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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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Related in: MedlinePlus

VAB-8 acts in the CAN neurons to regulate epidermal patterning.(A–C) Expression of vab-8s and CAN promoters from the akEx923 transgenic array in wild-type L2 stage animals. Scale bar is 50 µm. The CANs are a pair of neurons, CANL and CANR, located on the left and right side of each animal, respectively. (A) DsRed2 channel. (B) YFP channel. (C) Merged images from (A) and (B). (D) CAN-specific expression of VAB-8S restores inhibition to vulval fate signaling in vab-8 mutants. vab-8 mutations displace cell bodies and affect axon outgrowth of a subset of neurons including the CANs (blue). VAB-8S was restored to vab-8 mutants with either the CAN-specific promoter (PCAN) or the control minimal pes-10 promoter (Ppes-10). Vulval fates: number of vulval progenitor cells adopting vulval fates. Wild-type is 3.00. p-Value was calculated using a two-tailed Student's t test. Transgenic arrays were dyEx24 (Ppes-10::vab-8s) or dyEx20 (PCAN::vab-8s). CAN-specific expression of VAB-8S from the dyEx20 transgenic array rescues patterning defects in the posterior and anterior epidermis (E and F). p-Value was calculated using a two-tailed Fisher's exact test.
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pbio-1001465-g003: VAB-8 acts in the CAN neurons to regulate epidermal patterning.(A–C) Expression of vab-8s and CAN promoters from the akEx923 transgenic array in wild-type L2 stage animals. Scale bar is 50 µm. The CANs are a pair of neurons, CANL and CANR, located on the left and right side of each animal, respectively. (A) DsRed2 channel. (B) YFP channel. (C) Merged images from (A) and (B). (D) CAN-specific expression of VAB-8S restores inhibition to vulval fate signaling in vab-8 mutants. vab-8 mutations displace cell bodies and affect axon outgrowth of a subset of neurons including the CANs (blue). VAB-8S was restored to vab-8 mutants with either the CAN-specific promoter (PCAN) or the control minimal pes-10 promoter (Ppes-10). Vulval fates: number of vulval progenitor cells adopting vulval fates. Wild-type is 3.00. p-Value was calculated using a two-tailed Student's t test. Transgenic arrays were dyEx24 (Ppes-10::vab-8s) or dyEx20 (PCAN::vab-8s). CAN-specific expression of VAB-8S from the dyEx20 transgenic array rescues patterning defects in the posterior and anterior epidermis (E and F). p-Value was calculated using a two-tailed Fisher's exact test.

Mentions: In contrast to mutations that disrupt both VAB-8 isoforms, the vab-8(ev411) mutation, which only affects VAB-8L [31], did not cause ectopic vulval fates in P8.p, did not increase the frequency of P3.p becoming a vulval progenitor, and did not suppress the underinduced phenotypes of egf/lin-3 or egfr/let-23 mutants (Figure 2A and 2B; Table 1). Thus, VAB-8S is sufficient to inhibit vulval fates. To identify cells whose position might influence epidermal development, we examined promoter activity in the smallest genomic fragment previously shown to encode functional VAB-8S activity [31]. This promoter drove reporter expression in eight head neurons and the pair of CANs (CANL and CANR) (Figure 3A). To determine whether any of these neurons regulate epidermal development, we used this promoter to restore VAB-8S (Pvab-8s::vab-8s) to all of these cells in egfr/let-23(lf); vab-8(gm138) double mutants. VAB-8S expression in these cells fully restored the egfr/let-23(lf) vulvaless phenotype (Table S1), suggesting that the positioning of one or more of these cells inhibits vulval fate signaling in P6.p. We then focused on the CANs, since (1) of the ten neurons, only the CAN axons span the entire anterior–posterior axis, (2) vab-8 mutations severely displace CAN cell bodies and shorten posterior axons [26], and (3) the vab-8(ev411) mutation, which does not affect epidermal development, does not affect CAN cell body position, and only weakly affects CAN axon outgrowth [26]. Since VAB-8 acts cell autonomously in the CANs to promote their posterior migration and axon outgrowth [31],[32], we used a previously described CAN-specific enhancer [34] to create an expression vector to specifically restore VAB-8S and proper positioning only to the CANs. This vector drove YFP expression only in the CANs, which was also the only site of co-localization with vab-8s-driven DsRed2 (Figure 3B and 3C), confirming the cell-specificity of this vector. When expressed from the CAN-specific promoter (PCAN::vab-8s), but not the control minimal pes-10 promoter, VAB-8S also restored full inhibition to vulval fate signaling in P6.p in egfr/let-23(lf); vab-8(gm138) double mutants (Figure 3D; Table S1). This transgene also fully suppressed the other vab-8(gm138) epidermal phenotypes, including the ectopic vulval fates at P8.p (Figure 3E), the increased frequency of P3.p becoming a vulval progenitor (Figure 3F), and the P-Rvl phenotype (0%, n = 64). Thus, VAB-8 acts in the CANs to regulate epidermal development.


