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A non-canonical function of zebrafish telomerase reverse transcriptase is required for developmental hematopoiesis.

Imamura S, Uchiyama J, Koshimizu E, Hanai J, Raftopoulou C, Murphey RD, Bayliss PE, Imai Y, Burns CE, Masutomi K, Gagos S, Zon LI, Roberts TM, Kishi S - PLoS ONE (2008)

Bottom Line: On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction.These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis.The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words).

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from "authentic" telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words).

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Related in: MedlinePlus

The knockdown of TERT in the zebrafish embryo results in severe cytopenia and in the impaired differentiation of hematopoietic cells.(A) Induction of apoptosis in hematopoietic cells of the zebrafish embryo. Both a low magnification of the whole body and higher magnification of the trunk region are shown in (a) and (c), respectively. White squares in the low magnification images designate the regions shown in the higher magnification images in the adjoining right panels. (a) Apoptotic cells were detected by a TUNEL assay of the ventral wall of the dorsal aorta (VW-DA) (shown by green arrow heads in the right panel) at 28 hpf. (b) Quantification of the TUNEL-positive cells in the ventral wall of dorsal aorta (VW-DA) at 28 hpf. The number of TUNEL-positive cells was estimated within the gated area indicated by the green dashed rectangle at the upper yolk extension. (c) Apoptotic cells were detected by a TUNEL assay of the caudal venous plexus (CVP) (shown by green arrow heads in the right panel) at 48 hpf. (d) Quantification of the TUNEL-positive cells detected in the CVP at 48 hpf. The quantity of TUNEL-positive cells was assessed.within the gated area indicated by the green dashed rectangle at the anatomical CVP region. (e) Detection of apoptosis in gata-1GFP-positive hematopoietic cells. Transverse sections through the trunk region of 48-hpf gata-1GFP embryos with the dorsal up are shown. The caudal artery (CA; upper) and caudal vein (CV; lower) are shown by orange arrow heads in the panels. By TUNEL assay, gata-1GFP-positive apoptotic cells in the CV are evident and indicated by white arrows. (B) Lateral views of 48, 72 and 120 hpf embryos following the co-injection of zTERT-MO1 (or Cont-MO1) and GFP-zTERT-cDNA (or GFP-cDNA) expression vectors (a–l); black arrowheads indicate the heart regions. Bright field pictures of blood cells in trunks of 72 hpf embryos after co-injection of zTERT-MO1 or Cont-MO1 and either a GFP-cDNA or GFP-zTERT-cDNA vector (m–p). The upper vessel is the dorsal artery (from left to right arrows) and the lower vessel is the posterior cardinal vein (from right to left arrows). (C) Quantitation of the circulating blood cell number in zTERT-MO- (blue circle) versus Cont-MO- (black open circle) injected embryos during 28–72 hpf (a). (b) Calculation of the percentage of the control circulating blood cell numbers at 72 hpf after co-injection of zTERT-MO or Cont-MO and either a GFP-control or GFP-zTERT-cDNA vector. Blood cell numbers were determined for 10 embryos from each group. (D) Whole-mount o-dianisidine staining for heme detection in uninjected, Cont-MO1- and zTERT-MO1-injected embryos during 32–168 hpf. Blood flow over the yolk sac and in the tail vessels results in brown staining in wild type (data not shown) and Cont-MO1-injected embryos during 32–168 hpf (ventral view). (E) H&E staining of blood cells in tissue sections of the arteries or veins of Cont-MO1- and TERT-MO1-injected embryos at 33 and 48 hpf, and Wright-Giemsa staining of isolated blood cells from Cont-MO1- and TERT-MO1-injected embryos at 48 hpf.
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pone-0003364-g002: The knockdown of TERT in the zebrafish embryo results in severe cytopenia and in the impaired differentiation of hematopoietic cells.(A) Induction of apoptosis in hematopoietic cells of the zebrafish embryo. Both a low magnification of the whole body and higher magnification of the trunk region are shown in (a) and (c), respectively. White squares in the low magnification images designate the regions shown in the higher magnification images in the adjoining right panels. (a) Apoptotic cells were detected by a TUNEL assay of the ventral wall of the dorsal aorta (VW-DA) (shown by green arrow heads in the right panel) at 28 hpf. (b) Quantification of the TUNEL-positive cells in the ventral wall of dorsal aorta (VW-DA) at 28 hpf. The number of TUNEL-positive cells was estimated within the gated area indicated by the green dashed rectangle at the upper yolk extension. (c) Apoptotic cells were detected by a TUNEL assay of the caudal venous plexus (CVP) (shown by green arrow heads in the right panel) at 48 hpf. (d) Quantification of the TUNEL-positive cells detected in the CVP at 48 hpf. The quantity of TUNEL-positive cells was assessed.within the gated area indicated by the green dashed rectangle at the anatomical CVP region. (e) Detection of apoptosis in gata-1GFP-positive hematopoietic cells. Transverse sections through the trunk region of 48-hpf gata-1GFP embryos with the dorsal up are shown. The caudal artery (CA; upper) and caudal vein (CV; lower) are shown by orange arrow heads in the panels. By TUNEL assay, gata-1GFP-positive apoptotic cells in the CV are evident and indicated by white arrows. (B) Lateral views of 48, 72 and 120 hpf embryos following the co-injection of zTERT-MO1 (or Cont-MO1) and GFP-zTERT-cDNA (or GFP-cDNA) expression vectors (a–l); black arrowheads indicate the heart regions. Bright field pictures of blood cells in trunks of 72 hpf embryos after co-injection of zTERT-MO1 or Cont-MO1 and either a GFP-cDNA or GFP-zTERT-cDNA vector (m–p). The upper vessel is the dorsal artery (from left to right arrows) and the lower vessel is the posterior cardinal vein (from right to left arrows). (C) Quantitation of the circulating blood cell number in zTERT-MO- (blue circle) versus Cont-MO- (black open circle) injected embryos during 28–72 hpf (a). (b) Calculation of the percentage of the control circulating blood cell numbers at 72 hpf after co-injection of zTERT-MO or Cont-MO and either a GFP-control or GFP-zTERT-cDNA vector. Blood cell numbers were determined for 10 embryos from each group. (D) Whole-mount o-dianisidine staining for heme detection in uninjected, Cont-MO1- and zTERT-MO1-injected embryos during 32–168 hpf. Blood flow over the yolk sac and in the tail vessels results in brown staining in wild type (data not shown) and Cont-MO1-injected embryos during 32–168 hpf (ventral view). (E) H&E staining of blood cells in tissue sections of the arteries or veins of Cont-MO1- and TERT-MO1-injected embryos at 33 and 48 hpf, and Wright-Giemsa staining of isolated blood cells from Cont-MO1- and TERT-MO1-injected embryos at 48 hpf.

