- Email: [email protected]
Iron deficiency anemia’s effect on bone formation in zebrafish mutant
Accepted Manuscript Iron deficiency anemia's affect on bone formation in zebrafish mutant Lin Bo, Zhichun Liu, Yingbin Zhong, Jian Huang, Bin Chen, Han Wang, Youjia Xu PII:
S0006-291X(16)30768-9
DOI:
10.1016/j.bbrc.2016.05.069
Reference:
YBBRC 35819
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 2 May 2016 Accepted Date: 12 May 2016
Please cite this article as: L. Bo, Z. Liu, Y. Zhong, J. Huang, B. Chen, H. Wang, Y. Xu, Iron deficiency anemia's affect on bone formation in zebrafish mutant, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.05.069. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Iron deficiency anemia's affect on bone formation in zebrafish mutant Lin Bo1 MD, Zhichun Liu1 PhD, Yingbin Zhong2 PhD, Jian Huang2 PhD, Bin Chen3 MD, Han Wang2* PhD, Youjia Xu3* PhD Department of Rheumatology, The Second Affiliated Hospital of Soochow University,
RI PT
1
Suzhou 215004, Jiangsu, China; 2 Center for Circadian Clocks, Soochow University, Suzhou 215003, Jiangsu, China; 3 Department of Orthopaedics, The Second Affiliated
SC
Hospital of Soochow University, Suzhou 215004, Jiangsu, China
M AN U
Correspondence Youjia Xu ([email protected])
Department of Orthopaedics, The Second Affiliated Hospital of Soochow University 1055 Sanxiang Road,
TE D
Suzhou, Jiangsu 215123
Phone:+86-512-67783346
EP
Han Wang ([email protected])
AC C
Center for Circadian Clocks, Soochow University 199 Renai Road,
Suzhou, Jiangsu 215123 Phone: +86-512-65882115 This work was supported in part by the Natural Science Foundation of China (81200507), Clinical Medical Science and Technology Fond of Jiangsu Province (BL2014044), the Scientific and Technological Development Project of Suzhou
ACCEPTED MANUSCRIPT (SYS201341), and the Young Teachers’ Natural Science Foundation of Soochow University (SDY2013A34). All institutional and national guidelines for the care and
AC C
EP
TE D
M AN U
SC
RI PT
use of laboratory animals were followed.
ACCEPTED MANUSCRIPT Iron deficiency anemia affect bone formation in zebrafish mutant ABSTRACT Iron is one of the essential elements of life. Iron metabolism is related to bone
RI PT
metabolism. Previous studies have confirmed that iron overload is a risk factor for osteoporosis. But the correlation between iron deficiency and bone metabolism
SC
remains unclear. Ferroportin 1 is identified as a cellular iron exporter and required for normal iron cycling. In human, the mutation in Ferroportin 1 gene caused
M AN U
hemochromatosis (iron overload) syndrome. In zebrafish, the mutant of ferroportin 1 gene (fpn1), Wehtp85c exhibited the defective iron transport, leading to developing severe hypochromic anemia. To understand the mechanism of fpn1 in iron regulation and bone formation, we used Wehtp85c as a model for investigating iron deficiency and
TE D
bone metabolism. In this study, we examined the morphology of the developing cartilage and vertebrae of the Wehtp85 compared to the wild type siblings by staining
EP
the larvae with alcian blue for cartilage and alizarin red for the bone. In addition, we evaluated the expression patterns of the marker genes of bone development and cell
AC C
signaling in bone formation. Our results showed that wehtp85c mutant larvae exhibited the defects in bone formation, revealing by decreases in the number of calcified vertebrae along with decreased expression of osteoblast novel genes: alpl，runx2a and col1a1a and BMPs signaling genes in osteoblast differentiation: bmp2a and bmp2b. Our data suggest that iron deficiency anemia affects bone formation, potentially through the BMPs signaling pathway in zebrafish.
ACCEPTED MANUSCRIPT Keywords Fpn1
RI PT
Iron deficiency anemia Bone formation Zebrafish
SC
INTRODUCTION
M AN U
Bone is a dynamic connective tissue, which constantly remodels itself to accommodate growth and mechanical loads, and to maintain mineral homoeostasis. Bone metabolism is influenced by some trace elements in the body, such as calcium, phosphorus, magnesium, etc.
