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Neurons refine the Caenorhabditis elegans body plan by directing axial patterning by Wnts.

Modzelewska K, Lauritzen A, Hasenoeder S, Brown L, Georgiou J, Moghal N - PLoS Biol. (2013)

Bottom Line: Surprisingly, despite high levels of Ror expression in many other cells, these cells cannot substitute for the CAN axons in patterning the epidermis, nor can cells expressing a secreted Wnt inhibitor, SFRP-1.Thus, unmyelinated axon tracts are critical for patterning the C. elegans body.Our findings suggest that the evolution of neurons not only improved metazoans by increasing behavioral complexity, but also by expanding the diversity of developmental patterns generated by growth factors such as Wnts.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, USA.

ABSTRACT
Metazoans display remarkable conservation of gene families, including growth factors, yet somehow these genes are used in different ways to generate tremendous morphological diversity. While variations in the magnitude and spatio-temporal aspects of signaling by a growth factor can generate different body patterns, how these signaling variations are organized and coordinated during development is unclear. Basic body plans are organized by the end of gastrulation and are refined as limbs, organs, and nervous systems co-develop. Despite their proximity to developing tissues, neurons are primarily thought to act after development, on behavior. Here, we show that in Caenorhabditis elegans, the axonal projections of neurons regulate tissue progenitor responses to Wnts so that certain organs develop with the correct morphology at the right axial positions. We find that foreshortening of the posteriorly directed axons of the two canal-associated neurons (CANs) disrupts mid-body vulval morphology, and produces ectopic vulval tissue in the posterior epidermis, in a Wnt-dependent manner. We also provide evidence that suggests that the posterior CAN axons modulate the location and strength of Wnt signaling along the anterior-posterior axis by employing a Ror family Wnt receptor to bind posteriorly derived Wnts, and hence, refine their distributions. Surprisingly, despite high levels of Ror expression in many other cells, these cells cannot substitute for the CAN axons in patterning the epidermis, nor can cells expressing a secreted Wnt inhibitor, SFRP-1. Thus, unmyelinated axon tracts are critical for patterning the C. elegans body. Our findings suggest that the evolution of neurons not only improved metazoans by increasing behavioral complexity, but also by expanding the diversity of developmental patterns generated by growth factors such as Wnts.

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EGL-20/Wnt co-localizes with posterior CAN axons in vivo, in a Ror/CAM-1-dependent manner.CAN axons were visualized with the integrated Pceh-23::gfp transgene (kyIs4) (A–C and E), and EGL-20/Wnt was detected with the integrated Pegl-20::egl-20::protein A fusion transgene (huIs60) and injection of Cy5-conjugated rabbit IgG (IgG-Cy5) into living adult animals (B, C, and E). (A–C and E) The Cy5 channel is shown, as well as an overlay of the GFP channel, where the posterior CAN axon has been pseudo-colored blue. White arrows point to examples of EGL-20/Wnt punctae, and yellow arrows point to examples of variable background autofluorescence. In (C) and (E), numbers in the panels denote particular injected animals. Bottom panels are enlargements of red-boxed sections in the indicated animals. (D) Clustering of Ror/CAM-1 in the posterior CAN axon. Ror/CAM-1 was visualized as a functional translational GFP fusion from an extrachromosomal transgenic array (PCAN::cam-1::gfp; dyEx44) in animals that also carried an egf/lin-3(lf) mutation. Scale bars are 10 µm. (F) 37 independent measurements from 32 wild-type animals, and 26 independent measurements from 19 ror/cam-1 mutants were used to calculate the mean EGL-20/Wnt density along the posterior CAN axon. p-Value was calculated using a two-tailed Mann-Whitney U test versus wild-type animals.
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pbio-1001465-g008: EGL-20/Wnt co-localizes with posterior CAN axons in vivo, in a Ror/CAM-1-dependent manner.CAN axons were visualized with the integrated Pceh-23::gfp transgene (kyIs4) (A–C and E), and EGL-20/Wnt was detected with the integrated Pegl-20::egl-20::protein A fusion transgene (huIs60) and injection of Cy5-conjugated rabbit IgG (IgG-Cy5) into living adult animals (B, C, and E). (A–C and E) The Cy5 channel is shown, as well as an overlay of the GFP channel, where the posterior CAN axon has been pseudo-colored blue. White arrows point to examples of EGL-20/Wnt punctae, and yellow arrows point to examples of variable background autofluorescence. In (C) and (E), numbers in the panels denote particular injected animals. Bottom panels are enlargements of red-boxed sections in the indicated animals. (D) Clustering of Ror/CAM-1 in the posterior CAN axon. Ror/CAM-1 was visualized as a functional translational GFP fusion from an extrachromosomal transgenic array (PCAN::cam-1::gfp; dyEx44) in animals that also carried an egf/lin-3(lf) mutation. Scale bars are 10 µm. (F) 37 independent measurements from 32 wild-type animals, and 26 independent measurements from 19 ror/cam-1 mutants were used to calculate the mean EGL-20/Wnt density along the posterior CAN axon. p-Value was calculated using a two-tailed Mann-Whitney U test versus wild-type animals.