Mentions: With regard to the morphological phenotypes associated with a zTERT knockdown, zTERT-MO-injected embryos appear normal throughout embryogenesis, although a slight growth retardation was observed until 24 hpf (data not shown). However, after 24 hpf, TERT morphants do not display marked gross morphological changes, and their growth and development proceeds at the almost the same rate as in the control animals. We compared the number of apoptotic cells in zTERT-MO-injected embryos with wild-type animals. Accordingly, in zTERT morphants, TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling)-positive apoptotic cells can be observed throughout the head and trunk and in the ventral wall of the dorsal aorta (VW-DA) where the first definitive hematopoietic stem cells presumably emerge at 28 hpf (Figure 2A, a, b), with a gradual reduction after this time period. However, at 48 hpf (up to 72 hpf) in the caudal venous plexus (CVP), TUNEL-positive cells are detectable again (Figure 2A, c, d). The detection of TUNEL-positive cells in zebrafish gata-1-promoter-driven GFP (gata-1GFP) transgenic fish at 48 hpf revealed that apoptotic cells were indeed involved in the blood cell population (Figure 2A, e). These results thus suggest that a zTERT deficiency causes impaired hematopoiesis.


A non-canonical function of zebrafish telomerase reverse transcriptase is required for developmental hematopoiesis.

Imamura S, Uchiyama J, Koshimizu E, Hanai J, Raftopoulou C, Murphey RD, Bayliss PE, Imai Y, Burns CE, Masutomi K, Gagos S, Zon LI, Roberts TM, Kishi S - PLoS ONE (2008)