TE D
Iron is essential for growth and proliferation of cells because it act as a protein cofactor for hemoglobin synthesis, DNA synthesis and mitochondrial respiration. The
EP
regulation of iron content is important since iron overload prompt the generation of reactive oxygen species which damage DNA and proteins, while iron deficiency leads
AC C
to cell cycle arrest even cell death[1, 2]. Dysregulation of iron homeostasis may cause hematological, metabolic and neurological diseases. In recent years, the correlation between bone metabolism and iron is being a hotspot of research. Weinberg
[3]
firstly proposed that iron overload was a risk factor for
osteoporosis in 2006. Kim et al. [4] conducted a 3-year retrospective longitudinal study and demonstrated that iron overload is related to osteoporosis in healthy adults, especially in postmenopausal women. Further more, several previous studies have
ACCEPTED MANUSCRIPT proved that iron overload promoted osteoclast differentiation and bone reabsorption [5, 6]
. Iron overload has been recognized as an independent risk factor for osteoporosis.
However, besides iron overload, abnormal iron homeostasis also includes iron
RI PT
deficiency. Iron deficiency is one of widespread diseases in human beings with major consequences for human health and socioeconomic development. Globally, 1.5-1.7 billion people suffered by iron deficiency anemia, approximately 24.8% of the world
SC
population, while the highest prevalence is in preschool-age children (47.4%,
M AN U
283–303 million children) [7]. Previous studies have proved that iron deficiency, even in absence of anemia, adversely affects the physical growth and cognitive performance of children
[8]
. It can also affect the immune status of human beings
[9]
.
In terms of bone metabolism, it has been reported that iron deficiency diet can affect [10]
. However, the molecular mechanism of the iron deficiency effect
TE D
bone formation
on bone metabolism still remains unclear.
[11-13]
.
EP
Severe iron deficiency anemia has a negative effect on bone metabolism
However, the relationship between iron deficiency and bone loss remains unclear. The
AC C
zebrafish model is rapidly gaining prominence in the study of many aspects of biological and medical research. For example, zebrafish can serve as a good model for studying the development of the skeletal system due to its rapid bone development, simple bone structure, external embryonic development, and body transparence. The latter allows for easy observations of bone morphology in live embryos or larvea [14-16]. In
addition,
in
the
early stage
of
zebrafish
bone
development (<
20 dpf), osteoclast has not yet appeared while only osteoblast and bone formation
ACCEPTED MANUSCRIPT exist[17]. Zebrafish mutant, wehtp85may provide a useful tool for studies of diseases related to pathological bone formation, such as osteoporosis or osteomalacia. Wehtp85c is the autosomal recessive zebrafish mutants of ferroportin 1 gene (fpn1), in
between 10 and 14 days of age
[18]
RI PT
which homozygotes develop severe anemia within 48 hours after fertilization and dies . Fpn1 is the only non-heme iron exporter with
function in iron efflux, which is mainly expressed in duodenal mature enterocytes,
. Dietary iron exists mainly in form of ferric (Fe3+) and is reduced to ferrous (Fe2+)
M AN U
[2]
SC
reticuloendothelial macrophages, placental syncytiotrophoblast cells and hepatocytes
by duodenal cytochrome B (Dcyt B) prior to transport. The transport of non-heme iron across the apical membrane occurs via the divalent metal transporter 1 (DMT1). Cytosolic iron in intestinal enterocytes can be either stored in ferritin or exported into
TE D
plasma by the basolateral iron exporter FPN1
[19]
. In the plasma, iron binds with
transferrin and is imported into erythroid precursors and other cells via transferrinendocytosis.
Macrophages
also
play
an
important
role
EP
receptor-mediated
in iron delivery to plasma transferrin through phagocytosis of senescent red blood cell,
AC C
heme catabolism and recycling of iron [20]. In Wehtp85c mutant the hypochromia is caused by inadequate circulatory iron levels， although the erythroid cells are fully capable of haemoglobinization. Therefore, wehtp85c is one of the suitable models of iron deficiency anemia. In this study, we examined the morphology of developing cartilage and vertebrae of the Wehtp85c larvae by staining the fish with alcian blue and alizarin red, respectively.
ACCEPTED MANUSCRIPT In addition, we evaluated the gene expression patterns of bone development markers (e.g., alpl，runx2a，col1a1a and sox9b) and genes involved in cellular signaling during bone development, such as bmp2a and bmp2b. Our investigation may provide a basis
RI PT
for the research of diseases related to pathological bone metabolism, such as iron deficiency anemia.
SC
MATERIALS AND METHODS Animals
M AN U
Wehtp85c Heterozygous zebrafish used in this study were donated by Professor Zhou Yi (Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston,
TE D
Massachusetts 02115 USA). Breeding colonies were kept in 28.5℃with a 10/14h dark/light cycle and staged by hours post-fertilization (hpf) or days post-fertilization (dpf). Protocols for experimental procedures were according to NIH guidelines.
