![Page 1: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/1.jpg)
Deciphering HoxA9 function in multipotential progenitor
biologyand B cell development
Kay L. Medina, PhDAssociate Professor
Dept. of ImmunologyMayo Clinic College of Medicine
![Page 2: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/2.jpg)
HSC STRC
CMP
ILC2 NKP
GMP Mac PMN
MEP Ery Meg
Thymus
ETP
ELP
GMLP
ALP BLPPre-
Pro-BPro-B
Overview of adult hematopoiesis in the mouse
CDP pDC cDC
![Page 3: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/3.jpg)
Sequentially-acting regulatory circuits orchestrate B cell fatespecification and commitment
HSC STRC
ELP
GMLP
ALP BLPPre-
Pro-BPro-B
HoxA9IkarosPU.1
Flt3E2A
E2A, Flt3
IL-7R
c-Myb
EBF1
FOXO1
IL-7R
Pax5
IL-7R
Pax5
E2A B220+CD19-
B220+CD19+
DH-JH
VH-DJH
![Page 4: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/4.jpg)
Hoxa9 regulates Flt3 in lymphohematopoietic progenitors
Gwin and Medina, JI, 2010
![Page 5: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/5.jpg)
hoxa9
ebf1
Inverse expression of HoxA9 and EBF1 in lymphoid/B cell precursors
CLP/BCP
![Page 6: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/6.jpg)
Myeloid-Lymphoidprogenitor
All-Lymphoidprogenitor
B-Lymphoidprogenitor
Pro-B cell
HSC/MPP program Alternate lineage programs
B cell programTranscription factors, signaling molecules, chemokines, microRNAs
Hoxa9Flt3
Hoxa9Flt3IL7R
Hoxa9Flt3IL7
EBF1
IL7REBF1
![Page 7: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/7.jpg)
hoxa9-/- flt3l-/- mice exhibit a severe lympho-hematopoietic block
Gwin and Medina, JI, 2013
![Page 8: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/8.jpg)
HSC STRC GMLP
hoxa9-/- flt3l-/-
WT-RAG1-GFP+ Hoxa9-/- RAG1-GFP+ Flt3L-/- RAG1-GFP+ Hoxa9-/-Flt3L-/- RAG1-GFP+F
lt3
GFP GFP GFP GFP
ELP ELP ELP ELP
HoxA9 and Flt3 signaling function together to establish themultipotential progenitor pool competent to undergo lymphoid priming
![Page 9: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/9.jpg)
The role of HoxA9 in B cell fate specification.
ebf1 foxo1 gfi1b
EBF1FOXO1
GFI1b
![Page 10: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/10.jpg)
HoxA9-/- hematopoietic progenitors have impaired IL-7 signaling#
cells
x 1
05
B6 HoxA9-/- IL-7R
MF
I IL-
7R
Lin- ckit+ Flt3+
B6HoxA9
![Page 11: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/11.jpg)
Culture model to identify HoxA9 target genes critical for B cell development
WT
HoxA9-/-
c-kit Sca-1 CD34 CD150 Flt3 IL-7R
Lin- c-kit cells cultured for 17 days in SCF+FL+Tpo+IL-6
B
# ce
lls x
106
B22
0
CD19 CD19
WT MPPs HoxA9-/- MPPs
![Page 12: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/12.jpg)
Day 17 MPP
24 hr CLP
IL7,SCF,FL
Harvest
Switch
RNA-Seq Platform
Sequencing
WT MPP-1WT MPP-2HoxA9-/- MPP-1HoxA9-/- MPP-2
Sequencing
WT CLP-1WT CLP-2HoxA9-/- CLP-1HoxA9-/- CLP-2
2 independentexperiments
![Page 13: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/13.jpg)
Summary of RNA-Seq Platform
Hoxa9-/- MPP
42
Hoxa9-/- MPP
73
Hoxa9-/- CLP
16
Hoxa9-/- CLP
102
13
MPP CLP
29 3 17
MPP CLP
56 86
Sample Total Reads
Hoxa9ko-CLP-1 89,662,470
Hoxa9ko-CLP-2 86,812,194
Hoxa9ko-MPP-1 79,030,174
Hoxa9ko-MPP-2 102,001,936
WT-CLP-1 108,111,316
WT-CLP-2 118,748,182
WT-MPP-1 98,958,636
WT-MPP-2 99,110,526
Read length:50bp
40-50X depth of coverage/gene
Differential expression cutoff: absolute fold change 1.4
False discovery rate of ≥0.1; p>0.05
![Page 14: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/14.jpg)
Hoxa9-/- MPPs
64%HSC/MPP
myeloid/DC
Hoxa9-/- MPPs
54%Lymphoid
Hoxa9-/- CLPsHoxa9-/- CLPs
93%HSC/MPP
myeloid/DC
63%Lymphoid
Hoxa9-deficiency impairs lymphoid priming and downregulation of HSC/MPP and/or alternative lineage gene programs
