAX7, followed by Cy3-conjugated donkey anti-rabbit.

Upper arm blastemas (6 dpa) were injected with 2.5 mg/ml pCS2+ plasmid expressing a constitutively active form of β-catenin (kind gift of Gilbert Weidinger, Universität Ulm, Germany) or 2.5 mg/ml CAGGS:GFP as control. CAGGS:mCherry was added to each mix (final concentration 0.25 mg/ml). Limbs were electroporated at 300 V/cm, 50 ms pulse length, 5 pulses on a BTX-ECM-830 electroporator.

Fixed limbs were stained with mouse anti-MHC antibody, then Cy3-coupled Fab goat anti-mouse and cleared as previously described (Ertürk et al., 2012).

Whole-limb images were acquired on Olympus SZX-16. Whole-mount stainings and limb sections were imaged on Zeiss AxioImagerZ.2 LSM780, Zeiss AxioObserverZ.1 or Olympus-BX61VS. Images were processed with Fiji software for better visualisation and Volocity software (PerkinElmer) for 3D reconstruction.

In this study, we were interested in elucidating whether myogenic and connective tissue (CT)-derived blastema cells, both expressing MEIS and HOXA13, obey the rule of distal transformation, which is linked to the proximo-distal outcome of regeneration. In development, lateral plate mesoderm-derived cells are sufficient for forming a properly patterned limb (Christ et al., 1977; Grim and Wachtler, 1991). Therefore, we postulated that during regeneration CT cells, which are derived from lateral plate mesoderm, obey the rule of distal transformation.

To test this hypothesis, we first investigated whether MEIS, which can act as an upper arm identity regulator (Capdevila et al., 1999; Mercader et al., 1999; Mercader et al., 2005), is present in nuclei of CT cells only upon upper arm amputation. We generated, by embryonic transplantation, animals in which the vast majority of CT cells constitutively express GFP (GFP-CT) (supplementary material Fig. S1). In 10 dpa upper arm blastemas of GFP-CT limbs, we identified that 37-62% of GFP+ cells have nuclear MEIS, in contrast to 0.6% of GFP+ cells having nuclear MEIS+ in 10 dpa lower arm blastemas (Fig. 1A-F,M-Q; supplementary material Table S2). These results suggested that connective tissue cells could indeed obey the rule of distal transformation during regeneration.

To address the question at a cellular level, we set up the intercalation assay as previously described: a wrist blastema was transplanted onto an upper arm stump (Fig. 2A,C,E). If cells from the grafted blastema end up more proximally of their origin, those cells break the rule of distal transformation. When wrist blastemas from GFP-CT animals were transplanted onto upper arm stumps, the vast majority of GFP+ cells homed to the hand (Fig. 2C,D; supplementary material Fig. S2). In control experiments of upper arm GFP-CT blastema transplantations, GFP+ cells were found in upper arm, lower arm and hand (Fig. 2E,F; Table 1). These results show that CT-derived blastema cells obey the rule of distal transformation.

Next, we investigated whether myogenic blastema cells display nuclear MEIS signal only upon upper arm amputation by using animals in which myogenic somitic mesoderm derivatives constitutively express GFP (GFP-M) (supplementary material Fig. S1). We found that, in contrast to CT cells, myogenic blastema cells have nuclear MEIS after amputation through any limb segment: mid-upper arm (Fig. 1R-V), mid-lower arm (Fig. 1G-L), and metacarpals (supplementary material Fig. S3). Specifically, 51-80% of GFP+ myogenic blastema cells have nuclear MEIS in 10 dpa upper arm blastemas, and 23-86% in 10 dpa lower arm blastemas (supplementary material Table S2). Because myogenic blastema cells express PAX7 (supplementary material Table S2) (Kragl et al., 2009), we explored whether PAX7+ myogenic progenitors have nuclear MEIS. Double staining of MEIS and PAX7 revealed that 36.8-91.4% of PAX7+ cells in the blastema are MEIS+ (supplementary material Fig. S4, Table S3). These results suggested that during regeneration, myogenic blastema cells have a different proximo-distal determination system from connective tissues.

To follow muscle cells in the intercalation assay, we transplanted wrist blastemas from germline CarAct:GFP transgenic animals, which express GFP in muscle fibres, onto upper arm stumps (Mohun et al., 1986). Upon regeneration, GFP+ muscle fibres were observed in the upper arm, lower arm and hand, indicating that myogenic blastema cells break the rule of distal transformation (Fig. 2A,B; Table 1). GFP+ muscle fibres were observed in all muscles of the upper arm. The humeroantebrachialis and anconaeus humeralis medialis, which originate in the humerus (Walthall and Ashley-Ross, 2006), had the highest abundance of GFP+ muscle fibres (supplementary material Fig. S5, Tables S4, S5).

