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The C. elegans TPR Containing Protein, TRD-1, Regulates Cell Fate Choice in the Developing Germ Line and Epidermis.

Hughes S, Wilkinson H, Gilbert SP, Kishida M, Ding SS, Woollard A - PLoS ONE (2014)

Bottom Line: In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes.In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate.We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Correct cell fate choice is crucial in development. In post-embryonic development of the hermaphroditic Caenorhabitis elegans, distinct cell fates must be adopted in two diverse tissues. In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes. In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate. We have isolated a novel conserved C. elegans tetratricopeptide repeat containing protein, TRD-1, which is essential for cell fate determination in both the germline and the developing epidermis and has homologs in other species, including humans (TTC27). We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate. In the germline, trd-1 RNAi results in a strong masculinization phenotype, as well as defects in the mitosis to meiosis switch. Our data suggests that trd-1 acts downstream of tra-2 but upstream of fem-3 in the germline sex determination pathway, and exhibits a constellation of phenotypes in common with other Mog (masculinization of germline) mutants. Thus, trd-1 is a new player in both the somatic and germline cell fate determination machinery, suggestive of a novel molecular connection between the development of these two diverse tissues.

No MeSH data available.


Related in: MedlinePlus

TRD-1 regulates cell fate choice in seam lineages.(A) (i) Wild type late L4 animal carrying the integrated seam cell marker, scm::gfp (strain, JR667), has 16 seam cells per side. (ii) trd-1(RNAi) animals have a reduction in the number of seam cells. Seam cells are indicated by white arrowheads. (iii) trd-1(tm2764) homozygous mutants (strain, AW912) have a significant reduction in seam cell number. Scale bar, 20 µm. Anterior is to the left and dorsal up in all images. (B) Graph showing average seam cell number. Wild type animals were exposed to empty RNAi feeding vector as a control. n>100 for both control and trd-1(RNAi) animals. Heterozygotes of genotype hT2/trd-1(tm2764) have normal numbers of seam cells (n = 61), however, trd-1(tm2764) homozygotes have a significant reduction in seam cell number to 11.3 (n = 45). Error bars represent s.e.m. and ** indicate the 2-sample t-test where each strain was compared to the wild type, where p<0.01. (C) (i–iii) L4 Animal (strain, AW1015) carrying an integrated seam cell nuclear marker (scm::tdTomato) along with the pleckstrin homology domain PH::gfp outlining seam cells and a hyp7 nuclear marker dpy-7p::yfp. White arrowheads indicate seam cell nuclei. (iv–vi) trd-1(RNAi) animals carrying the same set of markers. There are obvious gaps in the seam cells, where the seam cell fate has been transformed to hypodermal. Asterisks indicate nuclei that inappropriately express dpy-7::yfp instead of scm::tdTomato. Note that these cells have lost their PH::gfp outline, also indicative of transformation towards the hypodermal fate. ii and v are images of the GFP/YFP channel. iii and vi is the red channel to show the tdTomato in the same animal. i and iv are merged imaged of both GFP/YFP and tdTomato channels. All scale bar, 20 µm. Anterior is to the left and dorsal up in all images. (D) Representative image of a trd-1(RNAi) animal displaying shallow or absent alae. Scale bar, 40 µm (E) Representative lineage trace showing the Vn lineage from hatching to late L2. In wild type animals at the L1 stage, Vn divides asymmetrically with the anterior daughter (Vn.a) adopting the hypodermal fate and the posterior daughter (Vn.p) retaining the proliferative fate. Vn.p will divide symmetrically during early L2 followed closely by a further asymmetric division. Vn.pap and Vn.ppp retain the ability to self-renew and will divide again at L3 and L4. We lineaged 7 trd-1(RNAi) animals, of which 5 had symmetrized the L1 asymmetric division of V2 (asterisk). In animals that divided in the wild type pattern at the L1 division (2 animals), the L2 asymmetric divisions were again symmetrized towards the hypodermal fate (§). Similar cell fate transformation events were observed in the other V lineages, albeit at lower frequency.
