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Cited by 36Genetic and Kidney Cyst DiseasesHedgehog Signaling Pathway StudiesCellular transport and secretion

The DOI matches its Crossref record; 126 of the 126 references it cites were found in a registry or an index; 1 of the 47 sources its claims cite were checked for support.

Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

Open access
Bhattacharyya, Sohinee, Rainey, Mark A, Arya, Priyanka, Mohapatra, Bhopal C., Mushtaq, Insha, Dutta, Samikshan, George, Manju, Storck, Matthew D., McComb, Rodney D., Muirhead, David, Todd, Gordon L., Gould, Karen, Datta, Kaustubh, Waes, Janee Gelineau-van, Band, Vimla, Band, Hamid·2016·Scientific Reports
DOI 10.1038/srep20727PMC PMC4756679
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Abstract

Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1 -null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1 -null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1 -null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1 -null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

EHD1 loss in mice on a predominantly B6 background causes mid-gestational embryonic lethality with neural tube closure defects, short primary cilia, and increased SHH signaling. EHD1 localizes to primary cilia upon SHH activation, co-traffics with and directly binds Smoothened, identifying EHD1 as a regulator of ciliary Smoothened trafficking and Hedgehog signaling.

Key findings

  • Ehd1 deletion on a predominantly B6 background (98% B6) is embryonic lethal: Ehd1-null embryos were present at or near expected Mendelian ratios at E9.5 and E10.5 but absent at E11.5, placing death between E10.5 and E11.5; E9.5 null embryos were smaller, had open neural tubes, failed axial rotation, and had fewer somites (12–18 vs. 21–25 in controls).
  • E9.5 Ehd1-null neuroepithelium had short, stubby primary cilia; by TEM, all imaged WT primary cilia looked normal, whereas only 2 of 30 imaged Ehd1-null cilia resembled WT. Ehd1-null MEFs also had significantly shorter cilia (mean ± SEM 1.710 ± 0.09775, N = 51 vs. 2.872 ± 0.1121, N = 39; p ≤ 0.0001).
  • SHH signaling was deregulated: Ehd1-null embryos had significantly reduced GLI3 repressor protein (p = 0.0002), comparable GLI1/GLI2 levels, no significant changes in Gli1/Gli2/Gli3 mRNA, increased ventral markers Foxa2 and Nkx6.1, and reduced dorsal markers Pax6 and Pax7. EHD1-null MEFs showed reduced GLI3 repressor after SAG stimulation (p < 0.05) and elevated GLI3R under unstimulated conditions (p = 0.0002).
  • EHD1 localized to primary cilia in 5% of serum-starved WT MEFs without SHH activation and in 90% after SAG treatment; it co-localized with SMO in 70% of WT MEF cilia after SHH activation. EHD1 co-trafficked with SMO into cilia, co-immunoprecipitated with SMO, and GST-EHD1 pulled down SMO, while GST-EHD1 lacking the EH domain did not; SMO localized to cilia without SHH stimulation in EHD1-null MEFs and was further enriched upon stimulation.

Methods

  • Ehd1 knockout mice were backcrossed to C57BL/6J for five generations and determined to be 98% B6 by whole-genome SNP analysis; timed matings, yolk sac PCR genotyping, and somite staging were used, with embryo analyses at E8.5–P1 and staged E9.5/E10.5/E11.5.
  • Primary cilia and SHH readouts used SEM/TEM, immunofluorescence, western blot, and qRT-PCR on E9.0–E9.5 embryos; MEFs from Ehd1flox/flox E9.5 embryos were rendered Ehd1−/− by Cre/GFP adenovirus and FACS, then serum-starved ± SAG for ciliogenesis and SHH activation.
  • SMO–EHD1 interaction/trafficking was tested by co-immunoprecipitation, GST-pulldown with GST-EHD1 or EH-domain-deleted GST-EHD1, and live-cell imaging in NIH3T3 cells stably expressing SMO-GFP plus DsRed EHD1; statistics used t-test or ANOVA with Tukey post-test, with p < 0.05 significant.

