.2013 Dec 9;27(5):504-15.

doi: 10.1016/j.devcel.2013.11.004.

Hemogenic endothelial cell specification requires c-Kit, Notch signaling, and p27-mediated cell-cycle control

Kathrina L Marcelo¹, Tiffany M Sills², Suleyman Coskun³, Hema Vasavada³, Supriya Sanglikar³, Lauren C Goldie¹, Karen K Hirschi⁴

Affiliations

¹ Interdepartmental Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Children's Nutrition Research Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
² Interdisciplinary Program in Cell and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Children's Nutrition Research Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
³ Yale Cardiovascular Research Center and Yale Stem Cell Center, Yale University School of Medicine, 300 George Street, New Haven, CT 06511, USA.
⁴ Interdepartmental Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Interdisciplinary Program in Cell and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Children's Nutrition Research Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Yale Cardiovascular Research Center and Yale Stem Cell Center, Yale University School of Medicine, 300 George Street, New Haven, CT 06511, USA. Electronic address: karen.hirschi@yale.edu.

PMID:24331925
PMCID: PMC3994666
DOI: 10.1016/j.devcel.2013.11.004

Hemogenic endothelial cell specification requires c-Kit, Notch signaling, and p27-mediated cell-cycle control

Kathrina L Marcelo et al. Dev Cell.2013.

.2013 Dec 9;27(5):504-15.

doi: 10.1016/j.devcel.2013.11.004.

Authors

Kathrina L Marcelo¹, Tiffany M Sills², Suleyman Coskun³, Hema Vasavada³, Supriya Sanglikar³, Lauren C Goldie¹, Karen K Hirschi⁴

Affiliations

¹ Interdepartmental Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Children's Nutrition Research Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
² Interdisciplinary Program in Cell and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Children's Nutrition Research Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
³ Yale Cardiovascular Research Center and Yale Stem Cell Center, Yale University School of Medicine, 300 George Street, New Haven, CT 06511, USA.
⁴ Interdepartmental Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Interdisciplinary Program in Cell and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Children's Nutrition Research Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Yale Cardiovascular Research Center and Yale Stem Cell Center, Yale University School of Medicine, 300 George Street, New Haven, CT 06511, USA. Electronic address: karen.hirschi@yale.edu.

PMID:24331925
PMCID: PMC3994666
DOI: 10.1016/j.devcel.2013.11.004

Abstract

Delineating the mechanism or mechanisms that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic specification, as well as cell-cycle control, of endothelium during embryogenesis. Herein, we define the molecular signals downstream of RA that regulate hemogenic endothelial cell development and demonstrate that cell-cycle control is required for this process. We found that re-expression of c-Kit in RA-deficient (Raldh2(-/-)) primordial endothelium induced Notch signaling and p27 expression, which restored cell-cycle control and rescued hemogenic endothelial cell specification and function. Re-expression of p27 in RA-deficient and Notch-inactivated primordial endothelial cells was sufficient to correct their defects in cell-cycle regulation and hemogenic endothelial cell development. Thus, RA regulation of hemogenic endothelial cell specification requires c-Kit, notch signaling, and p27-mediated cell-cycle control.

