Two origins of blastemal progenitors define blastemal regeneration of zebrafish lower jaw.
ABSTRACT: Zebrafish possess a remarkable ability to regenerate complicated structures by formation of a mass of undifferentiated mesenchymal cells called blastema. To understand how the blastema retains the original structural form, we investigate cellular transitions and transcriptional characteristics of cell identity genes during all stages of regeneration of an amputated lower jaw. We find that mesenchymal blastema originates from multiple sources including nucleated blood cells, fibroblasts, damaged muscle cells and pigment cells. These cells are transformed into two populations of blastemal progenitors: foxi1-expression and isl1-expression, before giving rise to cartilage, bone, and muscle. Time point- based transcriptomal analysis of 45 annotated Hox genes reveal that five 3'-end Hox genes and an equal number of 5'-end Hox genes are activated largely at the stage of blastema reformation. RNA in situ hybridization shows that foxi1 and pax3a are respectively expressed in the presumptive mandible skeletal region and regenerating muscle at 5 dpa. In contrast, hoxa2b and hoxa11b are widely expressed with different domain in chondrogenic blastema and blastema mesenchyme. Knockdown foxi1 changes the expression patterns of sox9a and hoxa2b in chondrogenic blastema. From these results we propose that two origins of blastemal progenitors define blastema skeleton and muscle respecifications through distinct signaling pathways. Meanwhile, the positional identity of blastema reformation is implicated in mesenchymal segmentation and characteristic expression pattern of Hox genes.
Project description:Appendage regeneration in salamanders and fish occurs through formation and maintenance of a mass of progenitor tissue called the blastema. A dedicated epidermis overlays the blastema and is required for its proliferation and patterning, yet this interaction is poorly understood. Here, we identified molecularly and functionally distinct compartments within the basal epidermal layer during zebrafish fin regeneration. Proximal epidermal subtypes express the transcription factor lef1 and the blastemal mitogen shh, while distal subtypes express the Fgf target gene pea3 and wnt5b, an inhibitor of blastemal proliferation. Ectopic overexpression of wnt5b reduced shh expression, while pharmacologic introduction of a Hh pathway agonist partially rescued blastemal proliferation during wnt5b overexpression. Loss- and gain-of-function approaches indicate that Fgf signaling promotes shh expression in proximal epidermis, while Fgf/Ras signaling restricts shh expression from distal epidermis through induction of pea3 expression and maintenance of wnt5b. Thus, the fin wound epidermis spatially confines Hh signaling through the activity of Fgf and Wnt pathways, impacting blastemal proliferation during regenerative outgrowth.
Project description:The blastema is a mass of progenitor cells that enables regeneration of amputated salamander limbs or fish fins. Methodology to label and track blastemal cell progeny has been deficient, restricting our understanding of appendage regeneration. Here, we created a system for clonal analysis and quantitative imaging of hundreds of blastemal cells and their respective progeny in living adult zebrafish undergoing fin regeneration. Amputation stimulates resident cells within a limited recruitment zone to reset proximodistal (PD) positional information and assemble the blastema. Within the newly formed blastema, the spatial coordinates of connective tissue progenitors are predictive of their ultimate contributions to regenerated skeletal structures, indicating early development of an approximate PD pre-pattern. Calcineurin regulates size recovery by controlling the average number of progeny divisions without disrupting this pre-pattern. Our longitudinal clonal analyses of regenerating zebrafish fins provide evidence that connective tissue progenitors are rapidly organized into a scalable blueprint of lost structures.
Project description:In vitro models represent a critical tool in cancer research to study tumor biology and to evaluate new treatment options. Unfortunately, there are no effective preclinical models available that represent Wilms tumor (WT) - the most common pediatric renal tumor. Especially the high-risk blastemal WT subtype is not represented by the few primary cell lines established until now. Here, we describe a new 3D approach for in vitro cultivation of blastemal WT cells, where primary cultures grown in suspension as spheroids could be propagated long-term. Besides blastemal cultures, we could generate spheroids representing epithelial and stromal WT. Spheroid cultures were analyzed by immunohistochemistry in comparison to corresponding tumor sections and were further characterized by RNA sequencing. Histological appearance of spheroids resembled the original tumor and they expressed marker genes characteristic of early renal development and blastemal WT elements. The cultures were amenable to genetic manipulation and they formed xenograft tumors, which resemble the primary human tumor. This collection of WT spheroids that carry different genetic drivers forms a long-sought tool for drug testing and in vitro modeling.