Limitations

  • Stated: SEM quantification was difficult because malformed Ehd1-null cilia were sparse and numerous filopodia-like extensions made it hard to distinguish cilia; TEM quantitation was also challenging, with only 30 Ehd1-null cilia imaged and only 2 resembling WT.
  • Stated: the mechanism of EHD1–SMO interaction remains to be tested, including future studies with NPF/DPF mutant SMO; the exact shared role of EHD1 and EHD4 in cilia formation and SHH signaling remains to be elucidated, and further ciliary proteome analysis is needed.
  • Evident: in vivo analyses are limited to pre-lethal stages because Ehd1-null embryos die between E10.5 and E11.5; EHD1-null MEFs could not be maintained beyond three passages, so isogenic Cre-deleted floxed MEFs were used, and the EHD4 reduction seen in Ehd1-null embryos was not observed in Ehd1-null MEFs, suggesting the cell model may not fully replicate the embryonic context.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/srep20727

Cites

126 of 126 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

1 of 47 cited sources checked · 0 supported · 0 flagged · accuracy not yet measured

  • Findingown data

    Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at E9.5.

    “We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5).”
    • Also stated in: § Aberrant primary cilia morphology in the Ehd1-null neuroepithelium · plain
      “In contrast, neuroepithelial cells of Ehd1-null embryos exhibited substantially shorter stubby and bulbous ciliary structures projecting from apparently normal ciliary pockets (Fig. 4A).”
    • Also stated in: § Aberrant primary cilia morphology in the Ehd1-null neuroepithelium · plain
      “In contrast, the cilia on Ehd1-null neuroepithelial cells were smaller and barely projected out of the ciliary pocket (Fig. 4B).”
    Quote in the paperSubstantial claim
  • Findingown data

    Ehd1-null embryos were present at or near expected Mendelian ratios at E9.5 and E10.5.

    “While Ehd1-null embryos were present at or near the expected Mendelian ratios at E9.5 and E10.5”
    Quote in the paperSubstantial claim
  • Findingown data

    EHD3 and EHD2 protein levels are comparable between Ehd1-null and control embryo lysates at E9.5.

    “Surprisingly, a significant reduction (p = 0.0038) in the level of EHD4 protein was seen in Ehd1-null compared to control embryo lysates, while the levels of EHD3 and EHD2 were comparable between Ehd1-null and control embryo lysates (Fig. 3B).”
    Quote in the paperNot a substantial claim
  • Findingown data

    EHD2 expression is first detectable at E9.5.

    “EHD1, EHD3 and EHD4 were detectable at E8.5 (the earliest time point examined), whereas the expression of EHD2 was first detectable at E9.5 (Fig. 2).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal.

    “In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal.”
    • Also stated in: § EHD1 deletion in a predominantly B6 background leads to mid-gestational embryonic lethality with major developmental defects · boosted
      “Collectively, these analyses demonstrated that Ehd1 deficiency on a predominantly B6 background is embryonic lethal around day E11, and major defects in neural and cardiac development, axial rotation and somitogenesis precede”
    Quote in the paperSubstantial claim
  • Probably not a substantial claim. 28
  • It may state two separate claims as one. 33

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    EHD1 can bind to the Rab8 effector MICAL-L1 and aid in membrane tubulation and vesicle scission.

    “EHD1 can bind to the Rab8 effector MICAL-L1 and aid in membrane tubulation and vesicle scission1119.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [11] Pant, S. et al. AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling. Nat Cell Biol 11, 1399–1410, 10.1038/ncb1986 (2009). source

      The Discussion (page unavailable in our copy) suggests that RME-1/EHD-family proteins function in membrane tubulation and potentially membrane fission, with AMPH-1 as the interacting partner. The paper does not identify MICAL-L1 or describe EHD1 binding to it; searches for “MICAL-L1” and “Rab8 effector” found no such passage. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [19] Sharma, M., Giridharan, S. S., Rahajeng, J., Naslavsky, N. & Caplan, S. MICAL-L1 links EHD1 to tubular recycling endosomes and regulates receptor recycling. Mol Biol Cell 20, 5181–5194, 10.1091/mbc.E09-06-0535 (2009). source

      Checked before — its earlier verdict is held back until a new check runs.