PubMed Disclaimer

Figures

Figure 1. Endothelial cells of the murine yolk sac respond to active RA signaling at the onset of definitive hematopoiesis, display enriched hematopoietic colony forming activityin vitro, and upregulated expression of hematopoietic genes
A. Cross sections of E8.5 yolk sac tissues showing blue-stained endothelial cells expressing β-galactosidase (β-gal) in concepti expressing the RARE-*lacZ* transgene (*Raldh2^+/+*; RARE-*lacZ^+/−*; andRaldh2^−/−; RARE-*lacZ^+/−* + RA). Blue staining is not observed in wildtype yolk sac tissues when thelacZ transgene is absent (*Raldh2^+/+*; RARE-*lacZ^−/−*), or when RA signaling is impaired (*Raldh2^−/−*; RARE-*lacZ^+/−*). VE: visceral endoderm. Scale bars in all panels represent 20 µm.B. FACS analysis of β-gal⁺ cells, primordial endothelial cells (CD31⁺ CD45⁻) and hemogenic endothelial cells (CD31⁺ CD45⁻ Flk-1⁺ c-Kit⁺) in E8.5Raldh2^+/+; RARE-*lacZ^+/−* yolk sac. All population data were calculated as a percentage of total live cell population ± SEM (n ≥ 3).C. Left chart indicates proportions of β-galactosidase-negative (β-gal−) and –positive (β-gal+) fractions withinRaldh2^+/+; RARE-*lacZ^+/−* yolk sac endothelial cells; values were calculated as percentage of CD31⁺ CD45⁻ live cells. Right chart depicts proportions of hemogenic and non-hemogenic endothelial cells within the β-gal⁺ subpopulation; values were calculated as percentage of β-gal⁺ CD31⁺ CD45⁻ live cells.D. qPCR analysis of hematopoietic gene expression in β-gal⁺ versus β-gal⁻ endothelial cells from pooled yolk sac tissues, calculated as gene expression relative to endogenous β-actin expression ± SEM (n ≥ 3).E. Hematopoietic potential of β-gal⁺ (β-gal⁺ CD31⁺ CD45⁻) versus β-gal⁻ endothelial cells from E8.5 yolk sacs. Total number of colony-forming units (CFU) or multi-lineage CFU-GEMM colonies generated are shown as percentage of seeded cells (300 cells) ± SEM (n ≥ 3).

**Figure 2. Infection ofRaldh2^−/− mutant yolk sac with lentivirus encodingc- Kit, Runx1 orc-Myb**
A. Schematic diagram of the bi-cistronic lentiviral expression constructs encoding candidate gene cDNA cloned upstream of anIRES-ZsGreen1 reporter.B. Control untreated (No LV) RA-deficient yolk sacs do not display ZsGreen1 fluorescence (green) 48 h post-infection in whole embryo culture (left panel).Raldh2^−/− embryos infected with lentivirus particles forc-Kit re-expression (c-Kit LV) display yolk sac endothelial cells expressing the ZsGreen1 reporter 48 h post-infection, as indicated by green cells lining the vasculature (right panel). Scale bars in all panels represent 20 µm.C. Proportions of CD31⁺ CD45⁻ endothelial, CD45⁺ blood, and CD31⁺ CD45⁻ Flk1⁺ c-Kit⁺ hemogenic endothelial cells within ZsGreen1-expressing yolk sac population. Values were calculated as percentage of ZsGreen1⁺ live cells.D. qPCR analysis ofc-Kit, Runx1 andc-Myb gene expression in endothelial cells isolated from control untreated (No LV), RA-treated (RA), and lentivirus-infected mutant (Mut) yolk sacs versus untreated wildtype yolk sacs (No LV WT). Data points were calculated as gene expression relative to endogenous β-actin expression ± SEM (n ≥ 3).

**Figure 3. Re-expression ofc-Kit inRaldh2^−/− mutants restores hemogenic endothelial cell development and its multi-lineage hematopoietic potential**
**A and D.** Quantitative analysis of Flk-1⁺ c-Kit⁺ CD45⁻ SP hemogenic endothelial cells(A) and Flk-1⁻ c-Kit⁺ CD45⁺ SP hematopoietic progenitors(D) from control untreated (No LV), RA-treated (RA), and lentivirus-infectedRaldh2^−/− (Mut) yolk sacs, compared with untreated wildtype yolk sac tissues (No LV WT). Data points were calculated as percentage of total live cell population relative to untreated wildtype control (No LV WT) ± SEM (n ≥ 3).B. Hematopoietic potential of hemogenic endothelial cells isolated from untreated, RA-treated, and lentivirus-infected pooled mutant yolk sacs versus those pooled from wildtype untreated yolk sacs. Total multi-lineage CFU-GEMM colonies generated are described as percentage of seeded cells (100 cells per well) ± SEM (n ≥ 3). See also Figure S2.C. qPCR analysis ofRunx1 gene expression in endothelial cells isolated from control untreated (No LV), RA-treated (RA), and lentivirus-infected mutant (Mut) yolk sacs versus untreated wildtype yolk sacs (No LV WT). Data points were calculated as gene expression relative to endogenous β-actin expression ± SEM (n ≥ 3). See also Figures S1 and S2.