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)

VAB-8 acts in the CAN neurons to regulate epidermal patterning.(A–C) Expression of vab-8s and CAN promoters from the akEx923 transgenic array in wild-type L2 stage animals. Scale bar is 50 µm. The CANs are a pair of neurons, CANL and CANR, located on the left and right side of each animal, respectively. (A) DsRed2 channel. (B) YFP channel. (C) Merged images from (A) and (B). (D) CAN-specific expression of VAB-8S restores inhibition to vulval fate signaling in vab-8 mutants. vab-8 mutations displace cell bodies and affect axon outgrowth of a subset of neurons including the CANs (blue). VAB-8S was restored to vab-8 mutants with either the CAN-specific promoter (PCAN) or the control minimal pes-10 promoter (Ppes-10). Vulval fates: number of vulval progenitor cells adopting vulval fates. Wild-type is 3.00. p-Value was calculated using a two-tailed Student's t test. Transgenic arrays were dyEx24 (Ppes-10::vab-8s) or dyEx20 (PCAN::vab-8s). CAN-specific expression of VAB-8S from the dyEx20 transgenic array rescues patterning defects in the posterior and anterior epidermis (E and F). p-Value was calculated using a two-tailed Fisher's exact test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539944&req=5

pbio-1001465-g003: VAB-8 acts in the CAN neurons to regulate epidermal patterning.(A–C) Expression of vab-8s and CAN promoters from the akEx923 transgenic array in wild-type L2 stage animals. Scale bar is 50 µm. The CANs are a pair of neurons, CANL and CANR, located on the left and right side of each animal, respectively. (A) DsRed2 channel. (B) YFP channel. (C) Merged images from (A) and (B). (D) CAN-specific expression of VAB-8S restores inhibition to vulval fate signaling in vab-8 mutants. vab-8 mutations displace cell bodies and affect axon outgrowth of a subset of neurons including the CANs (blue). VAB-8S was restored to vab-8 mutants with either the CAN-specific promoter (PCAN) or the control minimal pes-10 promoter (Ppes-10). Vulval fates: number of vulval progenitor cells adopting vulval fates. Wild-type is 3.00. p-Value was calculated using a two-tailed Student's t test. Transgenic arrays were dyEx24 (Ppes-10::vab-8s) or dyEx20 (PCAN::vab-8s). CAN-specific expression of VAB-8S from the dyEx20 transgenic array rescues patterning defects in the posterior and anterior epidermis (E and F). p-Value was calculated using a two-tailed Fisher's exact test.
Mentions: In contrast to mutations that disrupt both VAB-8 isoforms, the vab-8(ev411) mutation, which only affects VAB-8L [31], did not cause ectopic vulval fates in P8.p, did not increase the frequency of P3.p becoming a vulval progenitor, and did not suppress the underinduced phenotypes of egf/lin-3 or egfr/let-23 mutants (Figure 2A and 2B; Table 1). Thus, VAB-8S is sufficient to inhibit vulval fates. To identify cells whose position might influence epidermal development, we examined promoter activity in the smallest genomic fragment previously shown to encode functional VAB-8S activity [31]. This promoter drove reporter expression in eight head neurons and the pair of CANs (CANL and CANR) (Figure 3A). To determine whether any of these neurons regulate epidermal development, we used this promoter to restore VAB-8S (Pvab-8s::vab-8s) to all of these cells in egfr/let-23(lf); vab-8(gm138) double mutants. VAB-8S expression in these cells fully restored the egfr/let-23(lf) vulvaless phenotype (Table S1), suggesting that the positioning of one or more of these cells inhibits vulval fate signaling in P6.p. We then focused on the CANs, since (1) of the ten neurons, only the CAN axons span the entire anterior–posterior axis, (2) vab-8 mutations severely displace CAN cell bodies and shorten posterior axons [26], and (3) the vab-8(ev411) mutation, which does not affect epidermal development, does not affect CAN cell body position, and only weakly affects CAN axon outgrowth [26]. Since VAB-8 acts cell autonomously in the CANs to promote their posterior migration and axon outgrowth [31],[32], we used a previously described CAN-specific enhancer [34] to create an expression vector to specifically restore VAB-8S and proper positioning only to the CANs. This vector drove YFP expression only in the CANs, which was also the only site of co-localization with vab-8s-driven DsRed2 (Figure 3B and 3C), confirming the cell-specificity of this vector. When expressed from the CAN-specific promoter (PCAN::vab-8s), but not the control minimal pes-10 promoter, VAB-8S also restored full inhibition to vulval fate signaling in P6.p in egfr/let-23(lf); vab-8(gm138) double mutants (Figure 3D; Table S1). This transgene also fully suppressed the other vab-8(gm138) epidermal phenotypes, including the ectopic vulval fates at P8.p (Figure 3E), the increased frequency of P3.p becoming a vulval progenitor (Figure 3F), and the P-Rvl phenotype (0%, n = 64). Thus, VAB-8 acts in the CANs to regulate epidermal development.

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