  • It may state two separate claims as one.

    Findingown data

    EHD2 levels start to increase sharply at day 14.5 and continue to increase further through gestation.

    “EHD2 levels started to increase sharply at day 14.5 and continued to increase further through gestation.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Ehd1-null embryos display fewer somites than littermate controls (12-18 vs. 21-25), indicating impaired somitogenesis.

    “Ehd1-null embryos also displayed fewer somites than littermate controls (12–18 vs. 21–25 for control embryos), indicating impaired somitogenesis (Fig. 1A).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Ehd1-null embryos display delayed heart development; all E9.5 Ehd1-null embryos exhibited a beating heart, but heart chambers were not developed as well as in controls.

    “Ehd1-null embryos also displayed delayed heart development; while all E9.5 Ehd1-null embryos exhibited a beating heart, the heart chambers were not developed as well as in controls (data not shown).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    EHD3 shows a distinct perinuclear localization in both neuroepithelial and cranial mesenchyme cells.

    “In contrast, EHD3 shows a distinct, perinuclear localization in both the neuroepithelial and the cranial mesenchyme cells (Fig. 3A).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    The Ehd1-null allele was bred into the B6 background for five generations, and the mice were determined to be 98% B6 by whole genome strain-specific marker analysis.

    “We bred the Ehd1-null allele further into B6 background for 5 generations and determined these mice to be 98% B6 by whole genome strain-specific marker analysis (Supplementary Fig. 1).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Heterozygote crosses in the predominantly B6 background yielded only WT and heterozygous pups, with no Ehd1-null pups observed in multiple independent matings.

    “Heterozygote crosses in this background yielded only WT and heterozygous pups and no Ehd1-null pups were observed in multiple independent mating.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Ehd1-null embryos fail to complete neural tube closure and display an open neural tube.

    “Ehd1-null embryos were smaller, displayed an open neural tube and showed failure of axial rotation.”
    • Also stated in: § Abstract · plain
      “Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    EHD4 expression shows a pattern similar to that of EHD1.

    “EHD4 expression showed a pattern similar to that of EHD1.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    EHD1 levels progressively increase to peak at E14.5 and remain high through the remainder of gestation into P1.

    “EHD1 levels progressively increased to peak at E14.5 and remained high through the remainder of gestation into P1.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingcitation

    WT neuroepithelial cells have a large number of smaller, non-ciliary filopodia-like cytoplasmic extensions.

    “In addition, a large number of smaller, non-ciliary filopodia like cytoplasmic extensions were seen, as reported by others56.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Jacinto, A. et al. Dynamic actin-based epithelial adhesion and cell matching during Drosophila dorsal closure. Curr Biol 10, 1420–1426 (2000). source

      Checked before — its earlier verdict is held back until a new check runs.

  • It may state two separate claims as one.

    Findingown data

    The expression pattern of NKX2.2 showed marked increase in the expression domain at the lumbar arch level and some NKX2.2 positive cells were found dispersed dorsally in Ehd1-null compared to control embryos.

    “The expression pattern of V3 interneuron progenitor marker NKX2.2 also showed marked increase in the expression domain at the lumbar arch level and some NKX2.2 positive cells were found dispersed dorsally in Ehd1-null compared to the control embryos.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Ehd1-null embryos displayed severe developmental defects prior to death, including open neural tubes, failure to undergo axial rotation, delayed somitogenesis and defective cardiac development.

