Formación de patrón en una estructura dinámica. Desarrollo próximo-distal en la extremidad de los vertebrados

Tesis doctoral inédita. Universidad Autónoma de madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17/12/201

Diffusible signals, not autonomous mechanisms, determine the main proximodistal limb subdivision

ReportVertebrate limbs develop three main proximodistal (PD) segments (upper arm, forearm, and hand) in a proximal-to-distal sequence. Despite extensive research into limb development, whether PD specification occurs through nonautonomous or autonomous mechanisms is not resolved. Heterotopic transplantation of intact and recombinant chicken limb buds identifies signals in the embryo trunk that proximalize distal limb cells to generate a complete PD axis. In these transplants, retinoic acid induces proximalization, which is counteracted by fibroblast growth factors from the distal limb bud; these related actions suggest that the first limb-bud PD regionalization results from the balance between proximal and distal signals. The plasticity of limb progenitor cell identity in response to diffusible signals provides a unifying view of PD patterning during vertebrate limb development and regeneration.Supported by grants RD06/0010/0008, BFU2009-08331/BMC (M.T.) and BFU2008-00397 (M.A.R.), from the Spanish Ministry of Science and Innovation (MICINN) and fellowship CPI/0051/2007 (A. R.-D.) from Madrid’s Regional Government (CAM). The CNIC is supported by the MICINN and the Pro-CNIC Foundation.Peer reviewe

Altered paracrine signaling from the injured knee joint impairs postnatal long bone growth.

Regulation of organ growth is a poorly understood process. In the long bones, the growth plates (GPs) drive elongation by generating a scaffold progressively replaced by bone. Although studies have focused on intrinsic GP regulation, classic and recent experiments suggest that local signals also modulate GP function. We devised a genetic mouse model to study extrinsic long bone growth modulation, in which injury is specifically induced in the left hindlimb, such that the right hindlimb serves as an internal control. Remarkably, when only mesenchyme cells surrounding postnatal GPs were killed, left bone growth was nevertheless reduced. GP signaling was impaired by altered paracrine signals from the knee joint, including activation of the injury response and, in neonates, dampened IGF1 production. Importantly, only the combined prevention of both responses rescued neonatal growth. Thus, we identified signals from the knee joint that modulate bone growth and could underlie establishment of body proportions

Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice.

Catch-up growth after insults to growing organs is paramount to achieving robust body proportions. In fly larvae, injury to individual tissues is followed by local and systemic compensatory mechanisms that allow the damaged tissue to regain normal proportions with other tissues. In vertebrates, local catch-up growth has been described after transient reduction of bone growth, but the underlying cellular responses are controversial. We developed an approach to study catch-up growth in foetal mice in which mosaic expression of the cell cycle suppressor p21 is induced in the cartilage cells (chondrocytes) that drive long-bone elongation. By specifically targeting p21 expression to left hindlimb chondrocytes, the right limb serves as an internal control. Unexpectedly, left-right limb symmetry remained normal, revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an orchestrated response was triggered involving local enhancement of bone growth and systemic growth reduction that ensured that body proportions were maintained. The local response entailed hyperproliferation of spared left limb chondrocytes that was associated with reduced chondrocyte density. The systemic effect involved impaired placental function and IGF signalling, revealing bone-placenta communication. Therefore, vertebrates, like invertebrates, can mount coordinated local and systemic responses to developmental insults that ensure that normal body proportions are maintained

Mosaic local proliferation blockade in chondrocytes of the left limb results in systemic growth reduction.

