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Science 338 (6113): 1476-1480

Copyright © 2012 by the American Association for the Advancement of Science

Hox Genes Regulate Digit Patterning by Controlling the Wavelength of a Turing-Type Mechanism

Rushikesh Sheth1,*,{dagger}, Luciano Marcon2,3,*, M. Félix Bastida1,4, Marisa Junco1, Laura Quintana2,3, Randall Dahn5, Marie Kmita6,{ddagger}, James Sharpe2,3,7,{ddagger}, and Maria A. Ros1,4,{ddagger}

1 Facultad de Medicina, Instituto de Biomedicina y Biotecnología de Cantabria, Consejo Superior de Investigaciones Científicas–Sociedad para el Desarrollo Regional de Cantabria–Universidad de Cantabria, 39011 Santander, Spain.
2 European Molecular Biology Laboratory (EMBL)–Centre for Genomic Regulation (CRG) Systems Biology Research Unit, CRG, Doctor. Aiguader 88, 08003 Barcelona, Spain.
3 Universitat Pompeu Fabra, 08003 Barcelona, Spain.
4 Departamento de Anatomía y Biología Celular, Universidad de Cantabria, 39011 Santander, Spain.
5 7322 Countrywood Lane, Madison, WI 53719, USA.
6 Institut de Recherches Cliniques de Montréal, Université de Montréal, Montréal, Québec H2W 1R7, Canada.
7 Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys 23, 08010 Barcelona, Spain.

Figure 1
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Fig. 1. (A) Expression of Sox9 in E12.5 limbs of the Hoxa13;Gli3 allelic series. Note the delayed differentiation in the anterior mesoderm in the absence of Gli3. The curved white and yellow lines show the AP profiles used for the analysis of Sox9. The red arrowhead points to a digit bifurcation. WT, wild type. (B) Sox9 staining intensity along the yellow profile indicated by the curved arrow. AP length and the period of each digit (from minimum to minimum) are measured and shown for Hoxa13+/;Gli3XtJ/XtJ. (C) Chart showing the average digit periods versus AP lengths for each profile and limb. A linear relation is observed in controls and in the Gli3XtJ/XtJ background for either the normal or heterozygous dose of Hoxa13, whereas a flatter relation that correlates with bifurcations (red arrowhead) is observed in the Hoxa13/;Gli3XtJ/XtJ limbs (red line). The curved arrow marks the yellow point corresponding to the profile in (B). (D and E) Two simulations of the reaction-diffusion model inside an E12.5 Gli3 mutant limb shape. (D) The activator concentration obtained in the simulation with a uniform modulation of wavelength {omega} (shown in the graph) shows digit bifurcation (red arrowhead) similar to the Hoxa13/Gli3XtJ/XtJ mutants. (E) The simulation result when wavelength is modulated according to a suitable PD gradient (in this case, a 2D gradient of simulated FGF signaling activity) avoids bifurcations, because the wavelength increases with increasing AP length. Limbs shown in all figures are forelimbs with distal to the right and anterior to the top.


Figure 2
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Fig. 2. Representative skeletal phenotypes of newborns of the Hoxa13;Hoxd11-13;Gli3 allelic series. Digit number (indicated for the Gli3XtJ/XtJ condition) increases as distal Hox dose is reduced. When only one functional copy of Hoxa13 remains (right column), the tip of the digits is connected by a continuous band of ossified (red) and cartilaginous (blue) tissue rimming the distal border of the limb and becoming more conspicuous as Gli3 copies are removed.


Figure 3
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Fig. 3. The phenotypes of triple mutants can be replicated by the Turing model. (Top) The first three rows show Sox9 expression at E12.5 and E13.5 for different combinations of the triple Hoxa13;Hoxd11-13;Gli3 allelic series. As more Hox are removed, the general trend shows an increase in digit number and a decrease in digit thickness. The trend is most strongly evident in the complete absence of Gli3 (third row). (Bottom) A similar behavior is shown by the reaction-diffusion simulations, where a decrease of the PD gradient used to modulate wavelength is correlated with reduced Hox dose (khox). Additionally, the model predicts a narrower digital region along the PD axis, which eventually shrinks to zero, and no pattern is formed.


Figure 4
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Fig. 4. (A) Schematic representation of the network of a general activator-inhibitor Turing model. The four reaction kinetic parameters are shown: fu, fv, gu, and gv. Fgf promotes a PD-graded distribution of the parameter fu to drive stripe orientation (gray dashed arrow). Hox and Fgf inhibit the parameter gu to increase the wavelength in a PD-graded manner (bold line). U, activator; V, inhibitor. (B) Graphs of the average digit period of the triple mutants with Gli3–/– background at four equidistant positions along the PD axis of the digital region. With the exception of Hoxa13+/;HoxdDel11-13/Del11-13;Gli3XtJ/XtJ, a clear trend is observed: The PD gradient of wavelength is generally shallower as distal Hox genes are removed. (C) Graphs of average digit period (wavelength) versus distal Hox gene dose in the three different Gli3 backgrounds. A smooth positive correlation between Hox gene dose and wavelength is observed in all three cases.


Figure 5
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Fig. 5. Vertebrate limb evolution and distal Hox gene function. A phylogenetic tree of representative taxa and appendage skeletal patterns is shown, as well as corresponding distal Hox expression [in actinopterygians shown for Polyodon spathula (28)] and Gli3R gradient (when known). Genetically abrogating Shh signaling and reducing distal Hox function in mouse autopods (Hoxa13+/;HoxdDel11-13/Del11-13;Gli3XtJ/XtJ) reveals ancestral skeletal characteristics shared with the pectoral fins of sharks (Chiloscyllium punctatum) and primitive ray-finned fishes (Polypterus senegalus): numerous, densely packed, and iterative elements, with a distal cartilaginous band corresponding to the distal radials of fish fins (arrows). The periodic pattern of skeletal elements evident in fins and mutant limbs strongly suggests that a self-organizing Turing-type mechanism of chondrogenesis is deeply conserved in vertebrate phylogeny. Our results further indicate that distal Hox gene dose regulates the number and spacing of skeletal elements formed, implicating distal Hox gene regulatory networks as critical drivers of the evolution of the pentadactyl limb.


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