    “Ehd1-null embryos displayed severe developmental defects prior to death, including open neural tubes, failure to undergo axial rotation, delayed somitogenesis and defective cardiac development”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    WT neuroepithelium displays well-developed primary cilia projecting from ciliary pockets, with a well-defined basal body linking the ciliary membrane with the plasma membrane.

    “WT neuroepitheliam displayed well-developed primary cilia projecting from ciliary pockets, with a well-defined basal body linking the ciliary membrane with the plasma membrane57.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [57] Sorokin, S. P. Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J Cell Sci 3, 207–230 (1968). source

  • It may state two separate claims as one.

    Findingown data

    There was no significant change in GLI2 localization to the entire cilia under basal conditions and GLI2 enrichment at the ciliary tips after SHH pathway activation in EHD1-null MEFs.

    “there was no significant change in GLI2 localization to the entire cilia under basal conditions and GLI2 enrichment at the ciliary tips after SHH pathway activation (Fig. 10A) in EHD1-null MEFS”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    EHD2 and EHD3 staining intensity and localization are unchanged in Ehd1-null embryos compared to WT controls.

    “As noticed in Western blots, the intensity of EHD2 and EHD3 staining remained unchanged, and their localization was also comparable to the WT control embryos (Fig. 3A).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    In EHD1-null MEFs, SMO localizes to the primary cilium without SHH stimulation and is further enriched upon SHH stimulation.

    “found SMO localized to the cilium in null MEFS without SHH stimulation (Fig. 10B) with further enrichment upon SHH stimulation (Fig. 10B)”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    EHD family protein expression begins early during embryogenesis, with multiple members expressed as early as E8.5 and substantial developmentally regulated expression changes during the second half of gestation.

    “These findings indicate that the EHD family of protein expression begins early during embryogenesis and multiple members are expressed as early as E8.5, with substantial developmentally-regulated expression changes during the second half of gestation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    The early expression of EHD1 is consistent with a role in embryogenesis.

    “The early expression of EHD1 is consistent with a role in embryogenesis, as revealed by phenotypic studies mentioned above.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationown data

    EHD family members are prominently expressed in the neural tube of developing embryos, and EHD1 and EHD4, and to a certain extent EHD2, show polarized distribution underneath the apical membrane of polarized neuroepithelial cells.

    “These studies show that EHD family members are prominently expressed in the neural tube of developing embryos, and EHD1 and 4, and to a certain extent EHD2, show polarized distribution underneath the apical membrane of polarized neuroepithelial cells.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    Loss of EHD1 deregulates ciliary SHH signaling, with Ehd1-null embryos displaying features indicative of increased SHH signaling.

    “Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.

    Interpretationreasoning

    The elevated GLI3R in unstimulated EHD1-null MEFs presumably prevents hyperactivation of the pathway in the absence of ligand.

    “presumably to prevent the hyper activation of the pathway in the absence of ligand”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    Overexpressed EHD1 strongly interacts with EHD4, and a complex of endogenous EHD1 and EHD4 can be immuno-isolated from cells.

    “In keeping with this notion, it has been shown that overexpressed EHD1 strongly interacts with EHD4 and a complex of endogenous EHD1 and EHD4 can be immuno-isolated from cells41.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Sharma, M., Naslavsky, N. & Caplan, S. A role for EHD4 in the regulation of early endosomal transport. Traffic 9, 995–1018, 10.1111/j.1600-0854.2008.00732.x (2008). source

  • It may state two separate claims as one.

    Interpretationown data

    A prominent manifestation of EHD1 deficiency was a neural tube closure defect associated with defective ciliogenesis, aberrantly increased SHH signaling and altered dorso-ventral neural identities.

    “A prominent manifestation of EHD1 deficiency was a neural tube closure defect associated with defective ciliogenesis, aberrantly increased SHH signaling and altered dorso-ventral neural identities.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Premisecitation

    Aberrations in the structural, trafficking and signaling components of primary cilia lead to developmental disorders, including neural tube closure defects.

