Role of the HOXA cluster in HSC emergence and blood cancer

2021 ◽  
Author(s):  
Mays Abuhantash ◽  
Emma M. Collins ◽  
Alexander Thompson
Keyword(s):  
Embryonic Development ◽  
Hox Genes ◽  
Hoxa Cluster ◽  
Hematopoietic Stem ◽  
Blood Cancer ◽  
Non Coding Rna ◽  
Blood Formation ◽  
Peripheral Cells ◽  
Normal Embryonic Development

Hematopoiesis, the process of blood formation, is controlled by a complex developmental program that involves intrinsic and extrinsic regulators. Blood formation is critical to normal embryonic development and during embryogenesis distinct waves of hematopoiesis have been defined that represent the emergence of hematopoietic stem or progenitor cells. The Class I family of homeobox (HOX) genes are also critical for normal embryonic development, whereby mutations are associated with malformations and deformity. Recently, members of the HOXA cluster (comprising 11 genes and non-coding RNA elements) have been associated with the emergence and maintenance of long-term repopulating HSCs. Previous studies identified a gradient of HOXA expression from high in HSCs to low in circulating peripheral cells, indicating their importance in maintaining blood cell numbers and differentiation state. Indeed, dysregulation of HOXA genes either directly or by genetic lesions of upstream regulators correlates with a malignant phenotype. This review discusses the role of the HOXA cluster in both HSC emergence and blood cancer formation highlighting the need for further research to identify specific roles of these master regulators in normal and malignant hematopoiesis.

scholarly journals Activation of Hottip lncrna Perturbs HSC Function Leading to AML like Disease in Mice

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3877-3877
Author(s):  
Huacheng Luo ◽  
Ganqian Zhu ◽  
Jie Zha ◽  
Bowen Yan ◽  
Ying Guo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hox Genes ◽  
Chromatin Domain ◽  
Active Chromatin ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Chromatin Signature ◽  
Genes Expression ◽  
Hoxa Genes

Abstract Several HOX loci associated long noncoding RNAs (lncRNAs) have been shown to regulate transcription of HOX genes through influencing epigenetic landscape. Especially, the posterior HOXA domain associated lncRNA HOTTIP acts as an epigenetic regulator that recruits WDR5/MLL complex to coordinate active chromatin modifications and HOXA genes expression in the development of animal digits. Despite HOX genes, especially HOXA genes, are highly expressed in many acute myeloid leukemia (AML) patients, it remains largely unknown whether and how HOTTIP lncRNA regulates hematopoietic stem cell (HSC) function and contributes to leukemogenesis. We showed previously that disruption of the CTCF boundary located between HOXA7 and HOXA9 genes (CBS7/9) resulted in reduced lncRNA HOTTIP and HOXA genes expression in MLL rearranged AML suggesting that HOTTIP may play a role in ectopic expression of the posterior HOXA gene. We employed a pooled CRISPR-Cas9 KO library to specifically screen lncRNAs in four HOX gene loci and identify HOTTIP as acritical regulator in controlling oncogenic HOX chromatin signature and associated gene expression patterns in AML by collaborating with posterior HOXA chromatin boundary. HOTTIP is upregulated in AML patients with MLL-rearrangement or NPM1 mutation. AML patients with high HOTTIP expression exhibits a significantly shortened survival compared to low HOTTIP expressing patients. To test whether HOTTIP acts to coordinate posterior chromatin domain and HOXA genes activation in AML, we manipulated HOTTIP lncRNA expression levels in the MLL-AF9 rearranged MOLM13 by loss-of-function KO and gain-of function rescue, as well as carried out genome wide chromatin and transcriptomic analysis to intterrogate the role of HOTTIP in control of AML specific posterior HOXA chromatin domain. We found that knock-out of HOTTIP lncRNA led to a loss of active chromatin structure and invasion of repressive H3K27me3 mark over the posterior HOXA domain. HOTTIP KO attenuated progression of AML in the transplanted AML mouse model resembling the effect of CBS7/9 boundary disruption, while transcriptional activation of HOTTIP lncRNA in the CBS7/9 boundary-disrupted AML cells restored HOXA locus chromatin signature and gene expression as well as reversed the CBS7/9-mediated anti-leukemic effects. To further determine the role of HOTTIP lncRNA in regulating HSC function and leukemogenesis, we generated transgenic mice that expresses Hottip lncRNA under the control of the hematopoietic specific Vav1 enhancer and promoter. The Hottip transgenic (Tg) mice exhibited increased WBC and neutrophil counts and developed splenomegaly indicating that enforced expression of Hottip lncRNA resulted in perturbation of hematopoiesis. Furthermore, overexpression of Hottip lncRNA in mice bone marrow hematopoietic compartment strongly perturbed hematopoietic stem and progenitor cell (HSC/HPC) function by altering self-renewal and differentiation property of HSC/HPCs through affecting homeotic gene associated oncogenic transcription program. Approximately 20% of Hottip lncRNA transgenic mice developed abnormal hematopoietic phenotypes resembling AML-like disease. RNA-seq and ATAC-seq analysis indicated that overexpression of Hottip enhanced promoter chromatin accessibility and stimulates transcription of genes and pathways involved in HSC function and leukemogenesis, including WNT signaling, hematopoietic cell lineage, cell cycle, Hoxa9, Hoxa13, and Meis1, Runx1, and Twist1 genes. Thus, Hottip lncRNA overexpression acts as an oncogenic event to promote HSC self-renewal and HPC proliferation by reprograming leukemic associated chromatin signature and transcription programs. Disclosures No relevant conflicts of interest to declare.

