The WNT target SP5 negatively regulates WNT transcriptional programs in human pluripotent stem cells

Huggins, Ian J.; Bos, Tomas; Gaylord, Olivia; Jessen, Christina; Lonquich, Brianna; Puranen, Angeline; Richter, James; Rossdam, Charlotte; Brafman, David A.; Gaasterland, Terry; Willert, Karl

doi:10.1038/s41467-017-01203-1

Cited by 56 publications

(68 citation statements)

References 51 publications

(56 reference statements)

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“…The identification of motif consensus among the Sp5-enriched regions revealed the same core consensus motif GG(A/T/C)GG, centrally enriched within both the HySp5 and ZfSp5a bound sequences, very similar to the motif described for the human Sp5 protein (Huggins et al, 2017). Two other consensus motifs were found in the HySp5 dataset but neither in the ZfSp5a dataset, nor in the human or mouse Sp5 ChIPseq analyses (Figure 7D -Figure Supplement 9).…”

Section: Identification Of Dna Motifs Bound By the Hydra And Zebrafismentioning

confidence: 52%

An evolutionary-conserved Wnt3/β-catenin/Sp5 feedback loop restricts head organizer activity inHydra

Vogg

Beccari

Olle

et al. 2018

Preprint

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The Hydra polyp regenerates its head by transforming the gastric tissue below the wound into a head organizer made of two antagonistic cross-reacting components. The activator, previously characterized as Wnt3, drives apical differentiation by acting locally and auto-catalytically. The uncharacterized inhibitor, produced under the control of the activator, prevents ectopic head formation. By crossing RNA-seq data obtained in a β-catenin(RNAi) screen performed in planarians and a quantitative analysis of positional and temporal gene expression in Hydra, we identified Sp5 as a transcription factor that fulfills the head inhibitor properties: a Wnt/β-catenin inducible expression, a graded apical-to-basal expression, a sustained up-regulation during head regeneration, a multi-headed phenotype when knocked-down, a repressing activity on Wnt3 expression. In mammalian cells, Hydra and zebrafish Sp5 repress Wnt3 promoter activity while Hydra Sp5 also auto-activates its expression, possibly via β-catenin and/or Tcf/Lef1 interaction. This work identifies Sp5 as a novel potent feedback loop inhibitor of Wnt/β-catenin signaling across eumetazoans.

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Section: Identification Of Dna Motifs Bound By the Hydra And Zebrafismentioning

confidence: 52%

An evolutionary-conserved Wnt3/β-catenin/Sp5 feedback loop restricts head organizer activity inHydra

Vogg

Beccari

Olle

et al. 2018

Preprint

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“…Human embryonic stem (hES) cell H9 (WA09, NIH Registration number 062) cells were obtained from WiCell. Cells were maintained in Essential 8 (E8) media (Chen et al, 2011), with minor modifications, as previously described (Huggins et al, 2017). Plasmids encoding pools of shRNAs for FZD9 and WNT9A were obtains from ABM.…”

Section: Human Embryonic Stem Cell Culture and Hspc Differentiationmentioning

confidence: 99%

“…construct. APEX2-mediated proximity labeling was carried out essentially as described (Hung et al, 2016;Lam et al, 2015). Briefly, confluent 150mm plates of CMV:Fzd9b-5GS-APEX2 cells were treated with biotin-phenol for a total of 30 minutes each, ending at the time of hydrogen peroxide treatment.…”

Section: Apex2-mediated Proximity Labelingmentioning

confidence: 99%

“…Cells were treated with Wnt9a conditioned medium for 1 or 5 minutes, or with WT CHO conditioned medium for 5 minutes. Cells were treated with hydrogen peroxide for 1 minute, quenched, lysed, and biotinylated proteins enriched by streptavidin pulldown, as previously described (Hung et al, 2016).…”

Section: Apex2-mediated Proximity Labelingmentioning

confidence: 99%

“…A series of chimeric receptors between Fzd9b and a Fzd that does not promote Wnt9a signaling reveals that intracellular Fzd9b domains mediate the specificity of this Wnt-Fzd pairing, both in vitro and in vivo, indicating that a transmembrane spanning co-factor is involved in establishing specificity. Using a proximity ligation method followed by mass spectrometry (Hung et al, 2016;Lam et al, 2015), we identified the receptor tyrosine kinase EGFR as required for this specific signaling interaction for both zebrafish and human proteins. Altogether, these results demonstrate a conserved Wnt-Fzd pairing that mediates a precise Wnt cue required for hematopoiesis and opens the field for discovery of other Wnt-Fzd specificity co-regulators.…”

Section: Introductionmentioning

confidence: 99%

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EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

Grainger

Nguyen

Richter

et al. 2018

Preprint

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SummaryThe mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired with in vitro cell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9a in vivo and in vitro to elicit β-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCs in vitro.HighlightsAn in vitro signaling screen identifies Fzd9b as a Wnt9a-specific receptor.Fzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair.WNT9A and FZD9 are required for HSPC derivation from human pluripotent cells in vitro.EGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.

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Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells

et al. 2021

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Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.

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The WNT target SP5 negatively regulates WNT transcriptional programs in human pluripotent stem cells

Cited by 56 publications

References 51 publications

An evolutionary-conserved Wnt3/β-catenin/Sp5 feedback loop restricts head organizer activity inHydra

An evolutionary-conserved Wnt3/β-catenin/Sp5 feedback loop restricts head organizer activity inHydra

EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells

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