    “Prior studies have demonstrated that aberrations in the structural, trafficking and signaling components of primary cilia lead to developmental disorders, including neural tube closure defects434445.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites 434445

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    SHH, secreted by the notochord and floor plate, forms a morphogen gradient from ventral to dorsal neural tube and, in conjunction with other morphogens, specifies neuronal cell fates.

    “SHH, secreted by the notochord and floor plate, forms a morphogen gradient from ventral to dorsal neural tube and, in conjunction with concentration gradients of other morphogens such as Nodal and BMPs, specifies neuronal cell fates”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [59] Ruiz i Altaba, A., Nguyen, V. & Palma, V. The emergent design of the neural tube: prepattern, SHH morphogen and GLI code. Curr Opin Genet Dev 13, 513–521 (2003). source
    • cites [60] Wilson, L. & Maden, M. The mechanisms of dorsoventral patterning in the vertebrate neural tube. Dev Biol 282, 1–13, 10.1016/j.ydbio.2005.02.027 (2005). source
    • cites [61] Stamataki, D., Ulloa, F., Tsoni, S. V., Mynett, A. & Briscoe, J. A gradient of Gli activity mediates graded Sonic Hedgehog signaling in the neural tube. Genes Dev 19, 626–641, 10.1101/gad.325905 (2005). source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim.

    Premisecitation

    Recent studies have established that primary cilia are hubs for SHH signaling.

    “Recent studies have established that primary cilia are hubs for SHH signaling96.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [96] Goetz, S. C. & Anderson, K. V. The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet 11, 331–344, 10.1038/nrg2774 (2010). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Genetic background has long been known to influence observed phenotypic expression across organisms.

    “genetic background has long been known to influence observed phenotypic expression across organisms”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [71] Remold, S. K. & Lenski, R. E. Pervasive joint influence of epistasis and plasticity on mutational effects in Escherichia coli. Nat Genet 36, 423–426, 10.1038/ng1324 (2004). source
    • cites [72] Dowell, R. D. et al. Genotype to phenotype: a complex problem. Science 328, 469, 10.1126/science.1189015 (2010). source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [73] Threadgill, D. W. et al. Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 269, 230–234 (1995). source

  • It may state two separate claims as one.

    Interpretationown data

    EHD protein expression is completely absent in Ehd1-null embryonic neural tubes, and EHD4 expression in the neural tube is markedly reduced.

    “The staining and blotting data together reveal that EHD protein expression is completely absent in Ehd1-null embryonic neural tubes, and for reasons that are not understood, the expression of EHD4 in the neural tube is markedly reduced.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    EHD1 is a novel player in regulating ciliogenesis and SHH signaling.

    “establishing EHD1 as a novel player in regulating ciliogenesis and SHH signaling”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Endocytic proteins including members of the Rab and Arf/Arl subfamilies of small GTPases, such as Rab8, Rab11, Rab23, Arl6 and Arl13b, are implicated in ciliary function.

    “direct links between vesicle trafficking and primary cilium formation and function have emerged recently from studies that have implicated endocytic proteins including members of the Rab and Arf/Arl subfamilies of the Ras superfamily of small GTPases, such as Rab8, Rab11, Rab23, Arl6, Arl13b in ciliary function46474849505152535455.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites 46474849505152535455

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim.

    Premisecitation

    Primary cilia function as a hub of SHH signaling during neural tube development.

    “Primary cilia, non-motile microtubule-based membrane projections found on nearly every vertebrate cell, function as a hub of SHH signaling during neural tube development4243.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4243]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Interpretationreasoning

    Loss of EHD1 selectively controls the generation or stability of GLI3 repressor, and the unchanged GLI1/2 levels in the face of reduced GLI3 suppressor levels is perceived by developing neurons as an increase in SHH signaling.