Project description:Several adult reptiles, such as Gekko japonicus, have the ability to precisely re-create a missing tail after amputation. To ascertain the associated acquisition of positional information from blastemal cells and the underlying molecular mechanism of tail regeneration, a candidate molecule CD59 was isolated from gecko. CD59 transcripts displayed a graded expression in the adult gecko spinal cord with the highest level in the anterior segment, with a stable expression along the normal tail. After tail amputation, CD59 transcripts in the spinal cord proximal to the injury sites increased markedly at 1 day and 2 weeks; whereas in the regenerating blastema, strong CD59 positive signals were detected in the blastemal cells anterior to the blastema, with a gradual decrease along the proximodistal (PD) axis. When treated with RA following amputation, CD59 transcripts in the blastema were up-regulated. PD confrontation assays revealed that the proximal blastema engulfed the distal one after in vitro culture, and rabbit-anti human CD59 antibody was able to block this PD engulfment. Overexpression of the CD59 during tail regeneration causes distal blastemal cells to translocate to a more proximal location. Our results suggest that position identity is not restricted to amphibian limb regeneration, but has already been established in tail blastema of reptiles. The CD59, a cell surface molecule, acted as a determinant of proximal-distal cell identity.
Project description:Wilms tumor (WT) is an embryonal malignant neoplasm of the kidney that accounts for 6-7% of all childhood cancers. WT seems to derive from multipotent embryonic renal stem cells that have failed to differentiate properly. Since mechanisms underlying WT tumorigenesis remain largely unknown, the aim of this study was to explore the expression of embryonic stem cell (ESC) markers in samples of WT patients after chemotherapy treatment SIOP protocol, as the gene expression patterns of ESC are like those of most cancer cells. We found that expression of ESC markers is heterogeneous, and depends on histological WT components. Interestingly, among ESC markers, HMGA2 was expressed significantly stronger in the blastemal component than in the stromal and the normal kidney. Moreover, two subsets of patients of WT blastemal type were identified, depending on the expression levels of HMGA2. High HMGA2 expression levels were significantly associated with a higher proliferation rate (p=0.0345) and worse patient prognosis (p=0.0289). The expression of HMGA2 was a stage-independent factor of clinical outcome in blastemal WT patients. Our multivariate analyses demonstrated the association between LIN28B-LET7A-HMGA2 expression, and the positive correlation between HMGA2 and SLUG expression (p=0.0358) in blastemal WT components. In addition, patients with a poor prognosis and high HMGA2 expression presented high levels of MDR3 (multidrug resistance transporter). Our findings suggest that HMGA2 plays a prominent role in the pathogenesis of a subset of blastemal WT, strongly associated with relapse and resistance to chemotherapy.
Project description:Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes, such as Msh homeobox (Msx) genes, are absent in this animal group. The lack of BCs and positional information in other cells is therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema-like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx-regulated genes (Bmp4, Fgf8, and keratin 14 (K14)) in BlC groups, including MSX1, MSX2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced the proliferation rate and osteogenesis of BlCs compared with mBMSCs and BCs via activation of Fgf8 and Bmp4 Histological analyses indicated full regrowth of digit tips in the Msx-overexpressing groups, particularly in MSX1/2, through endochondral ossification 6 weeks post-injection. In contrast, mBMSCs and BCs formed abnormal bone and nail. Full digit tip was regenerated only in the MSX1/2 group and was related to boosted Bmp4, Fgf8, and K14 gene expression and to limb-patterning properties resulting from Msx1 and Msx2 overexpression. We propose that Msx-transduced cells that can regenerate epithelial and mesenchymal tissues may potentially be utilized in limb regeneration.