EP
Mutant homozygotes screening
AC C
Ten female and twenty male (4-6 months old) heterozygotes were used for mating to generate the F1 fish, which contain wehtp85c homozygotes, heterozygotes and wild-type siblings. The wehtp85c homozygotes were identified in a morphologic screen for defects in circulating erythroid cells at 48 hpf [18]. All of the wehtp85c heterozygous zebrafish were identified by gene sequencing. O-dianisidine staining
ACCEPTED MANUSCRIPT The expression of the hemoglobin can be demonstrated by the O-dianisidine staining method. The zebrafish at 48 hpf were dechorionated and stained with O-dianisidine solution (0.6mg/ml), sodium acetate (0.01M, pH4.5), H2O2 (0.65%), ethanol (40%) in
RI PT
the dark for 15 minutes[21]. The O-dianisidine solution was removed and phosphatebuffered saline (PBS) was added to stop the reaction. The embryos were observed under the microscope. Brown precipitate is formed when the heme in the hemoglobin
SC
reacts with the O-dianisidine solution. Changes in the intensity of brown precipitate
M AN U
can be considered as a semi-quantitative estimation of erythroid development in zebrafish embryos.
Alizarin Red S and alcian blue staining
Bone and cartilage were stained by alcian blue (CAS No: 33864-99-2) and alizarin
specimen [22].
TE D
red S (CAS No: 130-22-3, Sigma-Aldrich, Diegem, Belgium) respectively on fixed
EP
Quantitative RT-PCR
AC C
Total RNA of larvae were isolated using Trizol (Ambion, Austin, TX; 15596-018), and cDNA was generated by reverse-transcription using reverse transcriptase (Invitrogen, Carlsbad, CA; C28025-014). Expression of alpl，runx2a，col1a1a, sox9b , catenin, bmp2a and bmp2b was examined by quantitative real-time PCR. Quantitative PCR was conducted on an ABI StepOnePlusTM real-time PCR system with SYBR Premix Ex Taq reagents (TaKaRa, Shiga, Japan): one cycle of 95 ℃for 1 min and 40 cycles of 94 ℃for 10 s, 59 ℃for 30 s, followed by melt curve analysis. Relative
ACCEPTED MANUSCRIPT expression was calculated using the formula 2-△△CT. Primers used for PCR are showed in Table 1.
Defect in hemoglobin synthesis of wehtp85c homozygotes
RI PT
RESULTS
Wehtp85c homozygotes were screened and identified by gene sequencing. Wild-type
SC
siblings were selected as control. Use O-dianisidine staining to detect the hemoglobincontaining cells. The mutation of the fpn1 gene in wehtp85c homozygotes
displayed
M AN U
the synthesis of hemoglobin reduced significantly, resulting in dysfunction of iron absorption, at 2 dpf (Fig.1). These results confirmed that wehtp85c homozygotes suffered from iron deficiency anemia.
TE D
Decreased bone calcification in wehtp85c homozygotes
Alizarin red is a dye that specifically binds to calcium salts. So it is widely used to
EP
detect and quantify mineralization of the bone. To examine skeletal development in wehtp85c homozygotes, we applied Alizarin Red S staining compared to wild-type
AC C
siblings. As shown in Figure 2, the defects in bone development of wehtp85c homozygotes at 5 dpf, 7 dpf and 14 dpf were detected. In wild-type larvae, mineralization of head skeleton occurs at 5 dpf, the axial skeleton at 7dpf and caudal fin rays at day 12. At 5 dpf, alizarin red stains in head skeleton both in wild-type and wehtp85c homozygotes, but the mutant fish displayed an evident decrease in staining area in head skeleton compared to controls (Fig.2 A and B). Morevoer, alizarin red stained vertebrae were observed in 75% of wild-type larvae
ACCEPTED MANUSCRIPT examined (n=24), but none of wehtp85c homozygotes examined (n = 21). At 7 dpf, in wild-type fish, 5.35±1.23 calcified vertebrae were detected (n=20), whereas in wehtp85c homozygotes, only 1.05±1.00 calcified vertebrae were detected (n=20) (Fig.2
RI PT
C and D). During development, the number of calcified vertebrae increased and the vertebral spur and coccyx appeared. But the formation of vertebral spur and coccyx of wehtp85c homozygotes was less and thinner than wild-type (Fig.2 E and F). These
SC
results indicated that wehtp85c homozygotes displayed defects in bone calcification.
M AN U
Normal cartilage development in wehtp85c homozygotes
The development of cartilages was evaluated by staining the larvae with alcian blue. Alcian blue is a cationic dye that binds to mucopolysaccharide under acidic conditions [22]
.