    “It is plausible that loss of EHD1 selectively controls the generation or stability of GLI3 repressor, and the unchanged GLI1/2 levels in the face of reduced GLI3 suppressor levels is perceived by developing neurons as an increase in SHH signaling.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    When SHH ligand is present, SHH binds Ptch1, causing it to move out of the cilium, and this allows SMO to enter the cilia.

    “When SHH ligand is present, SHH binds Ptch1, causing it to move out of the cilium, and this allows SMO to enter the cilia6567.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [65] Corbit, K. C. et al. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021, 10.1038/nature04117 (2005). source
    • cites [67] Rohatgi, R., Milenkovic, L. & Scott, M. P. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376, 10.1126/science.1139740 (2007). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Patched (Ptch1) is found in the ciliary membrane and inhibits pathway activation in the absence of ligand by repressing SMO.

    “The repressor for the SHH signaling pathway, Patched (Ptch1), is also found in the ciliary membrane, and inhibits pathway activation in the absence of ligand by repressing the downstream activator, SMO67.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [67] Rohatgi, R., Milenkovic, L. & Scott, M. P. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376, 10.1126/science.1139740 (2007). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    In the absence of ligand, GLI2 and GLI3 are localized to the tips of cilia and are processed to form transcriptional repressors.

    “In the absence of ligand, the GLI transcription factors GLI2 and GLI3 are localized to the tips of cilia and are processed to form transcriptional repressors66119120.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [66] Haycraft, C. J. et al. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet 1, e53, 10.1371/journal.pgen.0010053 (2005). source
    • cites [119] Huangfu, D. & Anderson, K. V. Cilia and Hedgehog responsiveness in the mouse. Proc Natl Acad Sci USA 102, 11325–11330, 10.1073/pnas.0505328102 (2005). source
    • cites [120] Liu, A., Wang, B. & Niswander, L. A. Mouse intraflagellar transport proteins regulate both the activator and repressor functions of Gli transcription factors. Development 132, 3103–3111, 10.1242/dev.01894 (2005). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationcitation

    The novel role of the endocytic recycling pathway regulator EHD1 in ciliary trafficking and signaling described here is consistent with emerging evidence implicating a number of vesicular trafficking proteins in primary ciliogenesis.

    “The novel role of the endocytic recycling pathway regulator EHD1 in ciliary trafficking and signaling we describe here is consistent with emerging evidence implicating a number of vesicular trafficking proteins in primary ciliogenesis”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    The N-terminal G domain of EHD1 is required for ATP binding and membrane recruitment.

    “The N-terminal G domain of EHD1 is required for ATP binding and membrane recruitment”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Grant, B. D. & Caplan, S. Mechanisms of EHD/RME-1 protein function in endocytic transport. Traffic 9, 2043–2052, 10.1111/j.1600-0854.2008.00834.x (2008). source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    EHD1 regulates the Rab11 endosome recycling compartment trafficking and binds to Rab11-FIP2, a Rab11 effector.

    “EHD1 regulates the Rab11 endosome recycling compartment trafficking and binds to Rab11-FIP2, a Rab11 effector118.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [118] Naslavsky, N., Rahajeng, J., Sharma, M., Jovic, M. & Caplan, S. Interactions between EHD proteins and Rab11-FIP2: a role for EHD3 in early endosomal transport. Mol Biol Cell 17, 163–177, 10.1091/mbc.E05-05-0466 (2006). source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim. It may state two separate claims as one.

    Premisecitation

    Almost all components of the Sonic hedgehog (SHH) signaling pathway are localized to the cilium, and their localization shifts in response to the SHH ligand.

    “Almost all components of the Sonic hedgehog (SHH) signaling pathway are localized to the cilium, and their localization shifts in response to the SHH ligand6566676869.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [65] Corbit, K. C. et al. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021, 10.1038/nature04117 (2005). source
    • cites [66] Haycraft, C. J. et al. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet 1, e53, 10.1371/journal.pgen.0010053 (2005). source
    • cites [67] Rohatgi, R., Milenkovic, L. & Scott, M. P. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376, 10.1126/science.1139740 (2007). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The central region of EHD1 is required for dimerization/oligomerization.