Project description:Blastema formation is a hallmark of limb regeneration that requires proliferation and migration of progenitors derived from many tissues to the amputation plane. To better understand the genetic programs that initiate limb regeneration, we reasoned that blastemal progenitors would be among early proliferating cells in the stump following amputation. Here we separately profiled dividing and non-dividing stump tissues, as well as the wound epidermis, during early axolotl limb regeneration to examine transcriptional programs of blastemal progenitors. We provide a description of the changes in gene expression specific to early dividing cells at the site of amputation, inclusive of progenitors for the regenerating limb. This work collectively demonstrates differential suppression/activation of core developmental signaling pathways in subsets of the early regenerating limb and further suggests that interleukin-8 (il-8) signaling is important for regeneration. Overall design: To identify transcripts that were differentially expressed and enriched within early dividing cells during axolotl limb regeneration, we separately sequenced stump-derived 4N, stump-derived 2N, and wound epidermis at 0, 4, and 5 days post-amputation (dpa). We sequenced a total of 27 samples, 3 biological replicates per timepoint. These samples were multiplexed and sequenced on 1 Illumina Nextseq 500 run and also across 6 lanes on a Hiseq 2500.
Project description:The limb blastemal cells of an adult salamander regenerate the structures distal to the level of amputation, and the surface protein Prod 1 is a critical determinant of their proximodistal identity. The anterior gradient protein family member nAG is a secreted ligand for Prod 1 and a growth factor for cultured newt blastemal cells. nAG is sequentially expressed after amputation in the regenerating nerve and the wound epidermis-the key tissues of the stem cell niche-and its expression in both locations is abrogated by denervation. The local expression of nAG after electroporation is sufficient to rescue a denervated blastema and regenerate the distal structures. Our analysis brings together the positional identity of the blastema and the classical nerve dependence of limb regeneration.
Project description:An open question remains in cancer stem cell (CSC) biology whether CSCs are by definition at the top of the differentiation hierarchy of the tumor. Wilms' tumor (WT), composed of blastema and differentiated renal elements resembling the nephrogenic zone of the developing kidney, is a valuable model for studying this question because early kidney differentiation is well characterized. WT neural cell adhesion molecule 1-positive (NCAM1(+)) aldehyde dehydrogenase 1-positive (ALDH1(+)) CSCs have been recently isolated and shown to harbor early renal progenitor traits. Herein, by generating pure blastema WT xenografts, composed solely of cells expressing the renal developmental markers SIX2 and NCAM1, we surprisingly show that sorted ALDH1(+) WT CSCs do not correspond to earliest renal stem cells. Rather, gene expression and proteomic comparative analyses disclose a cell type skewed more toward epithelial differentiation than the bulk of the blastema. Thus, WT CSCs are likely to dedifferentiate to propagate WT blastema.
Project description:The regeneration-competent adult animals have ability to regenerate their lost complex appendages with a near-perfect replica, owing to the positional identity acquired by the progenitor cells in the blastema, i.e. the blastemal cells. CD59, a CD59/Ly6 family member, has been identified as a regulator of positional identity in the tail blastemal cells of Gekko japonicus. To determine whether this function of CD59 is unique to the regenerative amniote(s) and how CD59 mediates PD axis patterning during tail regeneration, we examined its protective role on the complement-mediated cell lysis and intervened CD59 expression in the tail blastemal cells using an in vivo model of adenovirus transfection. Our data revealed that gecko CD59 was able to inhibit complement-mediated cell lysis. Meanwhile, CD59 functioned on positional identity through expression in cartilage precursor cells. Intervening positional identity by overexpression or siRNA knockdown of CD59 resulted in abnormal cartilaginous cone patterning due to the decreased differentiation of blastemal cells to cartilage precursor cells. The cartilage formation-related genes were found to be under the regulation of CD59. These results indicate that CD59, an evolutionarily transitional molecule linking immune and regenerative regulation, affects tail regeneration by mediating cartilage patterning.