Figure 1. Pipeline and Assessment of Read Representation for De Novo Axolotl Transcriptome
(A) Strategy for deriving a tissue-coded de novo transcriptome for axolotl.
(B) The count of the most highly expressed transcripts is plotted as a function of minimum expression value. 90% of the total expression (E90) is accounted for by the 26,378 most highly expressed transcripts. FPKM, fragments per kilobase of transcript per million mapped reads.
(C) The contig N50 value is computed for cumulative sets of the most highly expressed transcripts.
(D) Expression values for all tissue types were compared and Pearson correlation values were computed. Samples were clustered according to Pearson correlation values, indicating high similarity among sample replicates and between similar tissue types.
See also Figure S1, Table S1, and Data S1, S2, S3, S4, and S5.
Figure 2. Differential Gene Expression Analysis across Each Set of Tissues Identifies Transcripts Most Enriched in Specific Tissue Types
(A) Graph illustrating the methodology for the identification of genes that are tissue enriched in the context of all tissue pairwise comparisons using kazal-type serine peptidase inhibitor domain 1 (kazald1) as an example. Directed edges are drawn from upregulated to downregulated tissues, and fold changes in expression are indicated by edge colors.
(B) Heatmap showing all transcripts that are enriched in specific tissue types.
(C–J) RNA in situ hybridization performed on tissue sections.
(C) speriolin (spatc1) is enriched in the germ cells in the testes but is not detectable in adjacent support cells (asterisks).
(D) tropomyosin 1 (tpm1) is enriched in cardiomyocytes within the heart and not detectably expressed by other heart cell types such as epicardium (asterisks).
(E) titin (ttn) is enriched in limb skeletal muscle but is not detectable in adjacent cartilage (cart) and epidermis (epiderm).
(F) kelch repeat and BTB domain-containing protein 10 (klhl41) is highly enriched in skeletal muscle and not detectable in adjacent tissues such as epidermis (epiderm) and fascia.
(G) actin, alpha 1, skeletal muscle (acta1) mRNA is enriched in the very thin layer of vascular endothelial cells lining the blood vessels (arrowheads) and absent from adjacent dermis (derm). Asterisks mark red blood cell clumps in the vessel lumen.
(H) collagen type V alpha 1 (col5a1) expression is highly enriched in cartilage; shown are four carpals (outlined) within the wrist. Expression in joint (between carpals) and adjacent muscle is diminished.
(I) A bone-enriched marker, cathepsin k (ctsk), is highly expressed in ossified portions of the humerus and low in adjacent muscle.
(J) platelet-binding protein GspB (gspb) and mucin 1 (muc1) are detected in cartilage but not in bone by RT-PCR. eukaryotic translation elongation factor 1 alpha 1 (ef-1a) serves as the loading control.
Scale bars, 100 μm. See also Figure S7, Tables S2 and S3, and Data S1, S2, S3, S4, and S5.
Figure 3. Identification and Validation of Blastema-Enriched Transcripts
(A) We identified 159 transcripts (151 genes) enriched in the blastema (the combination of proximal and distal blastema tissue) as compared to all other tissues. Those predicted to encode proteins with RNA-binding/regulation properties are highlighted in yellow.
(B and C) In situ hybridization for six highly blastema-enriched transcripts at 23 days post-amputation (DPA (B) and on intact limbs (C). (B) Lower: higher magnifications of the boxed areas (upper). Yellow lines mark the WE/BL boundary. WE, wound epidermis; BL, blastema; mus, muscle; epi, epidermis; cart, cartilage; n, nerve. Scale bars, 500 μm.
See also Figure S3, Table S4, and Data S1, S2, S3, S4, and S5.
Figure 4. Axolotl Cold-Inducible RNA-Binding Protein Is a Cytoprotective Factor for Blastema Cells
(A–D) In situ hybridization for cirbp over the course of regeneration in regenerating limb tissue sections. (A) Three days post-amputation. (B) Five days post-amputation. (C) 15 days post amputation. (D) 35 days post-amputation. Asterisks denote newly differentiated cartilage in digits; arrowheads denote interdigital regions. WE, wound epidermis; BL, blastema; mus, muscle; epi, epidermis; cart, cartilage; n, nerve. Scale bars, 500 μm.
(E–F′) Tissue sections from regenerating limbs treated with standard morpholinos (MO) control (E and E′) and cirbp-targeting morpholino (F and F′) stained for nuclei (DAPI; E and F) and TUNEL (E′ and F′). Scale bars, 500 μm.
(G) Quantification of the percentage of TUNEL+ blastema cell nuclei in control and cirbp-MO-treated limbs. ∗p <0.05; n.s., not significant.
See also Figure S2 and Data S1, S2, and S3.
kazald1, the Most Robust Blastema Marker, Is Required for Limb Regeneration
(A) Differential tissue expression analysis identifies kazald1 as the most blastema-enriched transcript compared to all other tissues sequenced.
(B) RT-PCR performed on blastema cDNA samples throughout the course of regeneration for kazald1 expression. kazald1 was not detected in intact limbs and at 1 DPA, and has dramatically diminished by 60 DPA.
(C) In situ hybridization for kazald1 in the blastema over the course of regeneration (top panels). Kazald1 is not detectable in regenerated limbs (35 DPA), intact limbs, or developing limb buds (lower panels).
(D–F) Regenerating limbs at 19 DPA treated with control (D) or kazald1-targeting morpholino (E); quantified in (F).
(G–J′) Regenerating limbs at 28 DPA treated with control (G) or kazald1-targeting morpholino (H and H′).