Muscle fibres form by fusion of precursor cells; thus, the observed proximalisation could be the result of fusion of labelled myogenic blastema cells in the grafted blastema with pre-existing muscle fibres in the stump. To account for this, we examined whether satellite cells (mononucleate myogenic cells) were also proximalised. Upper arms of regenerates that were generated by transplanting CAGGS:GFP wrist blastemas (in which all cells express GFP) onto upper arm stumps, were analysed with an antibody against PAX7, which marks satellite cells. PAX7+GFP+ cells were present in the upper arms of the regenerates (seven out of seven limbs), indicating that myogenic blastema cells gave rise to proximalised satellite cells in the intercalation assay (Table 1; supplementary material Fig. S5, Table S5). These results show that myogenic cells break the rule of distal transformation.

Our results indicate that connective tissue cells, but not myogenic cells, guide the proximo-distal outcome of regeneration. In this regard, patterning in regeneration might recapitulate development, during which limb bud cells of lateral plate mesoderm origin direct the migration and patterning of naive myogenic progenitors (see Introduction). Next, we were interested in uncovering the molecules that might play a role in patterning muscle cells.

In limb development, TCF4 of the WNT/β-catenin pathway is expressed in early muscle connective fibroblasts and plays a crucial role in dictating the placement of limb muscle masses (Kardon et al., 2003; Mathew et al., 2011). Kardon and colleagues (Kardon et al., 2003) showed that expression of constitutively active β-catenin in chicken limb buds gives rise to ectopic muscle formation. We were therefore interested to determine whether β-catenin signalling affects muscle patterning in axolotl limb regeneration.

We overexpressed constitutively active β-catenin (Yost et al., 1996) in axolotl blastemas using electroporation. Five out of 11 limbs electroporated with β-catenin showed ectopic muscle formation in the regenerated upper arm, such as on the dorsal side of the limb close to the triceps (Fig. 3; supplementary material Fig. S6). Control GFP-electroporated regenerates showed defects in musculature, as occurs normally during regeneration (Diogo et al., 2013), but we did not observe in any of the ten samples ectopic muscle formation similar to that seen in β-catenin-electroporated limbs.

We show through intercalation assays that myogenic cells break the rule of distal transformation, whereas connective tissue cells obey it. These results suggest that, similarly to development, the connective tissue cells guide muscle patterning during regeneration. Therefore, it will be important in the future to study connective tissue cells in order to understand how the proximo-distal outcome of regeneration is established.

We show that myogenic blastema cells break the rule of distal transformation and display nuclear MEIS, which can functionally proximalise blastema cells (Mercader et al., 2005). We also found myogenic cells expressing HOXA11 and HOXA13 in 10 dpa lower arm blastemas (data not shown). It is possible that MEIS, HOXA11 and HOXA13 play a role in patterning of myogenic cells during regeneration; however, their expression is regulated by signals that myogenic cells receive from connective tissue cells. For example, in chicken, HOXA10 predisposes, in an autonomous way, the myogenic progenitors in the somite to a limb fate (Alvares et al., 2003). In Drosophila, the identity of muscle cells in different larval segments is believed to be the result of integration of extrinsic cues with autonomous expression of HOX homologues (Greig and Akam, 1993; Roy and Vijayraghavan, 1997).

Alternatively, MEIS and HOX genes might play a role not related to positional identity in myogenic cells. MEIS, in complex with PBX, binds DNA cooperatively with MYOD and marks genes for activation by MYOD (Knoepfler et al., 1999; Heidt et al., 2007). Also, HOXA11 and HOXA13 have been shown to repress MYOD and maintain the myogenic progenitors in a proliferative state during limb development (Yamamoto and Kuroiwa, 2003). Therefore, it is possible that MEIS, HOXA11 and HOXA13 are not involved in patterning, but have a role only in proliferation and differentiation of myogenic cells during regeneration. To distinguish between these hypotheses, it will be important to knockdown MEIS and assess the effect on muscle cell proximalisation in regeneration.

We further show that, similarly to development, activation of the β-catenin pathway influences muscle patterning, indicating that molecules involved in muscle patterning are likely to be conserved between regeneration and development. In addition to TCF4/β-catenin, TBX4 and TBX5 non-autonomously influence limb muscle patterning during mouse limb development (Hasson et al., 2010). Brand-Saberi and colleagues (Brand-Saberi et al., 1996) previously suggested that N-cadherin might participate in myoblast path finding. It will be interesting to investigate whether these molecules play a role in muscle patterning during regeneration also.

We thank Heino Andreas, Sabine Mögel and Beate Gruhl for axolotl care. We acknowledge Hajime Ogino for CarAct:GFP transgenesis construct and Gilbert Weidinger for the β-catenin construct. We thank Walter Bonacci and Shirley Galbiati for advice on the manuscript. We apologise for the limitation in possibility to extensively discuss and cite the literature on patterning during limb development and regeneration.