Department of Hematology School of Medical

Sciences Tarbiat Modares

University

May.28.2008

Najmaldin saki

Homeobox transcription factors of the Hox family are important regulators of hematopoiesis that control proliferation, differentiation, and self-renewal of hematopoietic stem cells (HSC) .

Introduction:

Several Hox genes, in particular HOXB3, HOXB4, HOXA5, HOXA9, and HOXA10, are expressed in the HSC compartment, but are subsequently downregulated upon differentiation.

Overexpression of HOXB4 causes striking expansion of HSCs in vitro and in vivo without causing overt leukemia or major lineage disturbances.

Enforced expression of HOXA9,HOXA10, and HOXB6 enhances proliferation of HSCs, expands the myeloid compartment and leads to fatal myeloid Leukemia .

Hoxa9 null mice display a small spleen, a modest reduction in white blood cell counts and a sevenfold reduction in competitive repopulating units, although the frequency ofimmunophenotypically defined stem cells by Lin-, c-kit + ,flk-2, Sca-1 + (KLFS) and homing of progenitor cells to the bone marrow appeared to be normal .

Hoxb4 null and compound hoxb3/hoxb4 null mice exhibit a proliferation defect in HSCs although the phenotype is considerably milder than seen in hoxa9 knockout mice

Hoxb4 and hoxb3/hoxb4 knockout mice displayed a mild reduction in spleen and bone marrow cellularity,a modest proliferation defect in primitive hematopoietic progenitors (Lin -,Sca-1 +,c-kit þ cells), normal homing of HSC and a significant reduction in repopulating ability in vivo.

Because deletion of hoxa9, hoxb3, and hoxb4 all contribute to an HSC defect,we asked whether a compound deficiency of hoxa9,hoxb3, and hoxb4 together would further increase the hematopoietic defect, particularly the HSC function?

Although the repopulating capacity of hoxa9/b3/b4 null HSC was very compromised, it was not more severe than the repopulating defect seen in hoxa9 null HSC.

These findings demonstrate that hoxa9, hoxb3,and hoxb4 are required for optimal HSC function, but are not essential for formation of all major blood lineages.

Materials and methods :

Generation and screening of null mice

Clonogenic assaysFor (CFU-GM)colonies, 30,000 bone marrow (BM) cells were cultured in 1 mL methylcellulose medium in duplicate samples containing :I. 50 ng/mL murine stem cell factorII. 10 ng/mL murine interleukin-3III.10ng/mL human interleukin-6 Colonies were scored on day 8.

Day 12 CFU-S assayBone marrow cells from wild-type (WT), hoxa9, and Hoxa9/b3/b4 null mice (100,000 cells) were injected into lethally irradiated recipients (9 cGy). The spleen was removed 12 days later,fixed in Telleyesniczky’s solution, and macroscopic surface colonies were counted.

FACS analysisphycoerythrin-conjugated anti-CD45.1 (Ly5.1)allophycocyanin-(biotin-) anti-CD45.2 (Ly5.2) For detection of B-, T-, myeloid-, and erythroid cells phycoerythrin-conjugated anti-B220, CD3, CD4, Mac1 CD71,and fluorescein isothiocyanate–conjugated anti-CD8, Mac1, Gr1,Ter119 were used .

All antibodies from BD Pharmingen , Stockholm , Sweden

Results

Hoxa9/b3/b4 null mice display amarked reduction in spleen size and cellularity

Hoxa9/b3/b4 null mice display amarked reduction in spleen size and cellularity

Hoxa9/b3/b4 and hoxa9 null CFU-Sprogenitors generate small spleen colonies

Hoxa9/b3/b4 null mice exhibit an increasein the frequency of immunophenotypic HSCs

Lin- , Sca+ , c-Kit+ , CD 150+ (LSKCD150)Lin- , Sca+ , c-Kit+ , CD 150+ (LSKCD150)Lin- , Sca+ , c-Kit+ , Cd34- , (LSKCD34-)

Loss of hoxb3 and hoxb4 does not exacerbatethe repopulating defect observed in

hoxa9¯’¯HSCs

The key question emerging from this study is why the deficiency of hoxa9 alone results in such dramatic reduction in repopulating capacity and is not significantly compromised by the additional deletions (hoxb3/b4) ?