<p>(A) Right femur and tibia length (normalized to the average <i>ePit-p21</i> littermate) from E17.5 embryos treated with Dox (<i>n =</i> 4 <i>ePit-p21</i> and <i>n</i> = 11 <i>ePit-Col-p21</i>) or untreated (<i>n =</i> 5 and <i>n</i> = 6). Comparison by 2-way ANOVA with Genotype and Bone identity as variables. <i>p-</i>Values for Genotypes are shown below graphs; <i>p-</i>values for Sidak’s post hoc test shown on graph. (B–C) Box and whisker plots for normalized bone length (panel B) and weight (panel C) of <i>ePit-Col-p21</i> and <i>Col2a1-rtTA; Igs7</i>^{<i>TRE-tdT/+</i>} embryos (<i>eCol-tdT</i>, expressing tdT in all cartilage elements), compared to their controls (<i>ePit-p21</i> and <i>ePit-tdT</i>) by unpaired <i>t</i> tests. <i>p-</i>Values corrected for multiple comparisons (Holm-Sidak method) are shown. For panel B, <i>n =</i> 22 <i>ePit-p21</i>, <i>n</i> = 26 <i>ePit-Col-p21</i>, <i>n</i> = 25 <i>ePit-tdT</i>, and <i>n</i> = 5 <i>eCol-tdT</i>). For panel C, <i>n =</i> 4, 11, 24, and 5. (D) Model of the systemic growth response after local chondrocyte arrest triggers a stress signal. (E–F) qRT-PCR (panel E) and in situ hybridisation (panel F) for the indicated transcripts in the proliferative plus pre-hypertrophic zone from <i>ePit-Col-p21</i> embryos. Panel E shows one of 2 independent experiments with 3 distinct biological replicates each (total <i>n</i> = 6). The –ΔCt (relative to <i>Gapdh</i>) for each stress-related transcript was compared by a paired <i>t</i> test (left versus right). In panel F, <i>n =</i> 2 E15.5, <i>n</i> = 4 E16.5, and <i>n</i> = 6 E17.5 embryos (arrowheads denote <i>Il6</i> expression). For panel A–C and E, see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005086#pbio.2005086.s003" target="_blank">S3 Data</a>. Dox, doxycycline; E, embryonic day; qRT-PCR, quantitative real-time polymerase chain reaction; tdT, tdTomato.</p

Mosaic proliferation blockade in the left limb cartilage slightly reduces bone growth but not left–right limb symmetry.

<p>(A) Absolute length of the indicated left bones of <i>ePit-p21</i> (<i>n =</i> 4–7 depending on the bone) and <i>ePit-Col-p21</i> (<i>n =</i> 11–15) E17.5 embryos. (B, C) Skeletal preparations (panel B) and quantification of the left/right ratio (panel C, mean ± SD) of the calcified region of the indicated bones at E17.5 (<i>n =</i> 4–7 <i>ePit-p21</i> and <i>n</i> = 11–15 <i>ePit-Col-p21</i> mice, depending on the bone) and P0 (<i>n =</i> 5–9, and 6–9). Dashed lines in panel B mark the ends of the mineralized region in control bones. In panel A and C, data were analysed by 2-way ANOVA with Genotype and Bone identity as variables. <i>p-</i>Values are shown below the graphs. For variables with significantly different measurements, Sidak’s post hoc test <i>p-</i>values are shown in the graph. For panel A and C, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005086#pbio.2005086.s003" target="_blank">S3 Data</a>. E, embryonic day; P, postnatal day.</p

An intersectional genetic approach enables inducible p21 misexpression primarily in left limb chondrocytes.

<p>(A) <i>DRAGON-p21</i> allele in the <i>Igs7</i> locus. (B) Schematic showing <i>p21</i> expression driven by the left-specific <i>Pitx2-Cre</i> and cartilage-specific <i>Col2a1-rtTA</i> (<i>Pit-Col-p21</i>). (C–E) Expression of tdT protein and <i>p21</i> mRNA (panel C and C’) and p21 protein and EdU (panel D and E) at E15.5, with Dox administered at E12.5. <i>n =</i> 3. Box in panel D is magnified in panel E. Asterisk indicate endogenous <i>p21</i> expression. Arrowheads indicate rare double-positive cells. (F) Quantification of p21⁺ cells in the PZ of <i>ePit-Col-p21</i> proximal tibias, at E15.5 (<i>n =</i> 3) and E17.5 (<i>n =</i> 5). The average left/right fold-change is indicated. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005086#pbio.2005086.s003" target="_blank">S3 Data</a>. (G) Quantification of EdU incorporation in p21⁺ and p21⁻ cells of left <i>ePit-Col-p21</i> PZ of the cartilage, at E15.5 and E17.5 (<i>n =</i> 3 and <i>n</i> = 5). Comparison by 2-way ANOVA with Cell population and Stage as variables (<i>p</i>-values below graphs). <i>p</i>-Values for Sidak’s multiple comparisons post hoc test (between cell populations) are shown on the graph. For panel F and G, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005086#pbio.2005086.s003" target="_blank">S3 Data</a>. 2xpA, transcriptional STOP; E, embryonic day; EdU, 5-ethynil-2’-deoxyuridine; Ins, insulator; PZ, proliferative zone; tdT, tdTomato; TRE, Tetracycline-responsive element; W, WPRE (mRNA-stabilizing sequence) followed by pA.</p