Shared upregulated Lcn2 – lipocalin 2 – neutrophilMgam – maltase-glucoamylase - neutrophilClec4b – ATP binding cassette - DCAdam23 – disintegrin and metallopeptidase - DCPglyrp1 – peptidoglycan recognition protein - neutrophilElane – elastase – macrophage/HSC/MPPAbca13 – ATP binding cassette - neutrophilEhd3 – EH domain containing 3 – T cellsDio2 – deiodinase – HSC/MPPMpo – myeloperoxidase - monocytesVcam1 – vascular cell adhesion molecule 1 – HSC/MPPDach1 – dachshund - neutrophilPpic – peptidylprolyl isomerase C – HSC/MPPRab38 – member of RAS family - macrophageMgl2 – macrophage galactose N-acetyl-galactosamine specific Ikzf2 – ikaros zinc finger 2; Helios – HSC/T cells/T regsGata2 – HSC/MPP
Shared downregulatedRag1 – recombinase activating gene – lymphocytesGfra1 – sperm stem cell self renewal - BCPsRag2 - recombinase activating gene - lymphocytesPpfia4 – protein tyrosine phosphatase – spl DC / MacsSlc15a2 – H+/peptide transporter - BCPsP2rx3 – purinergic receptor P2X - BCPsCdh17 – cadherin 17 - BCPsCecr2 – cat eye syndrome - BCPsGimap4 – GTPase - BCPsLin28b - regulator adult to fetal HSC; let7 biogenesis - BCPsCd27 – member of TNF receptor family – MPPs/BCPsIgfbp3 – insulin growth factor 2 binding protein - BCPs
![Page 15: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/15.jpg)
B22
0
CD19 CD19
WT MPPs HoxA9-/- MPPs
17 day MPP switched to SCF+FL+IL7 and cultured for an additional 6-8 days
Mac
-1
CD19 CD19
Gated onGFP+ cells Hoxa9-/- d17 MPP in SCF+FL+IL7
6-8 days after retroviral transduction
MigR1-GFP Mig-EBF-GFP
Ectopic expression of EBF1 is sufficient to restore the generation of BCPs from HoxA9-/- MPPs
![Page 16: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/16.jpg)
Linking RNA-Seq and Microarray data to the IL-7R signaling pathway
![Page 17: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/17.jpg)
Conclusions:
We developed a short-term in vitro culture model to expand MPPs from WT and HoxA9-/- mice that will be informative in the identification, characterization, and validation of HoxA9 regulated genetic circuits in early hematopoiesis.
RNA-Seq confirmed decreased lymphoid priming in MPPs and under B cell differentiation conditions due to HoxA9 deficiency independent of Flt3 signaling.
HoxA9 deficiency impairs IL-7R expression and signaling – enforced expressionof EBF1 can bypass the IL-7 signaling defect.
Gfi1b
Runx1 IL7R
E2A* → FOXO1→ EBF1→Pax5
Flt3 → PI3KMeis1Hoxa9
MAPK
STAT5
HSC program
c-Myb Gfra1Ctr9Ccr9Scdn4
![Page 18: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/18.jpg)
Underway:
1. Identify novel HoxA9 target genes : compare results to microarray platform of genes upregulated in EBF1-/- multipotent cells that express endogenous Hoxa9 - underexpressed in Hoxa9-/- MPPs in RNA-Seq platform (transcriptional activation) - overexpressed in EBF1-/- MPPs in Affymetrix Array Chd17, CD27, CCR9, Ppfia4, Sorcs2, RAG2, GPR25, Jup, Igf2bp3
- overexpressed in Hoxa9-/- MPPs in RNA-Seq platform (transcriptional repressor) - underexpressed in EBF1-/- MPPs in Affymetrix Array Mgst1, Tgm2, Enpp4, Plekha8, Muc13, Cpne2, Tmem176a, Mreg, Adcy6, STAT4, Sulf2, SpiB
2. Identify novel EBF1 target genes : compare results to microarray platform of genes underexpressed in EBF1-/- multipotent cells to genes underexpressed in HoxA9-deficient MPPs or CLPs P2rx3, RAG1, Rgs8, Cecr2, Egfl7, Uaca, Lax1, Dntt, Slc15a2, Gimap4
3. Perform gain-of-function and loss-of-function experiments in WT or HoxA9-/- MPPs to evaluate gene function in B cell development followed by molecular validation.
4. Determine if HoxA9 target genes are relevant to leukemogenesis.
![Page 19: Deciphering HoxA9 function in multipotential progenitor biology and B cell development](https://reader036.vdocument.in/reader036/viewer/2022062517/56812fc2550346895d954303/html5/thumbnails/19.jpg)
Acknowledgements
Medina LabKim GwinZhihui (Cherry) Xu, MDZhixin (Jason) Hua, PhDJoe Dolence, PhDMike BellElena FrankMariya Shapiro
Flow Cytometry CoreGene Expression Core
Funding by NHLBI R01096108Eagles FoundationConcern FoundationDept of Immunology