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pone-0114998-g002: TRD-1 regulates cell fate choice in seam lineages.(A) (i) Wild type late L4 animal carrying the integrated seam cell marker, scm::gfp (strain, JR667), has 16 seam cells per side. (ii) trd-1(RNAi) animals have a reduction in the number of seam cells. Seam cells are indicated by white arrowheads. (iii) trd-1(tm2764) homozygous mutants (strain, AW912) have a significant reduction in seam cell number. Scale bar, 20 µm. Anterior is to the left and dorsal up in all images. (B) Graph showing average seam cell number. Wild type animals were exposed to empty RNAi feeding vector as a control. n>100 for both control and trd-1(RNAi) animals. Heterozygotes of genotype hT2/trd-1(tm2764) have normal numbers of seam cells (n = 61), however, trd-1(tm2764) homozygotes have a significant reduction in seam cell number to 11.3 (n = 45). Error bars represent s.e.m. and ** indicate the 2-sample t-test where each strain was compared to the wild type, where p<0.01. (C) (i–iii) L4 Animal (strain, AW1015) carrying an integrated seam cell nuclear marker (scm::tdTomato) along with the pleckstrin homology domain PH::gfp outlining seam cells and a hyp7 nuclear marker dpy-7p::yfp. White arrowheads indicate seam cell nuclei. (iv–vi) trd-1(RNAi) animals carrying the same set of markers. There are obvious gaps in the seam cells, where the seam cell fate has been transformed to hypodermal. Asterisks indicate nuclei that inappropriately express dpy-7::yfp instead of scm::tdTomato. Note that these cells have lost their PH::gfp outline, also indicative of transformation towards the hypodermal fate. ii and v are images of the GFP/YFP channel. iii and vi is the red channel to show the tdTomato in the same animal. i and iv are merged imaged of both GFP/YFP and tdTomato channels. All scale bar, 20 µm. Anterior is to the left and dorsal up in all images. (D) Representative image of a trd-1(RNAi) animal displaying shallow or absent alae. Scale bar, 40 µm (E) Representative lineage trace showing the Vn lineage from hatching to late L2. In wild type animals at the L1 stage, Vn divides asymmetrically with the anterior daughter (Vn.a) adopting the hypodermal fate and the posterior daughter (Vn.p) retaining the proliferative fate. Vn.p will divide symmetrically during early L2 followed closely by a further asymmetric division. Vn.pap and Vn.ppp retain the ability to self-renew and will divide again at L3 and L4. We lineaged 7 trd-1(RNAi) animals, of which 5 had symmetrized the L1 asymmetric division of V2 (asterisk). In animals that divided in the wild type pattern at the L1 division (2 animals), the L2 asymmetric divisions were again symmetrized towards the hypodermal fate (§). Similar cell fate transformation events were observed in the other V lineages, albeit at lower frequency.

Mentions: We confirmed a significant reduction in seam cell number from 16 per side in wild type animals to around 13 in trd-1(RNAi) animals, p<0.01 (Fig. 2A, B). All animals hatched with 10 seam cells per side (data not shown), suggesting no embryonic defects in seam cell identity, prior to the point at which they start dividing. In males, trd-1 RNAi resulted in male tail abnormalities, including a significant reduction in the number of sensory rays from 9 rays in wild type to an average of 4 in trd-1(RNAi) animals, p<0.01, which are known to be derived from posterior seam lineages (S1 Figure). In addition, we observed a low penetrance molting defect in trd-1(RNAi) animals (S1 Figure), another phenotype that may be associated with seam cell defects [27]. RNAi knockdown was confirmed by quantitative real-time PCR (data not shown). Homozygous trd-1 mutants derived from the balanced strain AW912 also displayed a significant reduction in seam cell number in animals that made it through to adulthood, with an average of 11 seam cells per side, p<0.01 (Fig. 2A, B). The significant lethality of trd-1(tm2764) homozygous mutants precluded detailed analysis of the role of trd-1 in development in these animals, thus further analysis was performed on trd-1(RNAi) animals, in which the lethality was less penetrant.


