FoxO is a critical regulator of stem cell maintenance in immortal Hydra

Abstract

Hydra's unlimited life span has long attracted attention from natural scientists. The reason for that phenomenon is the indefinite self-renewal capacity of its stem cells. The underlying molecular mechanisms have yet to be explored. Here, by comparing the transcriptomes of Hydra's stem cells followed by functional analysis using transgenic polyps, we identified the transcription factor forkhead box O (FoxO) as one of the critical drivers of this continuous self-renewal. foxO overexpression increased interstitial stem cell and progenitor cell proliferation and activated stem cell genes in terminally differentiated somatic cells. foxO down-regulation led to an increase in the number of terminally differentiated cells, resulting in a drastically reduced population growth rate. In addition, it caused down-regulation of stem cell genes and antimicrobial peptide (AMP) expression. These findings contribute to a molecular understanding of Hydra's immortality, indicate an evolutionarily conserved role of FoxO in controlling longevity from Hydra to humans, and have implications for understanding cellular aging.

Fig. 1.

Stem cells in Hydra are continuously self-renewing. (A) Schematic phylogenetic tree showing the main branches in metazoan evolution. (B) Scheme of the stem cell compartment in Hydra with sharp boundaries towards terminally differentiated head and foot tissue. (C) The major cell types in Hydra. Stem cell lineages are colored, with derivatives of the interstitial cell lineage in gray. (D) The three independent stem cell systems in Hydra. Both epithelial cell lineages represent unipotent stem cells whereas interstitial stem cells exhibit multipotent features as they are able to differentiate into various derivatives. diff, differentiation ecto, ectoderm; ECM, extracellular matrix; ecto epi, ectodermal epithelial cell; endo, endoderm; endo epi, endodermal epithelial cell; gld, gland cell; i-cell, interstitial stem cell; nv, nerve cell; nem, nematocyte.

Fig. 2.

foxO is expressed in all three stem cell types in Hydra. (A) Venn diagram of conserved transcription factors being expressed in all three stem cell types according to 454 transcriptome sequencing. (B) Reads from 454 sequencing for foxO, cnvas1, and cniwi. (C–E) qRT-PCR reveals foxO expression in (C) all three stem cell lineages, (D) both tissue layers, and (E) in the body column (n = 2 replicates). Expression in endodermal epithelial cells, endodermal tissue layer, and foot tissue was used as calibrator. (F–H) In situ hybridization shows that foxO is expressed in both tissue layers along the body axis and absent from terminally differentiated cells in head, foot, and (H) gonads. (I) Maximum-parsimony phylogram of the forkhead domain from selected FoxO proteins rooted using Mus musculus FoxA1 and Hydra FoxA2. Numbers at nodes are bootstrap support values calculated by 1,000 replicates of maximum parsimony. Bootstrap values under 50 are not shown. Aa: Aedes aegypti; Ad: Acropora digitifera; Am: Acropora millepora, Aq: Amphimedon queenslandica; Bf: Branchiostoma floridae; Ce: C. elegans; Ch: Clytia hemisphaerica; Ci: Ciona intestinalis; Dm: Drosophila melanogaster; Dr: Danio rerio; Gg: Gallus gallus; Hs: Homo sapiens; HvAEP: H. vulgaris strain AEP; Mm: M. musculus; Ms: Metridium senile; Nv: Nematostella vectensis; Sp: Strongylocentrotus purpuratus; Ta: Trichoplax adhaerens; Xl: Xenopus laevis; and Xm: Xiphophorus maculates. (Scale bar in G: 20 µm.)