The knockdown of TERT in the zebrafish embryo results in severe cytopenia and in the impaired differentiation of hematopoietic cells.(A) Induction of apoptosis in hematopoietic cells of the zebrafish embryo. Both a low magnification of the whole body and higher magnification of the trunk region are shown in (a) and (c), respectively. White squares in the low magnification images designate the regions shown in the higher magnification images in the adjoining right panels. (a) Apoptotic cells were detected by a TUNEL assay of the ventral wall of the dorsal aorta (VW-DA) (shown by green arrow heads in the right panel) at 28 hpf. (b) Quantification of the TUNEL-positive cells in the ventral wall of dorsal aorta (VW-DA) at 28 hpf. The number of TUNEL-positive cells was estimated within the gated area indicated by the green dashed rectangle at the upper yolk extension. (c) Apoptotic cells were detected by a TUNEL assay of the caudal venous plexus (CVP) (shown by green arrow heads in the right panel) at 48 hpf. (d) Quantification of the TUNEL-positive cells detected in the CVP at 48 hpf. The quantity of TUNEL-positive cells was assessed.within the gated area indicated by the green dashed rectangle at the anatomical CVP region. (e) Detection of apoptosis in gata-1GFP-positive hematopoietic cells. Transverse sections through the trunk region of 48-hpf gata-1GFP embryos with the dorsal up are shown. The caudal artery (CA; upper) and caudal vein (CV; lower) are shown by orange arrow heads in the panels. By TUNEL assay, gata-1GFP-positive apoptotic cells in the CV are evident and indicated by white arrows. (B) Lateral views of 48, 72 and 120 hpf embryos following the co-injection of zTERT-MO1 (or Cont-MO1) and GFP-zTERT-cDNA (or GFP-cDNA) expression vectors (a–l); black arrowheads indicate the heart regions. Bright field pictures of blood cells in trunks of 72 hpf embryos after co-injection of zTERT-MO1 or Cont-MO1 and either a GFP-cDNA or GFP-zTERT-cDNA vector (m–p). The upper vessel is the dorsal artery (from left to right arrows) and the lower vessel is the posterior cardinal vein (from right to left arrows). (C) Quantitation of the circulating blood cell number in zTERT-MO- (blue circle) versus Cont-MO- (black open circle) injected embryos during 28–72 hpf (a). (b) Calculation of the percentage of the control circulating blood cell numbers at 72 hpf after co-injection of zTERT-MO or Cont-MO and either a GFP-control or GFP-zTERT-cDNA vector. Blood cell numbers were determined for 10 embryos from each group. (D) Whole-mount o-dianisidine staining for heme detection in uninjected, Cont-MO1- and zTERT-MO1-injected embryos during 32–168 hpf. Blood flow over the yolk sac and in the tail vessels results in brown staining in wild type (data not shown) and Cont-MO1-injected embryos during 32–168 hpf (ventral view). (E) H&E staining of blood cells in tissue sections of the arteries or veins of Cont-MO1- and TERT-MO1-injected embryos at 33 and 48 hpf, and Wright-Giemsa staining of isolated blood cells from Cont-MO1- and TERT-MO1-injected embryos at 48 hpf.
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pone-0003364-g002: The knockdown of TERT in the zebrafish embryo results in severe cytopenia and in the impaired differentiation of hematopoietic cells.(A) Induction of apoptosis in hematopoietic cells of the zebrafish embryo. Both a low magnification of the whole body and higher magnification of the trunk region are shown in (a) and (c), respectively. White squares in the low magnification images designate the regions shown in the higher magnification images in the adjoining right panels. (a) Apoptotic cells were detected by a TUNEL assay of the ventral wall of the dorsal aorta (VW-DA) (shown by green arrow heads in the right panel) at 28 hpf. (b) Quantification of the TUNEL-positive cells in the ventral wall of dorsal aorta (VW-DA) at 28 hpf. The number of TUNEL-positive cells was estimated within the gated area indicated by the green dashed rectangle at the upper yolk extension. (c) Apoptotic cells were detected by a TUNEL assay of the caudal venous plexus (CVP) (shown by green arrow heads in the right panel) at 48 hpf. (d) Quantification of the TUNEL-positive cells detected in the CVP at 48 hpf. The quantity of TUNEL-positive cells was assessed.within the gated area indicated by the green dashed rectangle at the anatomical CVP region. (e) Detection of apoptosis in gata-1GFP-positive hematopoietic cells. Transverse sections through the trunk region of 48-hpf gata-1GFP embryos with the dorsal up are shown. The caudal artery (CA; upper) and caudal vein (CV; lower) are shown by orange arrow heads in the panels. By TUNEL assay, gata-1GFP-positive apoptotic cells in the CV are evident and indicated by white arrows. (B) Lateral views of 48, 72 and 120 hpf embryos following the co-injection of zTERT-MO1 (or Cont-MO1) and GFP-zTERT-cDNA (or GFP-cDNA) expression vectors (a–l); black arrowheads indicate the heart regions. Bright field pictures of blood cells in trunks of 72 hpf embryos after co-injection of zTERT-MO1 or Cont-MO1 and either a GFP-cDNA or GFP-zTERT-cDNA vector (m–p). The upper vessel is the dorsal artery (from left to right arrows) and the lower vessel is the posterior cardinal vein (from right to left arrows). (C) Quantitation of the circulating blood cell number in zTERT-MO- (blue circle) versus Cont-MO- (black open circle) injected embryos during 28–72 hpf (a). (b) Calculation of the percentage of the control circulating blood cell numbers at 72 hpf after co-injection of zTERT-MO or Cont-MO and either a GFP-control or GFP-zTERT-cDNA vector. Blood cell numbers were determined for 10 embryos from each group. (D) Whole-mount o-dianisidine staining for heme detection in uninjected, Cont-MO1- and zTERT-MO1-injected embryos during 32–168 hpf. Blood flow over the yolk sac and in the tail vessels results in brown staining in wild type (data not shown) and Cont-MO1-injected embryos during 32–168 hpf (ventral view). (E) H&E staining of blood cells in tissue sections of the arteries or veins of Cont-MO1- and TERT-MO1-injected embryos at 33 and 48 hpf, and Wright-Giemsa staining of isolated blood cells from Cont-MO1- and TERT-MO1-injected embryos at 48 hpf.
Mentions: With regard to the morphological phenotypes associated with a zTERT knockdown, zTERT-MO-injected embryos appear normal throughout embryogenesis, although a slight growth retardation was observed until 24 hpf (data not shown). However, after 24 hpf, TERT morphants do not display marked gross morphological changes, and their growth and development proceeds at the almost the same rate as in the control animals. We compared the number of apoptotic cells in zTERT-MO-injected embryos with wild-type animals. Accordingly, in zTERT morphants, TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling)-positive apoptotic cells can be observed throughout the head and trunk and in the ventral wall of the dorsal aorta (VW-DA) where the first definitive hematopoietic stem cells presumably emerge at 28 hpf (Figure 2A, a, b), with a gradual reduction after this time period. However, at 48 hpf (up to 72 hpf) in the caudal venous plexus (CVP), TUNEL-positive cells are detectable again (Figure 2A, c, d). The detection of TUNEL-positive cells in zebrafish gata-1-promoter-driven GFP (gata-1GFP) transgenic fish at 48 hpf revealed that apoptotic cells were indeed involved in the blood cell population (Figure 2A, e). These results thus suggest that a zTERT deficiency causes impaired hematopoiesis.

Bottom Line: On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction.These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis.The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words).

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from "authentic" telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words).

Show MeSH
Related in: MedlinePlus