Targeted Gene Disruption of Kinesin-3 Family Motors Unveils a Negative Regulatory Mechanism for Mouse Erythroblast Enucleation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 371-371
Author(s):  
Toshihiko Hanada ◽  
Yunzhe Lu ◽  
Athar H Chishti
Keyword(s):  
Cell Division ◽  
Fusion Protein ◽  
Asymmetric Cell Division ◽  
Fetal Liver ◽  
Mutant Mice ◽  
Lacz Fusion ◽  
Erythroid Precursors ◽  
Lacz Activity ◽  
Mutant Mouse Line

Abstract Abstract 371 Terminal differentiation of mammalian erythroid precursors involves enucleation, a process required for the production of reticulocytes. The signaling pathways and the molecular components mediating the final enucleation step are not well understood, although the resemblance of enucleation to asymmetric cell division has been suggested. The enucleation of erythroblasts can be replicated in vitro; however, the enucleation efficiency is not optimal under in vitro culture conditions. A functional role of both microfilaments and microtubules has been suggested in the regulation of erythroblast enucleation. We hypothesized that molecular motors known to regulate asymmetric cell division might also play a functional role in erythroblast enucleation. GAKIN (also called KIF13B) is a kinesin-3 motor implicated in the regulation of cell polarity pathways. Its Drosophila homologue Khc-73 regulates polarity formation during the asymmetric cell division of neuroblasts. We generated a mutant mouse line of GAKIN and examined the efficiency of erythroblast enucleation using the fetal liver in vitro erythroid precursor culture system originally developed by the Lodish group. The GAKIN mutant mouse line was produced from the embryonic stem (ES) cells containing a genomic insertion of the beta-galactosidase-neomycin gene. The beta-geo insertion was mapped within intron 38 of the GAKIN gene, thus removing the CAP-Gly domain located at the C-terminus of GAKIN. This allowed us to visualize specific tissue expression of GAKIN, in addition to the subcellular localization of a GAKIN-lacZ fusion protein by lacZ activity staining. The lacZ activity revealed that the GAKIN-lacZ fusion protein is expressed in bone marrow macrophages and erythroblasts, while also highly concentrated at contact sites between macrophages and erythroblasts. GAKIN mutant mice are viable and fertile, and complete blood analysis did not reveal any discernible phenotype. To test for the enucleation efficiency, erythroid precursors were isolated from fetal liver stage at day 14.5 embryos and enucleation was quantified by flow cytometry after 2 days of in vitro culture. The GAKIN-lacZ fusion protein appeared as a single dot representing the microtubule organizing center (MTOC) in the erythroid precursors isolated from fetal liver (Fig. 1). However, GAKIN mutant erythroblasts enucleated at the same efficiency as wild type erythroblasts under steady state conditions in vitro. Since the mammalian genome contains a close homologue of GAKIN/KIF13B, termed KIF13A, a possibility exists that KIF13A functionally compensates for GAKIN mutation in critical pathways. To address this issue, we generated KIF13A null mice using a similar gene disruption strategy. Again, the KIF13A mutant erythroblasts did not show any measurable change in enucleation efficiency under similar conditions. However, fetal liver erythroblasts isolated from GAKIN and KIF13A double mutant mice exhibited a significant enhancement of enucleation efficiency. This finding suggests that together, GAKIN and KIF13A negatively regulate the erythroblast enucleation by modulating the microtubule-based cytoskeleton. We propose that efficient erythroid enucleation in vivo involves signaling mechanisms inhibiting both GAKIN and KIF13A motors. Since erythrocytes from adult double mutant mice appear to be hematologically normal, our findings raise the possibility of enhancing the in vitro production of functional erythrocytes by inhibiting GAKIN and KIF13A activity in CD34 positive stem cells. Disclosures: No relevant conflicts of interest to declare.

Unique Differentiation Programs of Human Fetal Liver Stem Cells Shown Both In Vitro and In Vivo in NOD/SCID Mice

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2686-2695 ◽  
Author(s):  
Franck E. Nicolini ◽  
Tessa L. Holyoake ◽  
Johanne D. Cashman ◽  
Pat P.Y. Chu ◽  
Karen Lambie ◽  
...  
Keyword(s):  
Cord Blood ◽  
Fetal Liver ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Human Fetal Liver ◽  
Human Erythropoietin ◽  
Erythroid Precursors ◽  
Lower Ratio