Fig. 3.

foxO overexpression. (A) Construct used for foxO overexpression. (B–D) foxO overexpressing (B) interstitial stem cells and (C and D) groups of nematoblasts. (E) foxO expression levels in ectodermal tissue layer of foxO⁺ polyps (A4 and C2 line), analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: A4 line = 0.014, C2 line = 0.0005. (F) BrdU-labeling index of interstitial stem cells (1s + 2s) and nematoblast progenitor cells (4s, 8s) in foxO⁺ (A4 line) and control polyps after 6 h exposure to BrdU (n^1s+2s = ∼1,000, n^4s = ∼200, n^8s = ∼100 per replicate; n = 3 replicates). Asterisks indicate significant changes in cell numbers (t test); P values: 1s + 2s = 0.0265, 4s = 0.0034, 8s = 0.0136. (G) Numbers of interstitial stem cells (1s + 2s) and nematoblasts (4s, 8s) per 100 epithelial cells in foxO⁺ (A4 line) and control polyps (n^{epithelial cells} = ∼1,000; n = 3 replicates). Asterisks indicate significant changes in cell numbers (t test); P values: 1s + 2s = 0.0016, 4s = 0.0078, 8s = 0.0474. (H) Expression levels of stem cell genes in ectodermal tissue layer of foxO⁺ polyps (A4 and C2 line) analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: cnvas1^{A4 line} = 0.0238, cnvas1^{C2 line} = 0.0031. (I–P) Douple in situ hybridization of cniwi and mc17 in (I–L) control and (M–P) foxO⁺ (A4 line) polyps. (I and J) Mc17-positive nematoblasts and cniwi-positive interstitial stem cells. (K) Mc17-positive nematoblasts. (L) Cniwi-negative nematocyte. (M–O) Coexpression of mc17 and cniwi in nematoblasts. (P) Cniwi-positive nematocyte. (Scale bars: B–D, K, and O—10 µm; J and N—20 µm; L and P—5 µm.) cyt, cytoplasm; ic, interstitial stem cells; nb, nematoblasts; nc, nematocyst.

Fig. 4.

foxO down-regulation. (A) Construct used for foxO down-regulation. (B) foxO expression levels in endodermal tissue layer of foxO⁻ endo polyps, ectodermal tissue layer of foxO⁻ ecto polyps, and total tissue of foxO⁻ ecto/endo polyps, analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: endo A6 line = <0.0001, endo D11a line = 0.0087, endo E1 line = 0.0032, ecto D11a line = 0.0123, and ecto/endo line = 0.0008. (C–F) In situ hybridization of pedibin in FoxO⁻ endo, foxO⁻ ecto, foxO⁻ ecto/endo and control polyps. (G) Foot length compared with body length in foxO⁻ and control polyps, as determined by pedibin labeling (n^{control endo} = 21, n^{control ecto} = 12, n^{endo A6 line} = 17, n^{endo D11a line} + n^{endo E1 line} = 22, n^{ecto D11a line} + n^{ecto D11b line} = 18, and n^{ecto/endo line} = 15). Asterisks indicate significant differences in foot length compared with control (t test); P values: all <0.0001. (H) Growth curves (n = 10 replicates à five polyps each) and (I) time of bud development (n = 10 replicates) of foxO⁻ endo (A6 line) and control polyps. Asterisks indicate significant differences in growth (t test); P value = <0.0001. (J) Expression levels of stem cell genes in foxO⁻ ecto/endo polyps analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: cnvas1 = 0.0021, cniwi = 0.0054. (K) Expression levels of AMPs in foxO⁻ endo polyps analyzed by qRT-PCR (n = 3 replicates). Asterisks indicate significant changes in expression levels (t test); P values: hydramacin^{A6 line} = 0.0001, periculin2b^{A6 line} = 0.0316, hydramacin^{D11a line} = 0.0031, hydramacin^{E1 line} = 0.0011, and periculin2b^{E1 line} = 0.0136.

Fig. 5.

Model of the role of FoxO in controlling longevity. Decline of foxO expression results in aging and death. Mutations in DAF-16 and dfoxO reduce life span in both C. elegans and flies. Increase of foxO expression delays aging by maintaining stem cell self-renewal and functionality of the immune system. Universally expressed foxO in Hydra results in a continuous self-renewal capacity of stem cells and immortality.