    “the central region for dimerization/oligomerization”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Grant, B. D. & Caplan, S. Mechanisms of EHD/RME-1 protein function in endocytic transport. Traffic 9, 2043–2052, 10.1111/j.1600-0854.2008.00834.x (2008). source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim.

    Premisecitation

    The C-terminal EH domain of EHD1 binds to NPF motifs in a number of proteins.

    “the C-terminal EH domain for binding to NPF motifs in a number of proteins18”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [18] Grant, B. D. & Caplan, S. Mechanisms of EHD/RME-1 protein function in endocytic transport. Traffic 9, 2043–2052, 10.1111/j.1600-0854.2008.00834.x (2008). source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Rab23 mutant cells have an accumulation of SMO into the cilium.

    “Rab23 mutant cells were shown to have an accumulation of SMO into the cilium123.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [123] Boehlke, C. et al. Differential role of Rab proteins in ciliary trafficking: Rab23 regulates smoothened levels. J Cell Sci 123, 1460–1467, 10.1242/jcs.058883 (2010). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    A recent study defined a role for EHD1 at a distinct step of ciliogenesis, in pre-ciliary vesicle formation during the very early stages of ciliogenesis, through interactions with Rab8/11 small GTPases.

    “A recent study defined a role for EHD1 at a distinct step of ciliogenesis, in pre-ciliary vesicle formation during the very early stages of ciliogenesis, through interactions with Rab8/11 small GTPases70.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [70] Lu, Q. et al. Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat Cell Biol 17, 228–240, 10.1038/ncb3109 (2015). source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Intraflagellar transport (IFT) is required for targeting and moving SHH signaling proteins in and out of the cilium.

    “Precisely how SHH signaling proteins are targeted and moved in and out of the cilium is not clear, but intraflagellar transport (IFT) is required66120122.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [66] Haycraft, C. J. et al. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet 1, e53, 10.1371/journal.pgen.0010053 (2005). source
    • cites [120] Liu, A., Wang, B. & Niswander, L. A. Mouse intraflagellar transport proteins regulate both the activator and repressor functions of Gli transcription factors. Development 132, 3103–3111, 10.1242/dev.01894 (2005). source
    • cites [122] Huangfu, D. et al. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426, 83–87, 10.1038/nature02061 (2003). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Rab23 negatively regulates the SHH signaling pathway during neural tube formation.

    “the ENU-induced Rab23 mutations in mice revealed that Rab23 negatively regulates the SHH signaling pathway during neural tube formation103”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [103] Eggenschwiler, J. T., Espinoza, E. & Anderson, K. V. Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway. Nature 412, 194–198, 10.1038/35084089 (2001). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisenone given

    In IFT122 mutant mice, retrograde transport is impaired and GLI2 and SMO accumulate in the cilia even in the absence of SHH signaling.

    “In these mice, retrograde transport is impaired and GLI2 and SMO accumulate in the cilia even in the absence of SHH signaling.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    SMO localization to the cilium inhibits GliR formation and, via an unknown mechanism, the full-length GLIs become GLI activators.

    “SMO localization to the cilium inhibits GliR formation and, via an unknown mechanism, the full-length GLIs become GLI activators121.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [121] McMahon, A. P., Ingham, P. W. & Tabin, C. J. Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol 53, 1–114 (2003). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds. It may state two separate claims as one.

    Premisenone given

    Anterograde IFT carries cargo toward the tip of the cilium, while retrograde transport carries turnover products out of the cilium; deletion of anterograde or retrograde IFT proteins results in distinct ciliary phenotypes.

    “Anterograde IFT carries cargo toward the tip of the cilium, while retrograde transport carries turnover products out of the cilium; deletion of anterograde or retrograde IFT proteins results in distinct ciliary phenotypes.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    EHD1 plays a role in the fine regulation of the balance between trafficking of SHH signaling proteins into and out of the cilium, which is critical for maintaining the correct ratio of GLI activators to GLI repressors and proper activation of the SHH pathway during development and other processes.

