The Haspin gene encodes an atypical serine/threonine mitotic kinase first discovered in mouse spermatocytes and preferentially expressed in tissues with a high rate of proliferating cells. Haspin acts at metaphase by phosphorylating threonine 3 of histone H3 (H3Thr3PH) and this modification allows the recruitment of the chromosomal passenger complex, a key factor required to orchestrate different steps of mitosis. In human cells, HASPIN depletion causes a decrease in H3Thr3 levels, resulting in premature loss of sister chromatid cohesion and in defects in chromosome alignment at metaphase. Haspin has been found in all eukaryotic organisms; however, up to know, its role during animal embryonic development has never been investigated. We decided to investigate its function and expression during zebrafish embryonic development and, to this aim, we took advantage of a morpholino (MO)-mediated knockdown approach and of the CRISPR-Cas9 knockout strategy. We identified and cloned the zebrafish haspin ortholog, together with a previously unknown splicing isoform, and we clarified its expression pattern during embryogenesis and in some adult tissues. We demonstrated a relevant maternal contribution for the haspin transcript and important levels of zygotic expression in tissues with a high rate of proliferating cells, such as the developing brain and hematopoietic tissues. We also detected haspin transcript in the adult gonads and found that its expression is significantly switched on after injury during adult fin tissue regeneration. Interestingly, after Haspin functional inactivation using two different MOs, a translation blocking (ATG MO) and a splicing one, we demonstrated that Haspin is involved in H3Thr3PH also in zebrafish. Moreover, microinjection of the haspin ATG MO results in high embryo mortality and severe defects during epiboly stages, indicating important alterations in cellular rearrangements and movements. A haspin stable mutant line was generated by using the CRISPR-Cas9 technology: we isolated three different mutant haspin alleles, all causing the formation of premature stop codons. Although they do not show evident phenotypic alterations during embryogenesis, embryos carrying a homozygous genotype for these mutations are not able to reach the adulthood stage, showing a high rate of mortality in the first three weeks of larval development, indicating that Haspin is fundamental for larval survival and growth. To conclude, we clarified various aspects of haspin expression pattern during zebrafish development and in adult organs. Even though we were not able yet to unambiguously define the phenotypic effect of Haspin functional inactivation by using a MO-mediated approach, we paved the way for the analysis of the effect of a complete haspin gene knockout during zebrafish development by generating a haspin stable KO line and by showing that this null mutant allele significantly affects larval survival and growth.

ANALYSIS OF THE IN VIVO FUNCTION OF HASPIN KINASE USING ZEBRAFISH AS A MODEL SYSTEM: KNOCKDOWN AND KNOCKOUT APPROACHES / G.r. Gallo ; tutor: Prof. Paolo Plevani. - : . Università degli Studi di Milano, 2016 May 25. ((28. ciclo, Anno Accademico 2015. [10.13130/gallo-guido-roberto_phd2016-05-25].

ANALYSIS OF THE IN VIVO FUNCTION OF HASPIN KINASE USING ZEBRAFISH AS A MODEL SYSTEM: KNOCKDOWN AND KNOCKOUT APPROACHES

GALLO, GUIDO ROBERTO
2016-05-25

Abstract

The Haspin gene encodes an atypical serine/threonine mitotic kinase first discovered in mouse spermatocytes and preferentially expressed in tissues with a high rate of proliferating cells. Haspin acts at metaphase by phosphorylating threonine 3 of histone H3 (H3Thr3PH) and this modification allows the recruitment of the chromosomal passenger complex, a key factor required to orchestrate different steps of mitosis. In human cells, HASPIN depletion causes a decrease in H3Thr3 levels, resulting in premature loss of sister chromatid cohesion and in defects in chromosome alignment at metaphase. Haspin has been found in all eukaryotic organisms; however, up to know, its role during animal embryonic development has never been investigated. We decided to investigate its function and expression during zebrafish embryonic development and, to this aim, we took advantage of a morpholino (MO)-mediated knockdown approach and of the CRISPR-Cas9 knockout strategy. We identified and cloned the zebrafish haspin ortholog, together with a previously unknown splicing isoform, and we clarified its expression pattern during embryogenesis and in some adult tissues. We demonstrated a relevant maternal contribution for the haspin transcript and important levels of zygotic expression in tissues with a high rate of proliferating cells, such as the developing brain and hematopoietic tissues. We also detected haspin transcript in the adult gonads and found that its expression is significantly switched on after injury during adult fin tissue regeneration. Interestingly, after Haspin functional inactivation using two different MOs, a translation blocking (ATG MO) and a splicing one, we demonstrated that Haspin is involved in H3Thr3PH also in zebrafish. Moreover, microinjection of the haspin ATG MO results in high embryo mortality and severe defects during epiboly stages, indicating important alterations in cellular rearrangements and movements. A haspin stable mutant line was generated by using the CRISPR-Cas9 technology: we isolated three different mutant haspin alleles, all causing the formation of premature stop codons. Although they do not show evident phenotypic alterations during embryogenesis, embryos carrying a homozygous genotype for these mutations are not able to reach the adulthood stage, showing a high rate of mortality in the first three weeks of larval development, indicating that Haspin is fundamental for larval survival and growth. To conclude, we clarified various aspects of haspin expression pattern during zebrafish development and in adult organs. Even though we were not able yet to unambiguously define the phenotypic effect of Haspin functional inactivation by using a MO-mediated approach, we paved the way for the analysis of the effect of a complete haspin gene knockout during zebrafish development by generating a haspin stable KO line and by showing that this null mutant allele significantly affects larval survival and growth.
PLEVANI, PAOLO
Settore BIO/11 - Biologia Molecolare
Settore BIO/06 - Anatomia Comparata e Citologia
ANALYSIS OF THE IN VIVO FUNCTION OF HASPIN KINASE USING ZEBRAFISH AS A MODEL SYSTEM: KNOCKDOWN AND KNOCKOUT APPROACHES / G.r. Gallo ; tutor: Prof. Paolo Plevani. - : . Università degli Studi di Milano, 2016 May 25. ((28. ciclo, Anno Accademico 2015. [10.13130/gallo-guido-roberto_phd2016-05-25].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/412957
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