Deletion of HoxA Genes Inhibits Proliferation and Differentiation of Myeloid Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1271-1271
Author(s):  
Janetta J. Bijl ◽  
Hayet Cherief ◽  
Denis-Claude Roy
Keyword(s):  
Hox Genes ◽  
Fetal Liver ◽  
Physiological Role ◽  
Myeloid Differentiation ◽  
Hoxa Cluster ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Hoxa Genes ◽  
Myeloid Progenitors

Abstract Mutants for single homeobox genes are showing neglible to mild phenotypes in the hematopoietic compartment. This is likely due to functional redundancy between Hox genes that has been demonstrated in embryonic development by compound mutant models. Interestingly, our previous data showed that hematopoietic stem cells (HSCs) lacking the majority of the HoxB genes (Hoxb1-Hoxb9) remained fully competent in replenishment of the hematopoietic compartment (Bijl, 2006). In addition we reported the expression of the majority of the HoxA cluster genes in HSC enriched E14.5 fetal liver fractions at levels at least one log higher than for HoxB cluster genes, suggesting that not HoxB but HoxA genes have a physiological role in hematopoiesis at that stage in the ontogeny. To investigate further whether HoxA genes are required for definitive hematopoiesis we used a conditional knock-out mouse model, in which the HoxA cluster was flanked by loxP sites (HoxAflox/flox). To excise the HoxA genes Cre recombinase in conjunction with GFP was retrovirally expressed in HoxAflox/flox fetal liver cells (Ly5.2). Flow cytometry showed transduction efficiencies in total fetal liver of 40% for Cre-GFP and 60% for MSCV-GFP control vector. Transduced cell composites were both plated in myeloid colony forming unit assays and transplanted in congenic (Ly5.1) mice to evaluate the transduction rate in progenitor populations and their capacity to proliferate and differentiate. Cre+ progenitor cells were able to grow out in colonies, and cultures showed a transduction efficiency of 69% for Cre-GFP and 99% for control vectors at the level of progenitors. The excision of the HoxA locus was confirmed by PCR for two Cre-GFP colonies. However, Cre-GFP+ colonies were much smaller than control colonies, indicating a defect in the proliferation potential of Cre+ myeloid progenitors. Further expansion of GM colonies in liquid culture was less efficient for Cre+ (1 out of 15), than for controls (5 out of 24), underlining a major defect in proliferation of early myeloid progenitors with granulocytic and monocytic differentiation potential in the absence of HoxA genes. All the colony types (GEMM, GM-CFU, G-CFU, M-CFU and BFU-E) were present in the Cre+ culture, but GM-CFUs and M-CFUs were proportional less represented than in control cultures (17% vs. 26% and 40% vs. 48%, respectively), indicating that HoxA genes have a function in early myeloid differentiation. Moreover, all colonies were in general poorly differentiated, showing that myeloid differentiation is sensible to reduced HoxA levels particularly in progenitors for the granulocytic/monocytic lineage. The capacity of HoxA−/− progenitors to induce small colonies might be due to some residual levels at the time of excision or in the case of mature progenitors a reduced requirement for HoxA genes, which is in agreement with the decreased expression of Hox genes with maturation of blood cells. No progeny of Cre-GFP+ cells was observed in irradiated recipients 4 weeks post-transplantation, which demonstrate complete failure of HoxA−/− progenitors to repopulate. However, it is too early to conclude whether deletion of HoxA genes affects the activity of HSCs. Altogether our in vitro and in vivo data show that HoxA genes are essential for the proliferation and differentiation of early myeloid progenitors that seems not to be compensated by other Hox gene members. The effect of HoxA deletion on HSC activity is currently under investigation.