Cartilage
is
a
strongly
sulfated
connective
tissue,
which
contains
TE D
mucopolysaccharide, and thus can be stained with alcian blue. The wild-type and wehtp85c homozygotes at 5dpf were stained. No obvious differences in alcian blue
EP
staining were observed between wild type and wehtp85c homozygotes larvae (Fig.2 G
AC C
and H), suggesting that the development of cartilage in the mutants was not affected. Gene expression of bone markers in wild-type and wehtp85c homozygotes To investigate the bone development, we examined the expression of alpl, runx2a, col1a1a and sox9b in developing wild-type and mutant larvae. Alpl, runx2a, col1a1a are involved in different stages of bone development[23]. In the early stages of bone development, sox9b plays an important role in chondrogenic differentiation[24].We performed qRT-PCR to examine the expression of alpl，runx2a，col1a1a and sox9b
ACCEPTED MANUSCRIPT using total RNA isolated from 7 dpf wild-type and wehtp85c homozygotes larvae (Fig.3 A-D; n=6 for each qRT-PCR).The expression of alpl，runx2a，col1a1a significantly decreased in wehtp85c homozygotes than in wild-type fish (p < 0.05). However, the
RI PT
expression of Sox9b in wehtp85c homozygotes slightly decreased in comparison to the expression in wild-type, with the difference being statistically insignificant (p = 0.09).
SC
Dextran iron rescued bone formation in wehtp85c homozygotes
To confirm the effect of iron deficiency on bone formation, dextran iron was given to
M AN U
wehtp85c homozygotes at 2 dpf, while phenol red was given as control. Due to the defect of iron absorption in intestine, dextran iron was given intravenously rather than by feeding. At 7 dpf, the number of calcified vertebrae was 5.96 ±1.52 (n = 23) in mutant injected with dextran iron and 1.00 ±0.69 (n = 18) in mutant injected with
TE D
phenol red (p < 0.05) (Fig.3 E, F). Furthermore, alpl and runx2a expression increased significantly (Fig.3 G, H). The results suggested that injection of dextran iron can
EP
rescue the bone formation defect of wehtp85c homozygotes.
AC C
Decreased BMPs signaling in wehtp85c homozygotes In order to explore the mechanism of iron deficiency affect on bone formation, we examined the expressions of the genes involved in bone formation related signal pathways, such as Wnt signaling and BMPs signalling pathways by qRT-PCR , using total RNA isolated from 7 dpf wild-type and wehtp85c homozygotes larvae. Results indicated that the expression of catenin, the key factor of the Wnt signaling pathways, had no obvious change in wehtp85c homozygotes compared with wild-type, while the
ACCEPTED MANUSCRIPT expressions of bmp2a, bmp2b which are the key factor genes of the BMPs signaling pathways were significantly lower than WT controls (p < 0.05). Hence, these results indicated that iron deficiency might down-regulate BMPs signaling pathways and
RI PT
affect bone formation in zebrafish (Fig.4). DISCUSSION
SC
The zebrafish mutant weh is known as the first animal model of fpn1 deficiency. Previous studies proved that this mutant exhibited severe embryonic anemia and
M AN U
would die between 10 and 14 days of age, due to a defect in iron transfer across the yolk syncytial layer, consistent with a block in intestinal iron export, which also caused nonheme iron retention in the intestinal epithelium
[18]
. The presence of
osteoclasts at endosteal surfaces of growing bones was observed only in zebrafish [17]
. So during the survival time of wehtp85c homozygotes, only
TE D
older than 20 days
osteoblast plays a role in bone formation, while osteoclasts have not yet appeared.
EP
Therefore, we used wehtp85c mutant zebrafish as the animal model to study the effect
AC C
of iron deficiency on bone formation in vivo. The defect in fpn1 function in wehtp85c homozygotes blocks iron export, which resulting in accumulation of iron in the reticuloendothelial system and the onset of hypochromic anemia
[25]
. Using O-dianisidine staining, we proved that the synthesis
of hemoglobin in the mutant was decreased significantly. Alizarin red staining was used to monitor the development of skeleton. Results indicated that bone formation decreased significantly in the development of wehtp85c homozygotes zebrafish,
ACCEPTED MANUSCRIPT suggesting that iron deficiency could affect bone metabolism. With osteogenesis being the generic term for bone formation, there are two basic types: intramembranous ossification and endochondral ossification.
