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A computational clonal analysis of the developing mouse limb bud.

Marcon L, Arqués CG, Torres MS, Sharpe J - PLoS Comput. Biol. (2011)

Bottom Line: However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward.Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time.The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis.

View Article: PubMed Central - PubMed

Affiliation: EMBL-CRG Systems Biology Research Unit, Center for Genomic Regulation (CRG), Universitat Pompeu Fabra, Barcelona, Spain. luciano.marcon@crg.es

ABSTRACT
A comprehensive spatio-temporal description of the tissue movements underlying organogenesis would be an extremely useful resource to developmental biology. Clonal analysis and fate mappings are popular experiments to study tissue movement during morphogenesis. Such experiments allow cell populations to be labeled at an early stage of development and to follow their spatial evolution over time. However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward. To overcome this problem, we develop a novel computational method that combines accurate quantification of 2D limb bud morphologies and growth modeling to analyze mouse clonal data of early limb development. Firstly, we explore various tissue movements that match experimental limb bud shape changes. Secondly, by comparing computational clones with newly generated mouse clonal data we are able to choose and characterize the tissue movement map that better matches experimental data. Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time. The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis. Lastly, we show that this comprehensive description can be used to reassess spatio-temporal gene regulations taking tissue movement into account and to investigate PD patterning hypothesis.

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Clonal analysis.(A) Four clones showing the quantification of the AP and PD clone lengths. (B) In order to compensate for the variation in developmental stage between different embryos each limb was staged and the tamoxifen injection time was adjusted accordingly. Large triangles represent the AP and PD clone expansion over space and time. PD and AP lengths were mapped at E12 (red line) considering prospective (dotted line) or retrospective lengths. (C) Each rectangle represents the AP and PD length of one clone. Clones were clustered into two groups: isotropically expanding clones, with comparable AP and PD length (blue rectangles), and an-isotropically expanding clones having the PD length greater than AP length (red and green rectangles). (D) A graph showing the degree of clone anisotropy in the limb, PD length over the AP length. Blue means low anisotropy and red high anisotropy. (E) Top: In-situ of Sox9, a known early skeletal marker showing the position of the three PD segments (S = stylopod, Z = zeugopod, A = autopod) Bottom: 16 clones showing the degree of overlap between clones spanning across different PD segments.
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pcbi-1001071-g003: Clonal analysis.(A) Four clones showing the quantification of the AP and PD clone lengths. (B) In order to compensate for the variation in developmental stage between different embryos each limb was staged and the tamoxifen injection time was adjusted accordingly. Large triangles represent the AP and PD clone expansion over space and time. PD and AP lengths were mapped at E12 (red line) considering prospective (dotted line) or retrospective lengths. (C) Each rectangle represents the AP and PD length of one clone. Clones were clustered into two groups: isotropically expanding clones, with comparable AP and PD length (blue rectangles), and an-isotropically expanding clones having the PD length greater than AP length (red and green rectangles). (D) A graph showing the degree of clone anisotropy in the limb, PD length over the AP length. Blue means low anisotropy and red high anisotropy. (E) Top: In-situ of Sox9, a known early skeletal marker showing the position of the three PD segments (S = stylopod, Z = zeugopod, A = autopod) Bottom: 16 clones showing the degree of overlap between clones spanning across different PD segments.

Mentions: We used the tamoxifen inducible Cre-line presented in [11] to conduct a mouse clonal analysis from stage E9 to stage E12 of development. The clones were induced by injecting low tamoxifen concentration at E8 (0.10mg) so that random recombination events would produce single cell labeling events within the embryos. 24 hind-limbs showing suitable monoclonal labeling were used for the clonal analysis. To compensate for the variation in development between embryos of the same litter and the uncertainty of the injection day, we staged each limb using the staging system presented in [28] and adjusted the estimation of the injection day accordingly. The PD and AP clone lengths relative to the maximum PD and AP length of the limb were measured as shown in Figure 3A (See also Figure S2). From the quantification of clone lengths, two graphs representing respectively PD and AP clone expansion were produced, an example is shown in Figure 3B. All clone lengths were mapped at stage E12 by considering prospective or retrospective lengths as shown in Figure 3B. PD and AP lengths at E12 were visualized representing each clone as a rectangle centered in its AP and PD midpoint (in Figure 3C). In this way we were able to cluster the clones in two groups according to their position and shape: a) isotropically expanding clones in the proximal and distal part of the limb that showed similar AP and PD expansion rate (highlighted in blue), b) anisotropic clones that expanded more along the PD axis than the AP axis (highlighted in green and red). Plotting the ratio between PD and AP lengths a similar behavior was revealed (Figure 3D). In accord with a previous study [11] we found no clear evidence for AP and PD compartments. Indeed, a high degree of cell mixing was observed across the whole limb. Consistently with previous studies in chick [8], [9] we found that clones expanded across one or two PD segments but never span across the whole PD axis of the limb, see Figure 3E. Remarkably, no clones were found restricted to the zeugopod alone – all clones found in this zone also overlapped with the autopod or the stylopod regions.