The C. elegans TPR Containing Protein, TRD-1, Regulates Cell Fate Choice in the Developing Germ Line and Epidermis.

Hughes S, Wilkinson H, Gilbert SP, Kishida M, Ding SS, Woollard A - PLoS ONE (2014)

TRD-1 regulates cell fate choice in seam lineages.(A) (i) Wild type late L4 animal carrying the integrated seam cell marker, scm::gfp (strain, JR667), has 16 seam cells per side. (ii) trd-1(RNAi) animals have a reduction in the number of seam cells. Seam cells are indicated by white arrowheads. (iii) trd-1(tm2764) homozygous mutants (strain, AW912) have a significant reduction in seam cell number. Scale bar, 20 µm. Anterior is to the left and dorsal up in all images. (B) Graph showing average seam cell number. Wild type animals were exposed to empty RNAi feeding vector as a control. n>100 for both control and trd-1(RNAi) animals. Heterozygotes of genotype hT2/trd-1(tm2764) have normal numbers of seam cells (n = 61), however, trd-1(tm2764) homozygotes have a significant reduction in seam cell number to 11.3 (n = 45). Error bars represent s.e.m. and ** indicate the 2-sample t-test where each strain was compared to the wild type, where p<0.01. (C) (i–iii) L4 Animal (strain, AW1015) carrying an integrated seam cell nuclear marker (scm::tdTomato) along with the pleckstrin homology domain PH::gfp outlining seam cells and a hyp7 nuclear marker dpy-7p::yfp. White arrowheads indicate seam cell nuclei. (iv–vi) trd-1(RNAi) animals carrying the same set of markers. There are obvious gaps in the seam cells, where the seam cell fate has been transformed to hypodermal. Asterisks indicate nuclei that inappropriately express dpy-7::yfp instead of scm::tdTomato. Note that these cells have lost their PH::gfp outline, also indicative of transformation towards the hypodermal fate. ii and v are images of the GFP/YFP channel. iii and vi is the red channel to show the tdTomato in the same animal. i and iv are merged imaged of both GFP/YFP and tdTomato channels. All scale bar, 20 µm. Anterior is to the left and dorsal up in all images. (D) Representative image of a trd-1(RNAi) animal displaying shallow or absent alae. Scale bar, 40 µm (E) Representative lineage trace showing the Vn lineage from hatching to late L2. In wild type animals at the L1 stage, Vn divides asymmetrically with the anterior daughter (Vn.a) adopting the hypodermal fate and the posterior daughter (Vn.p) retaining the proliferative fate. Vn.p will divide symmetrically during early L2 followed closely by a further asymmetric division. Vn.pap and Vn.ppp retain the ability to self-renew and will divide again at L3 and L4. We lineaged 7 trd-1(RNAi) animals, of which 5 had symmetrized the L1 asymmetric division of V2 (asterisk). In animals that divided in the wild type pattern at the L1 division (2 animals), the L2 asymmetric divisions were again symmetrized towards the hypodermal fate (§). Similar cell fate transformation events were observed in the other V lineages, albeit at lower frequency.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262444&req=5

