Zebrafish hematopoiesis has been shown to be very similar to that in mammals with evolutionary conserved genetic programs which drive the formation of analogous blood cell types. In Vertebrates blood cells formation consists in two successive waves. The primitive hematopoiesis produces predominantly erythrocytes and only some primitive macrophages while definitive hematopoiesis provides long term Hematopoietic Stem Cells (HSCs) able to give rise to mature blood lineages. The Notch signaling is an evolutionary conserved regulatory system implicated in regulating cell fate determination in various developmental processes included HSCs self renewal and blood lineages differentiation both in vitro and in vivo. One known inhibitor of Notch activity is the evolutionary conserved adaptor protein Numb. In mice, several lines of evidence suggest that Numb (Nb) and its homologue Numblike (Nbl) play redundant functions in specifying and maintaining neuronal differentiation. The expression of Nb and Nbl has been detected in most of the tissues of mouse embryos including the yolk sac and adult hematopoietic tissues in both mouse and human models. Hence, these findings rise the possibility that Nb and Nbl proteins could play a role also in the embryonic and adult hematopoietic system. It has been proposed that both Nb and Nbl are dispensable for hematopoiesis in adult mice but recent in vitro approaches provided evidences that Numb can modulate the specification of primitive erythrocytes through its interaction with Notch. Numb and Numblike homologues have been identified and cloned also in zebrafish where they are expressed in the whole embryo from the first cleavage stage to organogenesis stages and by 24hpf their expression become restricted to the anterior half of the embryo. Using morpholino antisense oligonucleotide (MO) knock-down of Numb and Numblike we observed embryos in which circulating blood cells are absent or severely reduced starting from the earliest time point that circulation can be detected. In order to assess the presence of differentiated primitive erythrocytes in nb/nbl morphants we analyzed the erythroid hemoglobin content by o-dianisidine staining in 48hpf morphants and we observed that only few differentiated red blood cells were detectable. Furthermore, to gain insight into the erythropoietic defects we injected the nb/nbl morpholino in the transgenic line Tg(gata1: DsRed). Injected embryos at about 28-30hpf showed that, at this stage of development, the overall fluorescence of nb/nbl morphants start to appear strongly reduced and at 48hpf and 72hpf only a little amount of red cells is present within the sinus venosus. Taking together, our results provide the first in vivo evidence of an involvement of Numb and Numblike in erythrocytes determination and differentiation during primitive hematopoiesis.

Zebrafish Numb and Numblike are involved in primitive erythrocytes differentiation / E. Bresciani, S. Confalonieri, S. Cimbro, E. Foglia, S. Carra, C. Lora Lamia, P.P. Di Fiore, F. Cotelli. ((Intervento presentato al 6. convegno European Zebrafish Genetics and Development Meeting tenutosi a Roma nel 2009.

Zebrafish Numb and Numblike are involved in primitive erythrocytes differentiation

E. Bresciani
Primo
;
S. Cimbro;E. Foglia;S. Carra;C. Lora Lamia;P.P. Di Fiore
Penultimo
;
F. Cotelli
Ultimo
2009

Abstract

Zebrafish hematopoiesis has been shown to be very similar to that in mammals with evolutionary conserved genetic programs which drive the formation of analogous blood cell types. In Vertebrates blood cells formation consists in two successive waves. The primitive hematopoiesis produces predominantly erythrocytes and only some primitive macrophages while definitive hematopoiesis provides long term Hematopoietic Stem Cells (HSCs) able to give rise to mature blood lineages. The Notch signaling is an evolutionary conserved regulatory system implicated in regulating cell fate determination in various developmental processes included HSCs self renewal and blood lineages differentiation both in vitro and in vivo. One known inhibitor of Notch activity is the evolutionary conserved adaptor protein Numb. In mice, several lines of evidence suggest that Numb (Nb) and its homologue Numblike (Nbl) play redundant functions in specifying and maintaining neuronal differentiation. The expression of Nb and Nbl has been detected in most of the tissues of mouse embryos including the yolk sac and adult hematopoietic tissues in both mouse and human models. Hence, these findings rise the possibility that Nb and Nbl proteins could play a role also in the embryonic and adult hematopoietic system. It has been proposed that both Nb and Nbl are dispensable for hematopoiesis in adult mice but recent in vitro approaches provided evidences that Numb can modulate the specification of primitive erythrocytes through its interaction with Notch. Numb and Numblike homologues have been identified and cloned also in zebrafish where they are expressed in the whole embryo from the first cleavage stage to organogenesis stages and by 24hpf their expression become restricted to the anterior half of the embryo. Using morpholino antisense oligonucleotide (MO) knock-down of Numb and Numblike we observed embryos in which circulating blood cells are absent or severely reduced starting from the earliest time point that circulation can be detected. In order to assess the presence of differentiated primitive erythrocytes in nb/nbl morphants we analyzed the erythroid hemoglobin content by o-dianisidine staining in 48hpf morphants and we observed that only few differentiated red blood cells were detectable. Furthermore, to gain insight into the erythropoietic defects we injected the nb/nbl morpholino in the transgenic line Tg(gata1: DsRed). Injected embryos at about 28-30hpf showed that, at this stage of development, the overall fluorescence of nb/nbl morphants start to appear strongly reduced and at 48hpf and 72hpf only a little amount of red cells is present within the sinus venosus. Taking together, our results provide the first in vivo evidence of an involvement of Numb and Numblike in erythrocytes determination and differentiation during primitive hematopoiesis.
2009
Settore BIO/06 - Anatomia Comparata e Citologia
Zebrafish Numb and Numblike are involved in primitive erythrocytes differentiation / E. Bresciani, S. Confalonieri, S. Cimbro, E. Foglia, S. Carra, C. Lora Lamia, P.P. Di Fiore, F. Cotelli. ((Intervento presentato al 6. convegno European Zebrafish Genetics and Development Meeting tenutosi a Roma nel 2009.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/153503
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