Comparative measurements of different types of hematopoietic progenitors present in human fetal liver, cord blood, and adult marrow showed a large (up to 250-fold), stage-specific, but lineage-unrestricted, amplification of the colony-forming cell (CFC) compartment in the fetal liver, with a higher ratio of all types of CFC to long-term culture-initiating cells (LTC-IC) and a lower ratio of total (mature) cells to CFC. Human fetal liver LTC-IC were also found to produce more CFC in LTC than cord blood or adult marrow LTC-IC, and more of the fetal liver LTC-IC–derived CFC were erythroid. Human fetal liver cells regenerated human multilineage hematopoiesis in NOD/SCID mice with the same kinetics as human cord blood and adult marrow cells, but sustained a high level of terminal erythropoiesis not seen in adult marrow-engrafted mice unless exogenous human erythropoietin (Epo) was injected. This may be due to a demonstrated 10-fold lower activity of murine versus human Epo on human cells, sufficient to distinguish between a differential Epo sensitivity of fetal and adult erythroid precursors. Examination of human LTC-IC, CFC, and erythroblasts generated either in NOD/SCID mice and/or in LTC showed the types of cells and hemoglobins produced also to reflect their ontological origin, regardless of the environment in which the erythroid precursors were generated. We suggest that ontogeny may affect the behavior of cells at many stages of hematopoietic cell differentiation through key changes in shared signaling pathways.

scholarly journals Erythropoiesis in vitro: enhancement by neuraminidase

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 483-490 ◽  
Author(s):  
PT Rowley ◽  
BM Ohlsson-Wilhelm ◽  
BA Farley
Keyword(s):  
Fetal Liver ◽  
Fetal Cell ◽  
Neuraminidase Treatment ◽  
Human Fetal Liver ◽  
Erythroid Precursors ◽  
Stimulation Of ◽  
Marrow Cells

Abstract Neuraminidase treatment of human fetal liver or adult marrow cells prior to culture results in an increased number of erythroid colonies and bursts. No increase occurs in the number of nonerythroid colonies. The number of bursts having more than eight subunits is increased preferentially. Individual burst subunits are also enlarged. Neuraminidase-treated cells yield erythroid bursts when cultured in concentrations of erythropoietin insufficient to produce bursts from untreated cells. It is proposed that (1) neuraminidase treatment of adult and fetal cell mixtures specifically stimulates differentiation of erythroid precursors, (2) the preferential stimulation of erythroid bursts having many subunits suggests a preferential susceptibility of more primitive BFU-Es, and (3) neuraminidase treatment enhances the response of erythroid precursors to erythropoietin.

Low concentrations of caffeine induce asymmetric cell division as observed in vitro by means of the CBMN-assay and iFISH

2015 ◽  
Vol 793 ◽  
pp. 71-78 ◽  
Author(s):  
Vasiliki I. Hatzi ◽  
Maria Karakosta ◽  
Katarzyna Barszczewska ◽  
Ioanna Karachristou ◽  
Gabriel Pantelias ◽  
...  
Keyword(s):  
Cell Division ◽  
Asymmetric Cell Division ◽  
Low Concentrations ◽  
Cbmn Assay

The microtubule modulator RanBP10 plays a critical role in regulation of platelet discoid shape and degranulation

Blood ◽  
2009 ◽  
Vol 114 (27) ◽  
pp. 5532-5540 ◽  
Author(s):  
Stefan Kunert ◽  
Imke Meyer ◽  
Silke Fleischhauer ◽  
Martin Wannack ◽  
Janine Fiedler ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Critical Role ◽  
Mutant Mice ◽  
Axis Ratio ◽  
Prolonged Bleeding Time ◽  
Normal Platelet ◽  
And Function ◽  
Granule Release

Abstract Terminally mature megakaryocytes undergo dramatic cellular reorganization to produce hundreds of virtually identical platelets. A hallmark feature of this process is the generation of an elaborate system of branched protrusions called proplatelets. We recently identified RanBP10 as a tubulin-binding protein that is concentrated along polymerized microtubules in mature megakaryocytes. RanBP10 depletion in vitro caused the disturbance of polymerized filaments. Here we study the function of RanBP10 in vivo by generating deficient mice using a gene-trap approach. Mutant mice show normal platelet counts, and fetal liver-derived megakaryocytes reveal only slightly reduced proplatelet formation. However, ultrastructural analysis unveiled a significantly increased geometric axis ratio for resting platelets, and many platelets exhibited disorders in microtubule filament numbers and localization. Mutant mice showed a markedly prolonged bleeding time. Granule release, a process that depends on internal contraction of the microtubule marginal coil, also was reduced. Flow cytometry analysis revealed reduced expression of CD62P and CD63 after PAR4-peptide stimulation. These data suggest that RanBP10 plays an essential role in hemostasis and in maintaining microtubule dynamics with respect to both platelet shape and function.