Hoxb4-YFP reporter mouse model: a novel tool for tracking HSC development and studying the role of Hoxb4 in hematopoiesis

Blood ◽  
2011 ◽  
Vol 117 (13) ◽  
pp. 3521-3528 ◽  
Author(s):  
David Hills ◽  
Ruby Gribi ◽  
Jan Ure ◽  
Natalija Buza-Vidas ◽  
Sidinh Luc ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hox Genes ◽  
Fetal Liver ◽  
Yellow Fluorescent Protein ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Hematopoietic Stem ◽  
Reporter Mouse

Abstract Hoxb4 overexpression promotes dramatic expansion of bone marrow (BM) hematopoietic stem cells (HSCs) without leukemic transformation and induces development of definitive HSCs from early embryonic yolk sac and differentiating embryonic stem cells. Knockout studies of Hoxb4 showed little effect on hematopoiesis, but interpretation of these results is obscured by the lack of direct evidence that Hoxb4 is expressed in HSCs and possible compensatory effects of other (Hox) genes. To evaluate accurately the pattern of Hoxb4 expression and to gain a better understanding of the physiologic role of Hoxb4 in the hemato-poietic system, we generated a knock-in Hoxb4–yellow fluorescent protein (YFP) reporter mouse model. We show that BM Lin−Sca1+c-Kit+ cells express Hoxb4-YFP and demonstrate functionally in the long-term repopulation assay that definitive HSCs express Hoxb4. Similarly, aorta-gonad-mesonephrous–derived CD45+CD144+ cells, enriched for HSCs, express Hoxb4. Furthermore, yolk sac and placental HSC populations express Hoxb4. Unexpectedly, Hoxb4 expression in the fetal liver HSCs is lower than in the BM, reaching negligible levels in some HSCs, suggesting an insignificant role of Hoxb4 in expansion of fetal liver HSCs. Hoxb4 expression therefore would not appear to correlate with the cycling status of fetal liver HSCs, although highly proliferative HSCs from young BM show strong Hoxb4 expression.

scholarly journals Regulation of mir-196b by MLL and its overexpression by MLL fusions contributes to immortalization

Blood ◽  
2009 ◽  
Vol 113 (14) ◽  
pp. 3314-3322 ◽  
Author(s):  
Relja Popovic ◽  
Laurie E. Riesbeck ◽  
Chinavenmeni S. Velu ◽  
Aditya Chaubey ◽  
Jiwang Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fusion Proteins ◽  
Hox Genes ◽  
Bone Marrow Cells ◽  
Embryonic Stem ◽  
Chimeric Proteins ◽  
Short Term ◽  
Hoxa Cluster ◽  
Hematopoietic Stem ◽  
Leukemia Development

Abstract Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here, we show that MLL normally regulates expression of mir-196b, a hematopoietic microRNA located within the HoxA cluster, in a pattern similar to that of the surrounding 5′ Hox genes, Hoxa9 and Hoxa10, during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage, mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b, while treatment of MLL-AF9 transformed bone marrow cells with mir-196–specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore, overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival, as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development.