(I–K) Same specimens stained with Alcian blue to visualize cartilage; (I) is the skeletal preparation of the limb shown in (G); (J) is the skeletal preparation of the limb shown in (H); (J′) is the skeletal preparation of the limb shown in (H′). Not that (G) and (I) are a control, while (H) and (J) and (H′) and (J′) are specimens treated with kazald1-targeting morpholino. Results are quantified in (K).
Scale bars, 500 μm (C) and 1 mm (D, E, and H–J′). ∗∗∗p <0.001. Error bars are SEM. Arrowheads mark the amputation plane in each image.
See also Figures S4–S6 and Data S1, S2, S3, S4, and S5.
Transcripts Differentially Expressed in Proximal versus Distal Elements
(A) Schematic illustrating specific elements of the hand and arm.
(B) Differential gene expression analysis identifies transcripts that are enriched in distinct sections of the intact limb.
(C) RT-PCR validation of select transcripts identified by differential expression analysis.
(D) Gradient gene expression analysis identifies transcripts enriched in a gradient from proximal to distal or distal to proximal.
(E) Schematic illustrating amputation planes for sampling of proximal and distal blastemas.
(F) Differential expression analysis identifies transcripts that are enriched in proximal versus distal blastemas.
(G) In situ and RT-PCR validation of computational predictions of differentially expressed transcripts in proximal and distal blastemas.
See also Figure S7, Table S5, and Data S1, S2, S3, S4, and S5.
Figure S1. Transcriptome read representation and comparison of methods for differential gene expression
analysis. Related to Figure 1 and Table 1. (A) Approximately 80% of RNA-Seq reads from each sample and
replicate are represented by the transcriptome assembly. (B) Most transcripts identified as significantly
differentially expressed were agreed upon by at least two different computational methods.
Figure S2. Administration of cirbp-targeting morpholino diminishes CIRBP protein level in axolotl tissue.
Related to Figure 4. Shown is a western blot labeled with anti-DsRed (top) and anti-myc (bottom) antibodies. On
the left are protein samples from HEK293T cells transfected with DNA constructs as shown for validation of protein
size and as positive controls. On the right are protein samples from blastemas electroporated with DNA constructs
and either the standard control morpholino (st. control MO) or the cirbp-targeting morpholino (cirbp-targeting MO).
Each lane was loaded with 2 μg of total protein.
Figure S3. Transcripts found globally repressed in blastemal tissue. Related to Figure 3. Many of the
transcripts repressed in blastemal tissue are found highly expressed in skeletal muscle tissue.
Figure S4. Administration of kazald1-targeting morpholinos diminish Kazd1 protein level in blastema cells
and a second kazald1-targeting morpholino also disrupts regeneration. Related to Figure 5. (A) Shown are
western blots probed with anti-myc (top) and anti-DsRed (bottom). Left blot is control morpholino (MO) and
kazald1-targeting MO for targeting sequence 1, right blot is control MO2 and kazald1-targeting MO2; left four lanes
are protein samples from limb blastemas electroporated with DNA constructs shown and with the kazald1-targeting
MO or the related control MO (inverted sequence). Empty lane separates blastema samples from 293T cell samples.
293T cell protein samples transfected with individual DNA constructs were used as the positive controls and to
verify size. (B-D) MO2 administration impairs blastema growth. (B-C) Regenerating limbs at 19 days postamputation
treated with control (B, inverted MO2 sequence) or a second kazald1-targeting morpholino (kazald1-
MO2) (C); quantified in (D). (D-H) MO2 administration causes a delay in chondrification. (D-G’) Whole-mount
brightfield images (top row, E-F’) of regenerating limbs with Alcian blue-stained skeleton pictured below (G-H’).
Limbs were harvested at 28 days post-amputation. (I) Quantification of cartilage area in control versus kazald1-
MO2-treated. Control refers to inverted MO sequence. Scale bar is 1 mm. *** indicates p<0.001 and error bars are
SEM. Arrowheads mark amputation plane in each image.
Figure S5. CRISPR-mediated deletions in kazald1 locus in embryos and unamputated limbs. Related to
Figure 5. (A) Kazald1 genomic locus and targeting scheme. Primers for PCR amplification for genotyping are
indicated. (B) Experimental schematic for targeting in embryos via injection. (C-D) Embryo genotyping via PCR
followed by T7 endonuclease assay for embryos injected with crRNA-1 (C) and crRNA-2 (D). (E) Experimental
schematic for targeting in unamputated limbs via electroporation. (F-G) EGFP expression in control (F, left limb)
and crRNA-1-treated (G, right limb) limbs at 25 days post-electroporation. Landmarks: el (elbow), tr (trunk). (H)
Limb genotyping via PCR followed by T7 endonuclease assay. For all genotyping, each lane indicates an individual
specimen. Editing efficiency is estimated by the indel percentage beneath each lane.
Figure S6. Expression of the most robust blastema marker must be temporally or spatially restricted to
achieve perfect regeneration. Related to Figure 5. (A) Experimental setup for virus-driven constitutive
misexpression. (B) Prolonged misexpression of kazald1 causes profound regenerative defects as compared to
controls (n=22 experimental, 18 control; p=0.004 with Fisher’s exact test). (C) The most common defect observed
was syndactyly (indicated by arrow). Scale bar is 500 microns.
Figure S7. (A) Expression of arm-gradient enriched transcripts across all tissue types. Related to Figure 2
and Figure 6. Many arm segment enriched transcripts have transcriptional enrichment that largely reflects the
tissue composition, such as relative skeletal muscle or cartilage content. Hand stands out as having a unique
transcriptional program with many hand-specific transcripts being expressed. (B) A paucity of differentially
expressed transcripts identified between wrist and long bone cartilage. Differences in expression between these two
tissue types are subtle, and this bulk analysis of these two very similar tissue types likely simply lacks the resolution
required to reveal key molecular differences between them.