**Figure 4. c-Kit re-expression inRaldh2^−/− mutants restores endothelial cell number, cell cycle distribution, and expression of cyclin-dependent kinase inhibitorp27**
A. Absence of blue-stained endothelial cells in X-gal-stainedRaldh2^−/−; RARE-*lacZ^+/−* yolk sac treated with c-Kit-expressing lentivirus. VE: visceral endoderm. Scale bar represents 20 µm.B. Quantitative FACS-based analysis of CD31⁺ CD45⁻ endothelial cells from control untreated (No LV), RA-treated (RA), and c-Kit lentivirus-infected (c-Kit LV) mutant yolk sacs versus control untreated wildtype tissues (No LV WT), calculated as percentage total live cell population relative to untreated wildtype control (No LV WT) ± SEM (n ≥ 3).C. FACS-based cell cycle analysis based on pyronin Y and Hoechst dye retention of endothelial cells from control and treated mutant yolk sacs in comparison to control wildtype tissues (No LV WT). Data points are calculated as percentage total live endothelial cell population ± SEM (n ≥ 3).D. qPCR analysis of cell-cycle related genes in CD31⁺ CD45⁻ endothelial cells from control and treated mutant yolk sacs versus control untreated wildtype yolk sac tissue. Data were calculated as gene expression relative to endogenous β-actin expression ± SEM (n ≥ 3). See also Figure S3.

**Figure 5. Re-expression ofp27 inRaldh2^−/− mutants rescues specification and function of hemogenic endothelial cells**
**A and B.** qPCR analysis ofp27(A) andc-Kit(B) gene expression in endothelial cells isolated from untreated (No LV) and lentivirus-infected (c-Kit and p27)*Raldh2^−/−* mutant (Mut) yolk sacs versus untreated wildtype yolk sac (No LV WT). Data points were calculated as gene expression relative to endogenous β-actin expression ± SEM (n ≥ 3).C. Quantitative FACS-based analysis of CD31⁺ CD45⁻ endothelial cells from control untreated (No LV) andp27 lentivirus-infected (p27) mutant yolk sacs versus control untreated wildtype tissues (No LV WT), calculated as percentage total live cell population relative to untreated wildtype control (No LV WT) ± SEM (n ≥ 3).D. FACS-based cell cycle analysis based on pyronin Y and Hoechst dye retention of endothelial cells from control andp27 lentivirus-infected mutant yolk sacs relative to endothelial cells from control wildtype tissues (No LV WT). Data points are calculated as percentage total live endothelial cell population ± SEM (n ≥ 3).E. Quantitative analysis of hemogenic endothelial cells from untreated (No LV) andp27 lentivirus-infected (p27 LV) mutant yolk sacs, in comparison to untreated wildtype yolk sac tissues (No LV WT). Data points were calculated as a percentage of total live cell population relative to untreated wildtype control (No LV WT) ± SEM (n ≥ 3).

