Regenerative capability of zebrafish mandible.

<p>The regenerated bone and cartilage are shown by Alcian blue staining. The first occurrence of the mandible regeneration was the chondrogenic differentiation indicated by cartilage blue-staining (10 dpa). At 21days after amputation, new bone formation began in an anterior direction and maintained the shape of the mandibular arch. In the following days, the cartilage blue-staining was reduced while red-skeletal stains increased. By two months the regenerated two ridges (mandibles) converged medially. Different from the uncut mandibular bones that were separated by a cartilaginous median symphysis, the regenerated mandibles appeared to fuse at the midline. Ms, mandibular symphysis; Ds, dorsal; V, ventral. Scale bars, 500 µm.</p

Homeostasis, inflammatory infiltration and reepitheliation at the early stage of lower jaw regeneration.

<p>As regular wound healing, homeostasis began immediately after amputation (0 hpa), followed by an inflammatory infiltration and epithelia regeneration (4 hpa). At 8 h post-amputation (8 hpa) as the infiltrated nucleated blood cells decreased under the provisional basement membrane, the epidermis-forming cells increased. After 12 hpa, the wound was closed by the stratified epithelia adhering to the underlying dermal matrix. Cut sites are indicated. Scale bars, 100 µm.</p

A schematic model of two origins of blastemal progenitors in lower jaw regeneration.

<p>Two origins of blastemal progenitors (<i>foxi1</i>-positive neural crest cells and <i>isl1</i>-positive mesodermal progenitors) arise in the lower jaw blastema. They undergo mesenchymalization, chondrogenic differentiation and tissue respecifications toward skeletal, connective tissues and muscle regeneration through specific signaling pathways. The possible signaling communications are indicated. For instance, positional signaling mediated by local cues orchestrates blastema reformation; the activation <i>hoxa2b</i> and <i>hoxa11b</i> is induced/enhanced by the second arch signaling and blood signaling respectively.</p

Two Origins of Blastemal Progenitors Define Blastemal Regeneration of Zebrafish Lower Jaw

<div><p>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: <i>foxi1</i>-expression and <i>isl1</i>-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 <i>in situ</i> hybridization shows that <i>foxi1</i> and <i>pax3a</i> are respectively expressed in the presumptive mandible skeletal region and regenerating muscle at 5 dpa. In contrast, <i>hoxa2b</i> and <i>hoxa11b</i> are widely expressed with different domain in chondrogenic blastema and blastema mesenchyme. Knockdown <i>foxi1</i> changes the expression patterns of <i>sox9a</i> and <i>hoxa2b</i> 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.</p></div

Anatomical structure and regenerative capability of zebrafish lower jaw.

<p>C–F, Alcian blue staining of bone and cartilage of lower jaw. The dotted line demarcates the amputation plane. Md, mandible; Ms, mandibular symphysis; Mc, Meckel's cartilage, Mm, mandibular muscle. Scale bars, 1000 µm.</p

Histological observation of blastema formation and reformation.

<p>Figure A shows that regeneration of the mandible retains the original mandibular arch. The cut arch (arrow) at 2 dpa was restored to the original shape (uncut) at 60 dpa. HE staining. Scale bars, 250 µm. Figure B shows blastemal structure and extracellular matrix composition. At 2 hpa, wound epidermis reconstitution started (blue arrow), and blastema was not yet formed between wound epidermis and the injured muscle. At 5 dpa, the blastemal ECM began to reorganize toward hypodermis (vertical red arrow) and the chondrogenic center (horizon red arrow). In comparison with HE stains, Alcian blue-Ponceau S staining shows hyaluronic acid and hyaline cartilage as blue, collagen and mineralized bone matrix as red; In a modification of Masson' trichromal staining, green or blue staining is collagen; brown staining is elastic fibers; red is cytoplasm, muscle, nerve sheathe, fibronectin and erythrocytes). Scale bars, 50 µm. Figure C shows two types of chondrogenic ossification. After blastema formation (8 dpa), chondrogenic blastema was composed of three chondrogenic ossification centers: two Meckel-lateral centers (Mlc) and one median symphysis center (Msc). In the Meckel-lateral centers, perichondral ossification was more like atypical or incomplete endochondral ossification. The surrounding matrix became calcified while the central portion of ossification center developed to bone cysts (BC), a similar structure like bone marrow cavity filled with little connective tissues (120 dpa). The median symphysis center, however, adopted perichondral ossification. Scale bars, 100 µm.</p

<i>foxi1</i> is important for reformation of chondrogenic blastema.

<p>In comparison with control morpholino-treated animals, <i>foxi1</i> vivo morpholino treatment disturbed the reformation of chondrogenic blastema at 5 dpa. In the control slides, <i>foxi1</i>, <i>sox9a and hoxa2b</i> were widely expressed in the chondrogenic blastema. Each gene had its distribution patterns (red dotted circles) and partly overlapped with other genes. After <i>foxi1</i> knockdown, <i>foxi1</i>- and <i>sox9a</i>-expression cells reduced (black dash-dotted circle) and appeared to be congregated in the areas close to the damaged mandible and the Meckel's cartilage. <i>Hoxa2b</i>-expressing cells were delocalized and particularly accumulated in the center of blastema (black dash-dotted circle). Scale bars, 50 µm.</p

Active and repressive histone modification markers were co-enriched in <i>Hoxa9</i> locus.

<div><p>Active histone markers including H4K16Ace, H3K4M2 and H3K4M3, were indicated as grey bars.</p> <p>Repressive markers including H3K4M1, H3K9M2 and H3K27M2 were indicated as black bars.</p> <p>(A) AT1 cells maintain both active and repressive histone modifications at amplicon 2.</p> <p>(B) AR1 cells show aberrant high expression of active histone modifications but low level of repressive histone modifications at amplicon 2.</p> <p>(C) and (D) show that MEF26 cells contain both active and repressive histone markers at amplicons 2 and 4, respectively.</p></div

Cdx4 is crucial for the expression of 5′ Hoxa cluster genes in hematopoietic AT1 cells.

<div><p>(A) Expression profile of <i>Men1</i>, <i>MLL</i>, <i>Cdx4</i> and <i>Hoxa9</i> was determined by quantitative real time RT-PCR.</p> <p>Ectopic expression of Cdx4 increased the expression of 5′ Hoxa cluster in AT1 cells (B) but not in AR1 cells (C).</p></div

<i>In vitro</i> detection of Cdx4 protein binding sites in the regulatory region of <i>Hoxa9</i>.

<div><p>(A) A diagram of <i>Hoxa9</i> locus, showing the corresponding locations between amplicons for qChIPs, reporter elements for luciferase analysis and oligo probes for EMSA.</p> <p>(B) The wild-type and mutation sequences of probe 2 (left) and probe 6 (right).</p> <p>The Cdx protein consensus binding sites were underlined.</p> <p>Wild type sequence of Probe 2 contains one potential Cdx consensus binding site (CBSs) while probe 6 contains two tandem CBSs.</p> <p>(C) Cdx4 protein was able to bind probe 2 wild type sequence but not the mutant sequence.</p> <p>Asterisk (*) denotes a band that likely resulted from partially degraded Cdx4.</p> <p>Their binding was inhibited by anti-Cdx4 antibody but not by a control IgG (left panel).</p> <p>The same <i>in vitro</i> binding assay was conducted for probe 6 and its mutant (right panel).</p></div