scholarly journals Erythropoiesis in vitro: enhancement by neuraminidase

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 483-490
Author(s):  
PT Rowley ◽  
BM Ohlsson-Wilhelm ◽  
BA Farley
Keyword(s):  
Fetal Liver ◽  
Fetal Cell ◽  
Neuraminidase Treatment ◽  
Human Fetal Liver ◽  
Erythroid Precursors ◽  
Stimulation Of ◽  
Marrow Cells

Neuraminidase treatment of human fetal liver or adult marrow cells prior to culture results in an increased number of erythroid colonies and bursts. No increase occurs in the number of nonerythroid colonies. The number of bursts having more than eight subunits is increased preferentially. Individual burst subunits are also enlarged. Neuraminidase-treated cells yield erythroid bursts when cultured in concentrations of erythropoietin insufficient to produce bursts from untreated cells. It is proposed that (1) neuraminidase treatment of adult and fetal cell mixtures specifically stimulates differentiation of erythroid precursors, (2) the preferential stimulation of erythroid bursts having many subunits suggests a preferential susceptibility of more primitive BFU-Es, and (3) neuraminidase treatment enhances the response of erythroid precursors to erythropoietin.

Inducible Expression of Leukemia-Associated Shp2 (Ptpn11) Affects Multiple Stages of Hematopoiesis and Causes a Fatal Myeloproliferative Disorder (MPD) in Mice

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1529-1529 ◽  
Author(s):  
Gordon Chan ◽  
Demetrios Kalaitzidis ◽  
M. Golam Mohi ◽  
Wentian Yang ◽  
Jeffery Kutok ◽  
...  
Keyword(s):  
Germ Line ◽  
Fetal Liver ◽  
Lymphoblastic Leukemia ◽  
Tyrosine Phosphatase ◽  
Specific Effect ◽  
Mutant Mice ◽  
Juvenile Myelomonocytic Leukemia ◽  
Increased Risk

Abstract Germ-line mutations in ptpn11, which encodes the protein tyrosine phosphatase Shp2, cause ∼50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ∼35% of sporadic JMML; nearly all other cases have either activating Ras mutations or homozygous Nf1 deficiency. Shp2 mutations are also found at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and B-cell acute lymphoblastic leukemia (B-ALL). NS and leukemia-associated Shp2 mutations can affect the same residue, but result in different substitutions. We showed previously that the leukemia-associated mutants E76K or D61Y, but not wild type (WT), Shp2 transform bone marrow (BM) or fetal liver cells. Transplantation of E76K- or D61Y-transduced BM evoked invasive MPD in ∼60% of recipients, with most dying at 6–7 months. In this model, mutant Shp2 is expressed under retroviral promoter control and the phenotype is variable and incompletely penetrant. To assess the effect of expressing a leukemogenic Shp2 allele under endogenous promoter control, we generated knock-in mice that can conditionally express Shp2D61Y (LSL-Shp2D61Y). Global expression of the D61Y heterozygous allele was embryonic lethal. Post-natal expression of D61Y, induced by treating Mx-1 Cre; LSL-Shp2D61Y mice with pI-pC, evoked fatal MPD with ∼50% mice dying within 5–7 months. Mutant mice showed a marked increase in WBC, expansion of the Gr-1+/Mac-1+population in BM and spleen, and histopathological evidence of infiltrating MPD. Unlike in our retroviral transduction/transplant model (and similar to JMML patients), most induced LSL-Shp2D61Y mice were also anemic. Mutant mice exhibited a marked depletion of quiescent LSK (Lin−Sca1+cKit+) cells in the BM, with a concomitant increase in LSK cells in the spleen. Interestingly, Shp2 mutant-expressing BM failed to promote long term reconstitution in BM transplant assays. Cells from mutant spleens did have some long term multi-lineage reconstitution activity, but the level was substantially less than predicted by their LSK content. Moreover, no transplant recipients develop MPD. In contrast to these effects on the stem cell compartment, D61Y directly allowed cytokine-independent differentiation of CMP and GMP in vitro. Macrophages from mutant mice showed enhanced GM-CSF-mediated proliferation and ERK activation. Our results showed that expression of leukemia-associated Shp2 at endogenous levels is sufficient to evoke MPD and has cell type-specific effect on different stages of hematopoiesis.