**Figure 6. Notch signaling regulates primordial endothelial cell proliferation and hemogenic specification in wildtype and RA-deficient mutants re-expressing c-Kit**
A. Quantitative FACS-based analysis of CD31⁺ CD45⁻ endothelial cells from control untreated (No LV),*c-Kit* lentivirus infected (c-Kit), and DAPT-treated,*c-Kit* lentivirus-infected (DAPT + c-Kit)*Raldh2^−/−* mutant, and DAPT-treated (DAPT) wild type yolk sacs versus control untreated wild type tissues (No LV WT), calculated as percentage total live cell population relative to untreated wildtype control (No LV WT) ± SEM (n ≥ 3).B. Quantitative analysis of Flk-1⁺ c-Kit⁺ CD45⁻ SP hemogenic endothelial cells from control untreated (No LV),*c-Kit* lentivirus infected (c-Kit), and DAPT-treated,*c-Kit* lentivirus-infected (DAPT + c-Kit)*Raldh2^−/−* mutant, and DAPT-treated (DAPT) wild type yolk sacs versus control untreated wild type tissues (No LV WT). Data points were calculated as a percentage of total live cell population relative to untreated wildtype control (No LV WT) ± SEM (n ≥ 3).C. qPCR analysis ofc-Kit expression in CD31⁺ CD45⁻ endothelial cells from untreated control (No LV),*c-Kit* lentivirus infected (c-Kit), and DAPT-treated +*c-Kit* lentivirus-infected (DAPT + c-Kit)*Raldh2^−/−* mutant (Mut) yolk sacs versus untreated wildtype yolk sac tissue (No LV WT).D. qPCR analysis ofp27 expression in CD31⁺ CD45⁻ endothelial cells from untreated (No LV),*c-Kit* lentivirus infected (c-Kit), DAPT-treated +*c-Kit* lentivirus-infected (DAPT + c-Kit)*Raldh2^−/−* mutant (Mut), and DAPT-treated (DAPT) wildtype yolk sacs versus untreated wildtype yolk sac tissue (No LV WT).E. qPCR analysis ofNotch1 and transcription factorsHes1 andHey1 in CD31⁺ CD45⁻ endothelial cells from control and treated mutant yolk sacs versus that from untreated wildtype yolk sac tissue.C-E. Data were calculated as gene expression relative to endogenous β-actin expression ± SEM (n ≥ 3).F and G. Quantitative analysis of Flk-1⁺ c-Kit⁺ CD45⁻ SP hemogenic endothelial cells(F) and Flk-1⁻ c-Kit⁺ CD45⁺ SP hematopoietic progenitor cells from untreated control (No LV), DAPT-treated (DAPT), and DAPT-treated +*p27* lentivirus-infected (DAPT + p27) wildtype yolk sacs versus untreated wildtype tissues (No LV WT). Data points were calculated as a percentage of total live cell population relative to untreated wildtype control (No LV WT) ± SEM (n ≥ 3).

**Figure 7. Hemogenic endothelial cells from E10.5 AGM are RA-responsive, require c-Kit for hematopoietic function, and are enriched for expression ofp27 and Notch signaling pathway components**
A. Hematopoietic potential of SP and non-SP (NSP) fractions isolated from E9.5-11.5 AGM relative to that E9.5 yolk sac SP cells, which serve as positive control, in methylcellulose colony formation assays. Individual colony types were scored as: erythroid (E), granulocyte-macrophage (GM), and granulocyte-erythroid-macrophage-megakaryocyte (GEMM) colony-forming units. Total number of colonies generated are reported per 1000 seeded cells ± SEM (n ≥ 3).B. Hematopoietic potential of selected cell surface marker-expressing cells within the SP fraction of the E10.5 AGM. Total multi-lineage GEMM colonies generated are reported per 100 seeded cells ± SEM (n ≥ 3).C. Clonal assay and time course of Flk-1⁺ c-Kit⁺ CD45⁻ SP (top panels) and Flk-1⁻ c-Kit⁺ CD45⁺ SP cell development (bottom panels). Scale bars represent 10 µm (days 1 and 3) and 50 µm (day 5–7).D. Proportions of β-galactosidase-negative (β-gal−) and –positive (β-gal+) fractions within E10.5Raldh2^+/+; RARE-*lacZ^+/−* AGM hemogenic endothelial cells; values were calculated as percentage of live Flk-1⁺ c-Kit⁺ CD45⁻ SP cells.E. qPCR analysis of RA signaling (*RAR*α andRARβ) and Notch signaling (*Notch1, Hey1*, andHes1) pathway genes, as well as of cell cycle-related (*p21* andp27) genes, in hemogenic endothelial cells (HemEC) versus non-HemEC cells isolated from E10.5 wildtype AGM tissues. Gene expression was calculated relative to endogenous β-actin expression ± SEM (n ≥ 3). See also Figure S4 and S5.