    “Our results point to a role of EHD1 in the fine regulation of the balance between trafficking of SHH signaling proteins into and out of the cilium which in turn is critical for maintaining the correct ratio of GLI activators to GLI repressors and proper activation of the SHH pathway during development and other processes.”
    Quote in the paperSubstantial claim
50 of 50

  • The paper relies on this without support.

    The antibodies used in immunostaining and immunoblotting specifically detect the intended proteins.

    “Immunostaining for Arl13B, a specific marker of primary cilia58, showed punctate staining of the embryonic neuroepithelium in WT embryos”

  • The paper relies on this without support.

    The primary cilia imaged by TEM in Ehd1-null embryos are representative of the primary cilia population despite sparse formation.

    “This too proposed a challenge due to sparse cilia formation in the Ehd1-null neuroepithelium but we were able to distinguish primary cilia based on their axonemal architecture.”

  • The paper relies on this without support.

    SAG treatment specifically activates the SHH pathway.

    “treated with SAG for another 24 h to activate the SHH pathway”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • All EHD proteins are said to be absent despite detected EHD2, EHD3 and EHD4

    Minor

    The paper says EHD protein expression is completely absent in Ehd1-null neural tubes, but the same paragraph reports unchanged EHD2 and EHD3 staining and remaining EHD4 staining.

1 of 1

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Metadata

Authors
Bhattacharyya, S., Rainey, M. A., Arya, P., Mohapatra, B. C., Mushtaq, I., Dutta, S., George, M., Storck, M. D., McComb, R. D., Muirhead, D., Todd, G. L., Gould, K., Datta, K., Waes, J. G.-V., Band, V., & Band, H.
Affiliations
  • University of Nebraska Medical Center (US)
  • Creighton University (US)
  • Fred and Pamela Buffett Cancer Center (US)
Corresponding author
Band, Hamid
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2016-02-17
Volume
6
Issue
1
Pages
20727-20727
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/srep20727
PMC
PMC4756679
OpenAlex
W2296334129
Semantic Scholar
c3c8a76b7cb67c53ed1064f6e871013406108c03
Cited by
36
References
126
Citations by year
2024: 2 · 2023: 3 · 2022: 4 · 2021: 2 · 2020: 5 · 2019: 4 · 2018: 10 · 2017: 4 · 2016: 2
FWCI
3.89
Type
journal article
Language
en
Update notices
correction (2017-03-23)
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • U.S. Department of Defense (W81XWH1110171, W81XWH-11-1-, W81XWH, W81XWH0710351)
  • U.S. Department of Health and Human Services (LB606, P30 GM106397, LB506)
  • Memorial Sloan-Kettering Cancer Center
  • Nebraska Department of Health and Human Services (18123-Y3, LB606, LB506, 2014-01)
  • National Institutes of Health (W81XWH, LB606)
  • National Cancer Institute (R01CA099163, R01CA096844, LB506, CA182435A, R01CA105489, R01CA087986, R01CA144027, R01CA182435, R01CA116552, R01CA140432)
  • National Institute of General Medical Sciences (P30GM106397)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic and Kidney Cyst Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Hedgehog Signaling Pathway Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
EHD1, primary cilia, Hedgehog signaling, Smoothened, endocytic recycling, neural tube closure, neural tube patterning, Gli3 repressor, neuroepithelium, embryonic lethality, receptor trafficking
MeSH terms
Genetic Background, Smoothened Receptor, Animals, Cilia, Female, Fibroblasts, Genes, Lethal, Multigene Family, Genotype, Male, Morphogenesis, Protein Binding, Signal Transduction, Gene Expression, Gene Deletion, Mice, Knockout, Protein Transport, Vesicular Transport Proteins, Embryonic Development, Mice, Hedgehog Proteins, Neural Tube

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

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