pone-0114998-g002: TRD-1 regulates cell fate choice in seam lineages.(A) (i) Wild type late L4 animal carrying the integrated seam cell marker, scm::gfp (strain, JR667), has 16 seam cells per side. (ii) trd-1(RNAi) animals have a reduction in the number of seam cells. Seam cells are indicated by white arrowheads. (iii) trd-1(tm2764) homozygous mutants (strain, AW912) have a significant reduction in seam cell number. Scale bar, 20 µm. Anterior is to the left and dorsal up in all images. (B) Graph showing average seam cell number. Wild type animals were exposed to empty RNAi feeding vector as a control. n>100 for both control and trd-1(RNAi) animals. Heterozygotes of genotype hT2/trd-1(tm2764) have normal numbers of seam cells (n = 61), however, trd-1(tm2764) homozygotes have a significant reduction in seam cell number to 11.3 (n = 45). Error bars represent s.e.m. and ** indicate the 2-sample t-test where each strain was compared to the wild type, where p<0.01. (C) (i–iii) L4 Animal (strain, AW1015) carrying an integrated seam cell nuclear marker (scm::tdTomato) along with the pleckstrin homology domain PH::gfp outlining seam cells and a hyp7 nuclear marker dpy-7p::yfp. White arrowheads indicate seam cell nuclei. (iv–vi) trd-1(RNAi) animals carrying the same set of markers. There are obvious gaps in the seam cells, where the seam cell fate has been transformed to hypodermal. Asterisks indicate nuclei that inappropriately express dpy-7::yfp instead of scm::tdTomato. Note that these cells have lost their PH::gfp outline, also indicative of transformation towards the hypodermal fate. ii and v are images of the GFP/YFP channel. iii and vi is the red channel to show the tdTomato in the same animal. i and iv are merged imaged of both GFP/YFP and tdTomato channels. All scale bar, 20 µm. Anterior is to the left and dorsal up in all images. (D) Representative image of a trd-1(RNAi) animal displaying shallow or absent alae. Scale bar, 40 µm (E) Representative lineage trace showing the Vn lineage from hatching to late L2. In wild type animals at the L1 stage, Vn divides asymmetrically with the anterior daughter (Vn.a) adopting the hypodermal fate and the posterior daughter (Vn.p) retaining the proliferative fate. Vn.p will divide symmetrically during early L2 followed closely by a further asymmetric division. Vn.pap and Vn.ppp retain the ability to self-renew and will divide again at L3 and L4. We lineaged 7 trd-1(RNAi) animals, of which 5 had symmetrized the L1 asymmetric division of V2 (asterisk). In animals that divided in the wild type pattern at the L1 division (2 animals), the L2 asymmetric divisions were again symmetrized towards the hypodermal fate (§). Similar cell fate transformation events were observed in the other V lineages, albeit at lower frequency.
Mentions: We confirmed a significant reduction in seam cell number from 16 per side in wild type animals to around 13 in trd-1(RNAi) animals, p<0.01 (Fig. 2A, B). All animals hatched with 10 seam cells per side (data not shown), suggesting no embryonic defects in seam cell identity, prior to the point at which they start dividing. In males, trd-1 RNAi resulted in male tail abnormalities, including a significant reduction in the number of sensory rays from 9 rays in wild type to an average of 4 in trd-1(RNAi) animals, p<0.01, which are known to be derived from posterior seam lineages (S1 Figure). In addition, we observed a low penetrance molting defect in trd-1(RNAi) animals (S1 Figure), another phenotype that may be associated with seam cell defects [27]. RNAi knockdown was confirmed by quantitative real-time PCR (data not shown). Homozygous trd-1 mutants derived from the balanced strain AW912 also displayed a significant reduction in seam cell number in animals that made it through to adulthood, with an average of 11 seam cells per side, p<0.01 (Fig. 2A, B). The significant lethality of trd-1(tm2764) homozygous mutants precluded detailed analysis of the role of trd-1 in development in these animals, thus further analysis was performed on trd-1(RNAi) animals, in which the lethality was less penetrant.

Bottom Line: In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes.In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate.We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Correct cell fate choice is crucial in development. In post-embryonic development of the hermaphroditic Caenorhabitis elegans, distinct cell fates must be adopted in two diverse tissues. In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes. In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate. We have isolated a novel conserved C. elegans tetratricopeptide repeat containing protein, TRD-1, which is essential for cell fate determination in both the germline and the developing epidermis and has homologs in other species, including humans (TTC27). We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate. In the germline, trd-1 RNAi results in a strong masculinization phenotype, as well as defects in the mitosis to meiosis switch. Our data suggests that trd-1 acts downstream of tra-2 but upstream of fem-3 in the germline sex determination pathway, and exhibits a constellation of phenotypes in common with other Mog (masculinization of germline) mutants. Thus, trd-1 is a new player in both the somatic and germline cell fate determination machinery, suggestive of a novel molecular connection between the development of these two diverse tissues.

No MeSH data available.


Related in: MedlinePlus