See this image and copyright information in PMC

Cited by

Developing HSCs become Notch independent by the end of maturation in the AGM region.
Souilhol C, Lendinez JG, Rybtsov S, Murphy F, Wilson H, Hills D, Batsivari A, Binagui-Casas A, McGarvey AC, MacDonald HR, Kageyama R, Siebel C, Zhao S, Medvinsky A.Souilhol C, et al.Blood. 2016 Sep 22;128(12):1567-77. doi: 10.1182/blood-2016-03-708164. Epub 2016 Jul 15.Blood. 2016.PMID:27421959Free PMC article.
Endothelial Cell Differentiation and Hemogenic Specification.
Aragon JW, Hirschi KK.Aragon JW, et al.Cold Spring Harb Perspect Med. 2022 Jul 29;12(7):a041164. doi: 10.1101/cshperspect.a041164.Cold Spring Harb Perspect Med. 2022.PMID:35193895Free PMC article.Review.
Rnd3 regulates lung cancer cell proliferation through notch signaling.
Tang Y, Hu C, Yang H, Cao L, Li Y, Deng P, Huang L.Tang Y, et al.PLoS One. 2014 Nov 5;9(11):e111897. doi: 10.1371/journal.pone.0111897. eCollection 2014.PLoS One. 2014.PMID:25372032Free PMC article.
Reprogramming human endothelial cells to haematopoietic cells requires vascular induction.
Sandler VM, Lis R, Liu Y, Kedem A, James D, Elemento O, Butler JM, Scandura JM, Rafii S.Sandler VM, et al.Nature. 2014 Jul 17;511(7509):312-8. doi: 10.1038/nature13547. Epub 2014 Jul 2.Nature. 2014.PMID:25030167Free PMC article.
MicroRNA-223 limits murine hemogenic endothelial cell specification and myelopoiesis.
Wu Y, Paila U, Genet G, Hirschi KK.Wu Y, et al.Dev Cell. 2023 Jul 24;58(14):1237-1249.e5. doi: 10.1016/j.devcel.2023.05.007. Epub 2023 Jun 8.Dev Cell. 2023.PMID:37295435Free PMC article.

See all "Cited by" articles

References

1. Argraves WS, Drake CJ. Genes critical to vasculogenesis as defined by systematic analysis of vascular defects in knockout mice. The anatomical record Part A, Discoveries in molecular, cellular, and evolutionary biology. 2005;286:875–884. - PubMed
1. Atkins GB, Jain MK, Hamik A. Endothelial differentiation: molecular mechanisms of specification and heterogeneity. Arteriosclerosis, thrombosis, and vascular biology. 2011;31:1476–1484. - PMC - PubMed
1. Bertrand JY, Chi NC, Santoso B, Teng S, Stainier DY, Traver D. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature. 2010;464:108–111. - PMC - PubMed
1. Bohnsack BL, Lai L, Dolle P, Hirschi KK. Signaling hierarchy downstream of retinoic acid that independently regulates vascular remodeling and endothelial cell proliferation. Genes & development. 2004;18:1345–1358. - PMC - PubMed
1. Boisset JC, van Cappellen W, Andrieu-Soler C, Galjart N, Dzierzak E, Robin C. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature. 2010;464:116–120. - PubMed

Publication types

Actions
Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Related information

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- scite Smart Citations
Medical
- MedlinePlus Health Information
Molecular Biology Databases
- Mouse Genome Informatics (MGI)

Movatterモバイル変換

Account

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Full text links

Actions

Share

Hemogenic endothelial cell specification requires c-Kit, Notch signaling, and p27-mediated cell-cycle control

Affiliations

Hemogenic endothelial cell specification requires c-Kit, Notch signaling, and p27-mediated cell-cycle control

Authors

Affiliations

Abstract

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases