Histology and BrdU analysis of injured innervated limbs, denervated limbs, and flank wounds
We histologically characterized normal innervated limbs (NL), denervated limbs (DL), and flank wounds (FW) over the first seven days post injury (dpi) in order to examine differences between each injury response at the cellular level (Fig. 2). Masson's Trichrome staining revealed that the structure of the uninjured skin in NL, DL, and FW were similar with one another (data not shown). Uninjured epithelium consisted of an outer apical layer of epithelial cells, an interstitial layer of mucous secreting Leydig cells interspersed with keratinocytes, and a basal layer of germinative basal keratinocytes (Fox, 1986;Kelly, 1966). The underlying uninjured dermis consisted of mucous and granular glands interspersed with a loose network of fibroblasts that overlies muscle (Seifert et al., 2012).
Fig. 2. Histology of NL, DL, and FW.
Masson's trichrome staining of sections of NL, DL, and FW at 1 dpi (A–F) and 7 dpi (G–L). Area of magnified images on right are boxed in images on left. (A,B) Denervated limb at 1 dpi showing injury closure by the WE and the hemostatic response under the WE. (C,D) Innervated limb 1 dpi showing high similarity to the denervated limb. Normal epidermis and dermis can be seen outside the wound margins (WM). (E,F) FW at 1 dpi showing that the WE has closed the wound directly over the muscle and that a small hemostatic response is taking place. (G–L) Injuries at 7 dpi showing the thickening of the WE in DL (G,H), NL (I,J), and FW (K,L). Scale bar in A,C,E,G,I,K = 200 µm. Scale bar in B,D,F,H,J,L = 100 µm.
For all three injury types, the wound re-epithelialized within 24 hours after injury by migration of surrounding epidermis, generating a WE comprised of Leydig cells and keratinocytes (Fig. 2A–F). Underneath the WE was an accumulation of plasma and blood cells, with more blood in NL and DL versus FW, possibly because amputation severed major vasculature in the limb (Fig. 2B,D,F). Additionally, the FW was almost exclusively composed of muscle, while the limbs included bone, peripheral nerves, vasculature and muscle. In all three cases, the WE appeared to behave similarly during the first 24 hours after injury, although the extent of the hemostatic response and complexity of the underlying tissue is greater in the amputated limb compared to the flank.
By 7 dpi, the WE had thickened in all three injury types, but a distinct mound of epidermal cells was apparent in the middle of NL and DL WE, which was not present in FW (Fig. 2G–L). The epidermal mound may represent the maturation of the WE into the AEC, which was likely due to continuous cell migration from the wound margins rather than cell proliferation within the WE because BrdU-positive cells were evident at the margins of NL and DL (Fig. 3A,D), but not within the center of the WE (Fig. 3B,E) (Chalkley, 1954; Hay and Fischman, 1961). The WE of the FW was in direct contact with the subjacent muscle with little muscle dedifferentiation (Fig. 2K,L). In contrast, muscle fibers and peripheral nerves were becoming disorganized due to degeneration in both NL and DL (Fig. 2G,I). Limbs also had more plasma, red blood cells, and inflammatory cells. All together, these results suggest similar processes were taking place in each injury type, but tissue histolysis was more complete by seven days after injury in limbs versus FW.
Fig. 3. BrdU staining of sections of injured limbs.
(A–C) BrdU staining of NL at 7 dpi. (A,D) Cell proliferation is present in the epidermis near the wound edge in both NL and DL. (B,E) Little DNA synthesis is present in the WE in NL and DL. (C,F) Some DNA synthesis is present in the limb mesenchyme of both NL and DL at 7 dpi. Scale bar in A–F = 100 µm.
Taken together our data show that both limbs exhibited a hemostatic response and were histologically similar, containing a thickened WE, osteoclasts surrounding the bone, and histolysed tissues (Fig. 2A,C,G,I). The WE was lying directly over the bone in DL, while cells were present between the WE and bone in NL, suggesting that blastema growth was beginning within NL. BrdU analysis showed that DNA synthesis was taking place within the WE margin and mesenchyme (Fig. 3A,C,D,F) of both NL and DL, which is in accordance with previous studies showing that mesenchymal cells and epidermal cells enter S phase and divide in both denervated and innervated limbs (Maden, 1978). Loss of cell cycling in the mesenchyme of denervated limbs likely takes place after 7 dpi in the large-sized animals used in this study. These findings demonstrate that the time frame chosen for our study encompassed blastema formation rather than blastema outgrowth.
Commonly changed genes following injury
In order to characterize transcription during regeneration, transcript abundances were estimated from total RNA collected from the WE and a few subjacent cells from all three treatments (Fig. 1A–C). A total of 6684 probe sets yielded expression estimates that differed significantly as a function of RNA source and sample time (supplementary material Table S1). These genes were parsed to identify similarities and differences between injury types. First, probe sets that changed significantly from baseline to 1 dpi, 1 dpi to 3 dpi, or from 3 dpi to 7 dpi in each injury were identified to examine the commonalities between the injuries. We found that transcription was more similar between NL, DL, and FW than it was different with 1840 genes up-regulated and 1667 genes down-regulated in all three injuries (Fig. 4A,B). This high degree of similarity suggested the presence of a general wound-healing response regardless of whether or not a limb will regenerate. The list of genes that presented higher transcript abundances above baseline was significantly enriched for genes that annotate to gene ontology terms associated with processes known to take place during mammalian skin wound healing including immune system response (n = 187), macrophage activation (n = 36), and response to stimulus (n = 124) (supplementary material Table S2). Down-regulated genes belonged to ontology categories including lipid metabolic process (n = 110), chromosome segregation (n = 30), metabolic process (n = 604), and response to stress (n = 48). These results suggest that many of the same processes that take place during mammalian wound healing also occur in the axolotl following injury. Indeed, our histological analysis supports this result as well as an in-depth study on flank wound healing in the axolotl, which demonstrated that inflammation and a hemostatic response occurs in axolotls, but is dampened compared to mammals (Seifert et al., 2012). Although the set of genes common to all three injuries provides insight about wound closure, inflammation, and immunity, we focus in the following paragraphs on gene expression patterns that associate specifically with limb regeneration.
Fig. 4. Summary of differentially regulated genes during limb regeneration.
(A,B) Venn diagram showing the number of probe sets that measured significantly higher (A) and lower (B) transcript abundances in injured tissues at either 1 dpi versus baseline, 3 dpi versus 1 dpi, or 7 dpi versus 3 dpi. The total number of differentially regulated genes is represented for each injury type. (C) A schematic representing the progression from the total number of probe sets with higher transcript abundance in injured NL tissues compared with baseline (red circle in A) to the identification of limb-specific and nerve-dependent genes during the first 7 dpi. Numbers outside parentheses represent the total number of probe sets identified and the numbers within parentheses represent unique probe sets that have presumptive human orthologs.
Limb-enriched gene expression patterns
Overall, 2941 probe sets measured higher transcript abundances above baseline during the first seven days in NL (supplementary material Table S1). This list was filtered to identify genes with higher transcript levels in NL versus FW at 0, 1, 3, or 7 dpi; this yielded a list of 570 injury-induced, limb-enriched genes, of which 377 annotate to unique presumptive human orthologs (Fig. 4C; supplementary material Table S1). Annotation of gene ontologies for these probe sets identified a substantial number of genes involved in biological processes linked to limb development includingdevelopmental process (n = 125), mesoderm development (n = 70), andectoderm development (n = 70) (supplementary material Table S3). In fact, mutations in 31 of these unique limb-enriched genes manifest human or mouse developmental limb defects when genetically disrupted, strongly suggesting that these genes play pivotal roles in vertebrate limb formation (Table 1). Furthermore, comparing our list to other genomic screens of limb regeneration (Campbell et al., 2011; Monaghan et al., 2009) we identified 73 genes that were commonly identified as highly expressed in amputated limbs (supplementary material Table S1).
Table 1. List of up-regulated, limb-enriched genes that cause limb defects in humans or mice when mutated.
Each of the 377 up-regulated, limb-enriched genes was queried against OMIM and Pubmed to identify published examples demonstrating that gene mutations cause congenital limb defects. Fold change differences between NL and FW are shown on the right.
Key regulatory genes involved in signaling pathways known to be necessary for limb development and limb regeneration were found in the limb-enriched list including genes integral to β-catenin-independent Wnt/planar cell polarity signaling (prickle1, prickle2, wnt5a, fzd2, fzd8, and ror2) (Stoick-Cooper et al., 2007), retinoic acid signaling (aldh1a3, crabp1, crabp2, and rdh10) (Blum and Begemann, 2012), insulin growth factor signaling (ctgf [igfbp8], cyr61[igfbp10], igfbp2, igfbp3, htra1, and kazald1 [igfbp-rP10]) (Chablais and Jazwinska, 2010), FGF signaling (dusp6, fgfr1, and pdlim7) (Lee et al., 2009), and BMP signaling (bmp2, id3, bmp2r) (Guimond et al., 2010). Overall, this list supports the hypothesis that some gene expression programs used in development are re-deployed during limb regeneration (Muneoka and Sassoon, 1992). Surprisingly, genes associated with limb patterning and growth were up-regulated before considerable increases in cell proliferation and blastemal outgrowth, suggesting that patterning of the limb blastema may occur in parallel or prior to blastema growth.
Limb-enriched and nerve-dependent gene expression patterns
To identify nerve-dependent genes, limb-enriched genes were filtered to identify probe sets that measured higher transcript levels in NL versus DL at 1, 3, or 7 dpi. This strategy identified a short list of 56 genes (41 unique transcripts with presumptive human orthologs) that were up-regulated after injury, had higher transcript abundance in NL versus FW, and had higher transcript abundance in NL versus DL (Fig. 4C; supplementary material Table S1). This list was significantly enriched for genes that annotate todevelopmental process (n = 19), ectoderm development (n = 6), cell cycle (n = 8), and neurological system process (n = 7) ontology terms (supplementary material Table S4). Further annotation through PubMed searches showed that 14 of the 40 genes are important in epithelial function (Table 2), including genes important in maintaining the structure of the epithelia (krt8, kera, krt15,cldn19, col29a1, eppk1, and tgm1), epithelial cell growth factors (ereg), and transcription factors involved in keratinocyte growth and differentiation (zfp36l2, ifit5), suggesting that these genes are necessary for the maturation of the WE into the AEC. We also identified 9 out of 40 genes that are highly expressed in the peripheral nervous system of vertebrates including four genes that are highly abundant in peripheral nervous system myelin Schwann cells (mbp, pmp22, gldn, and mpz) (Table 2). Overall, this list of genes suggests that denervation affects maturation of the WE and behavior of Schwann cells within the first week of regeneration.
No comments:
Post a Comment