Mentions: To visualize EGL-20/Wnt in living animals, we injected Cy5-labeled rabbit IgG, which has a high affinity for protein A, into double transgenic animals. This technique has been successfully used to label the extracellular portions of cell surface receptors, including protein-A-tagged receptors [49]. In single Pceh-23::gfp transgene control animals, IgG-Cy5 did not accumulate to a significant amount or label any cell surfaces (Figure 8A). In contrast, when injected into Pegl-20::egl-20::protein A; Pceh-23::gfp double transgenic animals, IgG-Cy5 labeled the surfaces of EGL-20/Wnt-producing rectal cells near the anus (Figure 8B), demonstrating specificity to the IgG-Cy5 signal. Cy5-labeled punctae were also enriched in other parts of the posterior body, but detection was variable, presumably because of variations in labeling between injected animals (e.g., Figure S12). However, the posterior enrichment of the Cy5-labeled punctae is consistent with the description of the EGL-20/Wnt gradient in fixed Pegl-20::egl-20::protein A L1 larvae stained with IgG-FITC [12]. Strikingly, Cy5 labeling was also detected along stretches of the posterior CAN axon, suggesting EGL-20/Wnt is bound to the axon (Figure 8C, three different animals are shown).


Neurons refine the Caenorhabditis elegans body plan by directing axial patterning by Wnts.

Modzelewska K, Lauritzen A, Hasenoeder S, Brown L, Georgiou J, Moghal N - PLoS Biol. (2013)

EGL-20/Wnt co-localizes with posterior CAN axons in vivo, in a Ror/CAM-1-dependent manner.CAN axons were visualized with the integrated Pceh-23::gfp transgene (kyIs4) (A–C and E), and EGL-20/Wnt was detected with the integrated Pegl-20::egl-20::protein A fusion transgene (huIs60) and injection of Cy5-conjugated rabbit IgG (IgG-Cy5) into living adult animals (B, C, and E). (A–C and E) The Cy5 channel is shown, as well as an overlay of the GFP channel, where the posterior CAN axon has been pseudo-colored blue. White arrows point to examples of EGL-20/Wnt punctae, and yellow arrows point to examples of variable background autofluorescence. In (C) and (E), numbers in the panels denote particular injected animals. Bottom panels are enlargements of red-boxed sections in the indicated animals. (D) Clustering of Ror/CAM-1 in the posterior CAN axon. Ror/CAM-1 was visualized as a functional translational GFP fusion from an extrachromosomal transgenic array (PCAN::cam-1::gfp; dyEx44) in animals that also carried an egf/lin-3(lf) mutation. Scale bars are 10 µm. (F) 37 independent measurements from 32 wild-type animals, and 26 independent measurements from 19 ror/cam-1 mutants were used to calculate the mean EGL-20/Wnt density along the posterior CAN axon. p-Value was calculated using a two-tailed Mann-Whitney U test versus wild-type animals.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539944&req=5