Extensively Self-Renewing Erythroid Precursors Emerge from the Embryo but Not from the Adult

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2455-2455
Author(s):  
Samantha England ◽  
Kathleen E. McGrath ◽  
James Palis
Keyword(s):  
Fetal Liver ◽  
Globin Gene ◽  
Yolk Sac ◽  
Erythroid Progenitor ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Erythroid Precursor ◽  
Stress Erythropoiesis ◽  
Erythroid Precursors

Abstract The only cell in the hematopoietic hierarchy thought to be capable of long-term selfrenewal is the stem cell. An erythroid progenitor derived from mammalian hematopoietic tissue, fetal or adult, is capable of limited proliferation (103–106 fold expansion; Bauer, 1999; Panzenböck, 1998; von Lindern, 1999). Here we report that an erythroid precursor derived from the mouse embryo is capable not only of limited, but also of extensive proliferation (~1030 fold expansion). These cells resemble proerythroblasts and basophilic erythroblasts based on their morphology, their globin gene expression profile, and their immunophenotype. While aneuploidy is not necessary for extensive proliferation, it sporadically begins to accumulate after prolonged culture. These cells are capable of massive (>100 days) daily proliferation in vitro in the presence of Epo, SCF, IGF-1, and dexamethasone. Examination of cultures lacking each of these factors support that glucocorticoids play an important role in this expansion by uncoupling erythroid precursor proliferation from maturation. Despite prolonged in vitro culture, these cells preserve their potential to fully differentiate into enucleated red blood cells with the removal of dexamethasone. Differentiation occurs over 2–3 days and is characterized by the accumulation of adult (α, β1, and β2), but not embryonic (ζ, εy, and βH1), globins. The retention of full differentiation potential despite >1030 fold expansion indicates that this proliferation represents self-renewal. To determine the developmental origin of these extensively self-renewing erythroblasts (ESREs), we initiated in vitro cultures from staged mouse embryos as well as adult tissues. E7.5 embryos, that contain primitive but not definitive erythroid progenitors, failed to generate ESREs. In contrast, ESREs can be derived from E8.5–E10.5 yolk sac and E11.5–E14.5 fetal liver. These findings along with the globin expression pattern indicate that erythroblast self-renewal is associated with definitive, but not primitive, erythropoiesis. Surprisingly, marrow from adult steady-state hematopoiesis failed to yield ESREs. Furthermore, despite the characteristics shared by stress erythropoiesis (adult spleen) and fetal erythropoiesis (liver), stress erythropoiesis only yielded erythroblasts with limited, and not extensive, self-renewal capacity. This result suggests that extensive self-renewal potential is linked either to the transient yolk sac-derived definitive erythroid lineage or to the fetal hematopoietic microenvironment. We are currently investigating the mechanisms responsible for the extensive self-renewal capacity of such lineage-restricted and mature hematopoietic precursors. Our findings raise the possibility that the expansive cellular output of the erythron within the midgestation mammalian embryo may be regulated, in part, at the level of late stage erythroid precursors.

Asymmetric segregation and self-renewal of hematopoietic stem and progenitor cells with endocytic Ap2a2

Blood ◽  
2012 ◽  
Vol 119 (11) ◽  
pp. 2510-2522 ◽  
Author(s):  
Stephen B. Ting ◽  
Eric Deneault ◽  
Kristin Hope ◽  
Sonia Cellot ◽  
Jalila Chagraoui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Division ◽  
Asymmetric Cell Division ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Definitive Evidence

Abstract The stem cell–intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions.

scholarly journals A transient definitive erythroid lineage with unique regulation of the β-globin locus in the mammalian embryo

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4600-4608 ◽  
Author(s):  
Kathleen E. McGrath ◽  
Jenna M. Frame ◽  
George J. Fromm ◽  
Anne D. Koniski ◽  
Paul D. Kingsley ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Expression Patterns ◽  
Yolk Sac ◽  
Globin Genes ◽  
Mammalian Embryo ◽  
Hematopoietic Stem ◽  
Erythroid Precursors ◽  
Low Levels ◽  
Definitive Erythropoiesis

Abstract A transient erythromyeloid wave of definitive hematopoietic progenitors (erythroid/myeloid progenitors [EMPs]) emerges in the yolk sac beginning at embryonic day 8.25 (E8.25) and colonizes the liver by E10.5, before adult-repopulating hematopoietic stem cells. At E11.5, we observe all maturational stages of erythroid precursors in the liver and the first definitive erythrocytes in the circulation. These early fetal liver erythroblasts express predominantly adult β-globins and the definitive erythroid-specific transcriptional modifiers c-myb, Sox6, and Bcl11A. Surprisingly, they also express low levels of “embryonic” βH1-, but not εy-, globin transcripts. Consistent with these results, RNA polymerase and highly modified histones are found associated with βH1- and adult globin, but not εy-globin, genes. E11.5 definitive proerythroblasts from mice transgenic for the human β-globin locus, like human fetal erythroblasts, express predominately human γ-, low β-, and no ε-globin transcripts. Significantly, E9.5 yolk sac–derived EMPs cultured in vitro have similar murine and human transgenic globin expression patterns. Later liver proerythroblasts express low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. We conclude that yolk sac–derived EMPs, the first of 2 origins of definitive erythropoiesis, express a unique pattern of globin genes as they generate the first definitive erythrocytes in the liver of the mammalian embryo.

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