A computational clonal analysis of the developing mouse limb bud.

Marcon L, Arqués CG, Torres MS, Sharpe J - PLoS Comput. Biol. (2011)

Clonal analysis.(A) Four clones showing the quantification of the AP and PD clone lengths. (B) In order to compensate for the variation in developmental stage between different embryos each limb was staged and the tamoxifen injection time was adjusted accordingly. Large triangles represent the AP and PD clone expansion over space and time. PD and AP lengths were mapped at E12 (red line) considering prospective (dotted line) or retrospective lengths. (C) Each rectangle represents the AP and PD length of one clone. Clones were clustered into two groups: isotropically expanding clones, with comparable AP and PD length (blue rectangles), and an-isotropically expanding clones having the PD length greater than AP length (red and green rectangles). (D) A graph showing the degree of clone anisotropy in the limb, PD length over the AP length. Blue means low anisotropy and red high anisotropy. (E) Top: In-situ of Sox9, a known early skeletal marker showing the position of the three PD segments (S = stylopod, Z = zeugopod, A = autopod) Bottom: 16 clones showing the degree of overlap between clones spanning across different PD segments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037386&req=5

pcbi-1001071-g003: Clonal analysis.(A) Four clones showing the quantification of the AP and PD clone lengths. (B) In order to compensate for the variation in developmental stage between different embryos each limb was staged and the tamoxifen injection time was adjusted accordingly. Large triangles represent the AP and PD clone expansion over space and time. PD and AP lengths were mapped at E12 (red line) considering prospective (dotted line) or retrospective lengths. (C) Each rectangle represents the AP and PD length of one clone. Clones were clustered into two groups: isotropically expanding clones, with comparable AP and PD length (blue rectangles), and an-isotropically expanding clones having the PD length greater than AP length (red and green rectangles). (D) A graph showing the degree of clone anisotropy in the limb, PD length over the AP length. Blue means low anisotropy and red high anisotropy. (E) Top: In-situ of Sox9, a known early skeletal marker showing the position of the three PD segments (S = stylopod, Z = zeugopod, A = autopod) Bottom: 16 clones showing the degree of overlap between clones spanning across different PD segments.
Mentions: We used the tamoxifen inducible Cre-line presented in [11] to conduct a mouse clonal analysis from stage E9 to stage E12 of development. The clones were induced by injecting low tamoxifen concentration at E8 (0.10mg) so that random recombination events would produce single cell labeling events within the embryos. 24 hind-limbs showing suitable monoclonal labeling were used for the clonal analysis. To compensate for the variation in development between embryos of the same litter and the uncertainty of the injection day, we staged each limb using the staging system presented in [28] and adjusted the estimation of the injection day accordingly. The PD and AP clone lengths relative to the maximum PD and AP length of the limb were measured as shown in Figure 3A (See also Figure S2). From the quantification of clone lengths, two graphs representing respectively PD and AP clone expansion were produced, an example is shown in Figure 3B. All clone lengths were mapped at stage E12 by considering prospective or retrospective lengths as shown in Figure 3B. PD and AP lengths at E12 were visualized representing each clone as a rectangle centered in its AP and PD midpoint (in Figure 3C). In this way we were able to cluster the clones in two groups according to their position and shape: a) isotropically expanding clones in the proximal and distal part of the limb that showed similar AP and PD expansion rate (highlighted in blue), b) anisotropic clones that expanded more along the PD axis than the AP axis (highlighted in green and red). Plotting the ratio between PD and AP lengths a similar behavior was revealed (Figure 3D). In accord with a previous study [11] we found no clear evidence for AP and PD compartments. Indeed, a high degree of cell mixing was observed across the whole limb. Consistently with previous studies in chick [8], [9] we found that clones expanded across one or two PD segments but never span across the whole PD axis of the limb, see Figure 3E. Remarkably, no clones were found restricted to the zeugopod alone – all clones found in this zone also overlapped with the autopod or the stylopod regions.

Bottom Line: However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward.Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time.The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis.

View Article: PubMed Central - PubMed

Affiliation: EMBL-CRG Systems Biology Research Unit, Center for Genomic Regulation (CRG), Universitat Pompeu Fabra, Barcelona, Spain. luciano.marcon@crg.es

ABSTRACT
A comprehensive spatio-temporal description of the tissue movements underlying organogenesis would be an extremely useful resource to developmental biology. Clonal analysis and fate mappings are popular experiments to study tissue movement during morphogenesis. Such experiments allow cell populations to be labeled at an early stage of development and to follow their spatial evolution over time. However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward. To overcome this problem, we develop a novel computational method that combines accurate quantification of 2D limb bud morphologies and growth modeling to analyze mouse clonal data of early limb development. Firstly, we explore various tissue movements that match experimental limb bud shape changes. Secondly, by comparing computational clones with newly generated mouse clonal data we are able to choose and characterize the tissue movement map that better matches experimental data. Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time. The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis. Lastly, we show that this comprehensive description can be used to reassess spatio-temporal gene regulations taking tissue movement into account and to investigate PD patterning hypothesis.

Show MeSH