Deletion of Hoxa Genes in Adult Mice Affects Hematopoietic Stem Cell and B Cell Progenitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2359-2359
Author(s):  
Charles-Etienne Lebert-Ghali ◽  
Marilaine Fournier ◽  
Heloise Frison ◽  
Janetta Jacoba Bijl
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
Proliferative Potential ◽  
Hoxa Cluster ◽  
Hematopoietic Stem ◽  
Hoxa Genes ◽  
Definitive Hematopoiesis

Abstract Abstract 2359 BACKGROUND AND OBJECTIVE: Functional compensation between homeodomain proteins has hindered the ability to unravel their role in hematopoiesis using single gene knock-outs. Although several Hox genes can expand hematopoietic stem cells (HSC) when overexpressed, it remains unclear whether these genes are required for proper adult hematopoiesis. Moreover, it has been shown that HoxB genes are dispensable for hematopoiesis, and that expression of most HoxA genes is ten-fold superior to genes from other Hox clusters in HSC enriched fetal liver populations (Bijl, 2006). Using a haploinsufficient mice for the entire HoxA cluster (HoxA+/−), we have shown that adult HSCs and progenitors are particularly sensitive to HoxA gene levels (Lebert-Ghali, 2010). Thus, we hypothesize that HoxA genes have a crucial function in definitive hematopoiesis. MATERIALS AND METHODS: To assess the role of HoxA genes in definitive hematopoiesis, we used a conditional mutant mouse model for the entire HoxA cluster in combination with an inducible Mx-Cre model. The functional effect of HoxA cluster deletion on hematopoietic cells was analysed by culture and repopulation assays. RESULTS: Highly efficient excision of HoxA cluster was achieved by 7 doses of poly(I):poly(C) treatment (91–100%). Mice (control, n=3 and Mx-CreHoxAflox/flox, n=3) were sacrificed and analysed three days after the last injection. Immunophenotyping showed a 3 to 4 fold increase of CD150+/CD48-/CD244-/Sca+/c-kit+/Lin- hematopoietic stem cells. Despite the enhancement of the HoxA−/− HSC population, single cell cultures showed that their proliferative potential in response to growth factors was significantly reduced (p=0.036) as growth was observed only for 16.6 ± 14.4% of HoxA−/− compared to 42.4 ±10.3% of control HSCs after 3-weeks of culturing. In contrast, the number of multipotent progenitor (MPP) cells (CD34+/CD135+/Sca+/c-kit+/Lin-) was reduced, indicating a partial block from the short-term HSC (CD34+/CD135-/Sca+/c-kit+/Lin-) to the MPP transition. Colony forming cell assays showed a dramatic decrease of B-cell progenitors in the bone marrow (BM) (10-fold, p=0.0079), while myeloid progenitors were not affected by the deletion. Transplantation assays demonstrated that grafts composed of > 91% HoxA−/− HSCs have slower repopulation kinetics compared to control HSCs and strongly reduced long-term engraftment (37 ± 28% and 92 ± 6% for HoxA−/− and control, respectively, 20 weeks post-transplantation). Genotyping of engrafted donor cells is currently analyzed to confirm repopulation by HoxA−/− cells. Consistent with the observations in primary mice, peripheral blood analysis revealed also a dramatic reduction of B220+ B-cell population in mice transplanted with HoxA−/− BM cells compared to control (7.1 ± 8.5% and 56.2 ±4.8% respectively p=0,000002) Altogether, in vitro assays and transplantation assays revealed that the functions of HoxA−/− HSC seem to be impaired. CONCLUSION: Together, these results show that HoxA cluster genes are required for both HSC function and B cell development, indicating that these genes are important regulators of adult hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The stem cell–specific long noncoding RNA HOXA10-AS in the pathogenesis of KMT2A-rearranged leukemia

2019 ◽  
Vol 3 (24) ◽  
pp. 4252-4263
Author(s):  
Sina Al-Kershi ◽  
Raj Bhayadia ◽  
Michelle Ng ◽  
Lonneke Verboon ◽  
Stephan Emmrich ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Target Genes ◽  
Hox Genes ◽  
Locked Nucleic Acid ◽  
Monocytic Differentiation ◽  
Hoxa Cluster ◽  
Hematopoietic Stem ◽  
Human Cd34