pbio-1001465-g008: EGL-20/Wnt co-localizes with posterior CAN axons in vivo, in a Ror/CAM-1-dependent manner.CAN axons were visualized with the integrated Pceh-23::gfp transgene (kyIs4) (A–C and E), and EGL-20/Wnt was detected with the integrated Pegl-20::egl-20::protein A fusion transgene (huIs60) and injection of Cy5-conjugated rabbit IgG (IgG-Cy5) into living adult animals (B, C, and E). (A–C and E) The Cy5 channel is shown, as well as an overlay of the GFP channel, where the posterior CAN axon has been pseudo-colored blue. White arrows point to examples of EGL-20/Wnt punctae, and yellow arrows point to examples of variable background autofluorescence. In (C) and (E), numbers in the panels denote particular injected animals. Bottom panels are enlargements of red-boxed sections in the indicated animals. (D) Clustering of Ror/CAM-1 in the posterior CAN axon. Ror/CAM-1 was visualized as a functional translational GFP fusion from an extrachromosomal transgenic array (PCAN::cam-1::gfp; dyEx44) in animals that also carried an egf/lin-3(lf) mutation. Scale bars are 10 µm. (F) 37 independent measurements from 32 wild-type animals, and 26 independent measurements from 19 ror/cam-1 mutants were used to calculate the mean EGL-20/Wnt density along the posterior CAN axon. p-Value was calculated using a two-tailed Mann-Whitney U test versus wild-type animals.
Mentions: To visualize EGL-20/Wnt in living animals, we injected Cy5-labeled rabbit IgG, which has a high affinity for protein A, into double transgenic animals. This technique has been successfully used to label the extracellular portions of cell surface receptors, including protein-A-tagged receptors [49]. In single Pceh-23::gfp transgene control animals, IgG-Cy5 did not accumulate to a significant amount or label any cell surfaces (Figure 8A). In contrast, when injected into Pegl-20::egl-20::protein A; Pceh-23::gfp double transgenic animals, IgG-Cy5 labeled the surfaces of EGL-20/Wnt-producing rectal cells near the anus (Figure 8B), demonstrating specificity to the IgG-Cy5 signal. Cy5-labeled punctae were also enriched in other parts of the posterior body, but detection was variable, presumably because of variations in labeling between injected animals (e.g., Figure S12). However, the posterior enrichment of the Cy5-labeled punctae is consistent with the description of the EGL-20/Wnt gradient in fixed Pegl-20::egl-20::protein A L1 larvae stained with IgG-FITC [12]. Strikingly, Cy5 labeling was also detected along stretches of the posterior CAN axon, suggesting EGL-20/Wnt is bound to the axon (Figure 8C, three different animals are shown).

Bottom Line: Surprisingly, despite high levels of Ror expression in many other cells, these cells cannot substitute for the CAN axons in patterning the epidermis, nor can cells expressing a secreted Wnt inhibitor, SFRP-1.Thus, unmyelinated axon tracts are critical for patterning the C. elegans body.Our findings suggest that the evolution of neurons not only improved metazoans by increasing behavioral complexity, but also by expanding the diversity of developmental patterns generated by growth factors such as Wnts.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, USA.

ABSTRACT
Metazoans display remarkable conservation of gene families, including growth factors, yet somehow these genes are used in different ways to generate tremendous morphological diversity. While variations in the magnitude and spatio-temporal aspects of signaling by a growth factor can generate different body patterns, how these signaling variations are organized and coordinated during development is unclear. Basic body plans are organized by the end of gastrulation and are refined as limbs, organs, and nervous systems co-develop. Despite their proximity to developing tissues, neurons are primarily thought to act after development, on behavior. Here, we show that in Caenorhabditis elegans, the axonal projections of neurons regulate tissue progenitor responses to Wnts so that certain organs develop with the correct morphology at the right axial positions. We find that foreshortening of the posteriorly directed axons of the two canal-associated neurons (CANs) disrupts mid-body vulval morphology, and produces ectopic vulval tissue in the posterior epidermis, in a Wnt-dependent manner. We also provide evidence that suggests that the posterior CAN axons modulate the location and strength of Wnt signaling along the anterior-posterior axis by employing a Ror family Wnt receptor to bind posteriorly derived Wnts, and hence, refine their distributions. Surprisingly, despite high levels of Ror expression in many other cells, these cells cannot substitute for the CAN axons in patterning the epidermis, nor can cells expressing a secreted Wnt inhibitor, SFRP-1. Thus, unmyelinated axon tracts are critical for patterning the C. elegans body. Our findings suggest that the evolution of neurons not only improved metazoans by increasing behavioral complexity, but also by expanding the diversity of developmental patterns generated by growth factors such as Wnts.

Show MeSH
Related in: MedlinePlus