Abstract HOX genes are highly conserved, and their precisely controlled expression is crucial for normal hematopoiesis. Accordingly, deregulation of HOX genes can cause leukemia. However, despite of intensive research on the coding HOX genes, the role of the numerous long noncoding RNAs (lncRNAs) within the HOX clusters during hematopoiesis and their contribution to leukemogenesis are incompletely understood. Here, we show that the lncRNA HOXA10-AS, located antisense to HOXA10 and mir-196b in the HOXA cluster, is highly expressed in hematopoietic stem cells (HSCs) as well as in KMT2A-rearranged and NPM1 mutated acute myeloid leukemias (AMLs). Using short hairpin RNA– and locked nucleic acid-conjugated chimeric antisense oligonucleotide (LNA-GapmeR)–mediated HOXA10-AS-knockdown and CRISPR/Cas9-mediated excision in vitro, we demonstrate that HOXA10-AS acts as an oncogene in KMT2A-rearranged AML. Moreover, HOXA10-AS knockdown severely impairs the leukemic growth of KMT2A-rearranged patient-derived xenografts in vivo, while high HOXA10-AS expression can serve as a marker of poor prognosis in AML patients. Lentiviral expression of HOXA10-AS blocks normal monocytic differentiation of human CD34+ hematopoietic stem and progenitor cells. Mechanistically, we show that HOXA10-AS localizes in the cytoplasm and acts in trans to induce NF-κB target genes. In total, our data imply that the normally HSC-specific HOXA10-AS is an oncogenic lncRNA in KMT2A-r AML. Thus, it may also represent a potential therapeutic target in KMT2A-rearranged AML.

Leukemogenic transformation by HOXA cluster genes

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2910-2918 ◽  
Author(s):  
Christian Bach ◽  
Sebastian Buhl ◽  
Dorothée Mueller ◽  
María-Paz García-Cuéllar ◽  
Emanuel Maethner ◽  
...  
Keyword(s):  
Hox Genes ◽  
Kinetic Studies ◽  
Cellular Transformation ◽  
Hoxa Cluster ◽  
Oncogenic Potential ◽  
Hematopoietic Malignancy ◽  
Permanent Cell ◽  
Hoxa Genes ◽  
Hoxa Gene

Abstract HOX homeobox genes are important regulators of normal and malignant hematopoiesis. Abdominal-type HOXA genes like HOXA9 are highly leukemogenic. However, little is known about transformation by anterior HOXA genes. Here we performed a comprehensive assessment of the oncogenic potential of every HOXA gene in primary hematopoietic cells. With exception of HOXA2 and HOXA5, all HOXA genes caused a block or delay of hematopoietic differentiation and cooperated with Meis1. No evidence for the alleged tumor-suppressor function of HOXA5 could be found. Whereas all active HOXA genes immortalized mixed granulocytic/monocytic populations, HOXA13 preferentially specified monocytoid development. The anterior HOXA genes HOXA1, HOXA4, and HOXA6 transformed cells, generating permanent cell lines, although they did so less potently than HOXA9. Upon transplantation these lines induced myeloproliferation and acute myeloid leukemia in recipient animals. Kinetic studies with inducible HOX derivatives demonstrated that anterior HOXA genes autonomously contributed to cellular transformation. This function was not mediated by endogenous Hoxa9, which was persistently expressed in cells transformed by anterior HOX genes. In summary our results demonstrate a hitherto unexpected role of anterior HOXA genes in hematopoietic malignancy.

scholarly journals Hox genes in the coordination of embryonic development. Model of hourglass in the description of vertebrate ontogenesis

2021 ◽  
Vol 11 (12) ◽  
pp. 24-37
Author(s):  
Sergey Dolomatov ◽  
Vera Kazakova ◽  
Walery Zukow
Keyword(s):  
Embryonic Development ◽  
Tissue Regeneration ◽  
Target Genes ◽  
Hox Genes ◽  
Development Model ◽  
Tissue Morphogenesis ◽  
Hox Proteins ◽  
Temporal And Spatial

The paper analyzes the role of HOX genes in the processes of embryonic development of vertebrates. Based on the analysis, it is concluded that HOX genes are the most important regulators of embryonic development. The HOX genes predominantly realize their influence through specific HOX proteins that have the ability to regulate the expression of target genes. The order of expression of the HOX genes, as a rule, obeys the rule of temporal and spatial colinearity. This mechanism determines the temporal and spatial course of tissue morphogenesis during embryonic development and tissue regeneration in organisms that have reached the stage of maturity. The process of embryo morphogenesis, determined by highly conserved HOX genes, explains the appearance of the phylotypic period - the stage of embryonic development of vertebrates, at which embryos of different classes of vertebrates have distinct morphological similarities.

scholarly journals Transplacental delivery of retinoid: the role of retinol-binding protein and lipoprotein retinyl ester

2004 ◽  
Vol 286 (5) ◽  
pp. E844-E851 ◽  
Author(s):  
Loredana Quadro ◽  
Leora Hamberger ◽  
Max E. Gottesman ◽  
Vittorio Colantuoni ◽  
Rajasekhar Ramakrishnan ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Congenital Malformations ◽  
Binding Protein ◽  
Retinol Binding Protein ◽  
Wild Type ◽  
Maternal Circulation ◽  
Retinyl Ester ◽  
Retinyl Esters ◽  
Normal Embryonic Development

Retinoids are required for normal embryonic development. Both embryonic retinoid deficiency and excess result in congenital malformations. There is little understanding of the physiology underlying retinoid transfer from the maternal circulation to the embryo. We now report studies that explore this process using retinol-binding protein-deficient (RBP−/−) mice and mice that express human RBP on the RBP−/−background. Our studies establish that dietary retinoid, bound to lipoproteins, can serve as an important source for meeting tissue retinoid requirements during embryogenesis. Indeed, retinyl ester concentrations in the circulations of pregnant RBP−/−mice are significantly elevated over those observed in wild-type mice, suggesting that lipoprotein retinyl esters may compensate for the absence of retinol-RBP during pregnancy. We also demonstrate, contrary to earlier proposals, that maternal RBP does not cross the placenta and cannot enter the fetal circulation. Overall, our data indicate that both retinol-RBP and retinyl esters bound to lipoproteins are able to provide sufficient retinoid to the embryo to allow for normal embryonic development.

Inhibition of Retinoic Acid Signaling by the cdx-hox Pathway Is Essential for Blood Cell Formation during Embryogenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 134-134
Author(s):  
Alan J. Davidson ◽  
Yuan Wang ◽  
George Q. Daley ◽  
Leonard I. Zon
Keyword(s):  
Retinoic Acid ◽  
Time Course ◽  
Hox Genes ◽  
Embryoid Bodies ◽  
Zebrafish Embryos ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Critical Determinant ◽  
Hematopoietic Stem ◽  
Blood Formation

Abstract The zebrafish mutant kugelig (kgg) is caused by a defect in the caudal-related homeobox gene cdx4 and has a deficit in both ‘primitive’ and ‘definitive’ hematopoietic stem cells. The embryonic anemia in the mutants can be rescued by overexpressing hox genes such as hoxb7a and hoxa9a, but not hoxb8a. This suggests that specific hox genes are required to make mesoderm competent to form blood. To further explore this, we undertook a microarray analysis to identify differentially expressed genes in kgg mutants and wild-type embryos. We found that raldh2, an enzyme required for retinoic acid (RA) production, is overexpressed in kgg mutants during the early stages of blood formation. This data led us to hypothesize that RA may act to suppress blood formation and that the cdx-hox pathway functions to limit RA production, thereby permitting blood formation to occur. To test this, we treated wild-type zebrafish embryos with RA and found that they became severely anemic. Treating kgg embryos with DEAB, a chemical that blocks raldh2 activity, restored hematopoiesis in kgg mutants. Expression of hoxa9a was not rescued in these treated embryos, indicating that RA acts downstream of the hox genes. DEAB also induced an expansion of erythroid cells in wild-type embryos, thus supporting the notion that the levels of RA during development are a critical determinant for blood formation. By performing a time-course rescue experiment, we determined that DEAB is effective when scl + hematopoietic progenitors are first formed from mesoderm, suggesting that RA acts upstream of the blood-inducer scl. In support of this, SCL overexpression rescues GATA-1 expression in embryos treated with RA. We next looked at the effects of DEAB and RA on the formation of mouse hematopoietic progenitors arising from ES cell-derived embryoid bodies (EBs). Addition of DEAB to EBs between days 2 to 3 of development resulted in a 5–8 fold increase in ‘primitive’ erythroid colonies (CFU-Ep), analogous to our results in zebrafish embryos. In contrast, RA treatment caused a general inhibition in the growth of all colony types. Taken together, these results suggest a new model in which suppression of RA by the cdx-hox pathway is necessary for yolk sac hematopoiesis to occur. This model provides an explanation for how hox genes control the spatiotemporal formation of hematopoietic tissue during organogenesis and may shed new light on the pathogenesis of leukemias involving translocations of the cdx , hox, and retinoic acid receptors.

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