Chondrocytes are
RI PT
involved in the process of endochondral ossification, while osteoblasts crucial role in both processes. We examined the expression of genes related to differentiation and mineralization of osteoblasts such as alpl, runx2a and col1a1a, and found all of these
SC
genes were down regulated. On the other hand, sox9b involved in chondrogenesis has
M AN U
no remarkable change in wehtp85c homozygotes. So we conclude that iron deficiency may affect bone formation through differentiation and mineralization of osteoblasts. Furthermore, our study suggested that the defect of bone formation of wehtp85c homozygotes could be rescued by injection with iron dextran. We propose that
TE D
injecting wehtp85c homozygotes with iron dextran rescues the mutants by bypassing the block in intestinal iron absorption.
EP
To further explore the probable mechanism involved in the correlation between iron deficiency and bone formation, we tested the expression of genes of BMPs and Wnt
AC C
signaling pathway, which are the main signaling pathways related to bone formation. The qPCR results suggested genes involved in BMPs signaling pathway were significantly down-regulated, while the expression of key factor genes of Wnt signaling pathway did not change obviously. So we inferred that iron deficiency may affect bone formation through the BMPs signaling pathways. In this work, we have presented evidences implicating iron deficiency may affect bone formation in BMPs signaling pathway that regulates differentiation and
ACCEPTED MANUSCRIPT mineralization of osteoblasts. Our study laid a foundation of studying the effects of iron deficiency on bone development, osteoporosis and bone metabolic disease. In addition, our research provides the experimental data and theoretical basis to seek
RI PT
new targets for the prevention and treatment of osteoporosis, and helps to find the treatment of the osteoporosis caused by iron deficiency. Future investigation will be
relationship to metabolic bone diseases.
M AN U
ACKNOWLEDGEMENTS
SC
needed to further understand the mechanisms underlying bone remodeling and its
This work was supported in part by the Natural Science Foundation of China (81200507), Clinical Medical Science and Technology Fond of Jiangsu Province (BL2014044), the Scientific and Technological Development Project of Suzhou
TE D
(SYS201341), and the Young Teachers’ Natural Science Foundation of Soochow University (SDY2013A34). All institutional and national guidelines for the care and
AC C
References
EP
use of laboratory animals were followed.
[1]
Pietrangelo A. Hepcidin in human iron disorders: therapeutic implications. J
Hepatol. 2011. 54(1): 173-81.
[2]
Zhang Z, Zhang F, An P, et al. Ferroportin1 deficiency in mouse macrophages impairs iron homeostasis and inflammatory responses. Blood. 2011. 118(7): 1912-22.
ACCEPTED MANUSCRIPT [3]
Weinberg ED. Iron loading: a risk factor for osteoporosis. Biometals. 2006. 19(6): 633-5.
[4]
Kim BJ, Ahn SH, Bae SJ, et al. Iron overload accelerates bone loss in healthy
RI PT
postmenopausal women and middle-aged men: a 3-year retrospective longitudinal study. J Bone Miner Res. 2012. 27(11): 2279-90.
Tsay J, Yang Z, Ross FP, et al. Bone loss caused by iron overload in a murine
SC
[5]
[6]
M AN U
model: importance of oxidative stress. Blood. 2010. 116(14): 2582-9.
Jia P, Xu YJ, Zhang ZL, et al. Ferric ion could facilitate osteoclast differentiation and bone resorption through the production of reactive oxygen species. J Orthop Res. 2012. 30(11): 1843-52.
Worldwide Prevalence of Anaemia 1993???¡ì?C2005. WHO Global Database
TE D
[7]
on Anaemia. WHO Library Cataloguing-in-Publication Data_ 2008 .
Iron Deficiency Anaemia Assessment, Prevention, and Control. A Guide for
EP
[8]
Programme Managers; United Nations Children??s Fund; United Nations
AC C
University; World Health Organization .
[9]
Jonker FA, van Hensbroek M B. Anaemia, iron deficiency and susceptibility to
infections. J Infect. 2014. 69 Suppl 1: S23-7.
[10]
Katsumata S, Tsuboi R, Uehara M, Suzuki K. Dietary iron deficiency decreases serum osteocalcin concentration and bone mineral density in rats. Biosci Biotechnol Biochem. 2006. 70(10): 2547-50.
ACCEPTED MANUSCRIPT [11]
Medeiros DM, Stoecker B, Plattner A, Jennings D, Haub M. Iron deficiency negatively affects vertebrae and femurs of rats independently of energy intake and body weight. J Nutr. 2004. 134(11): 3061-7.
Katsumata S, Katsumata-Tsuboi R, Uehara M, Suzuki K. Severe iron
RI PT
[12]
deficiency decreases both bone formation and bone resorption in rats. J Nutr.
[13]
SC
2009. 139(2): 238-43.
Diaz-Castro J, Lopez-Frias MR, Campos MS, et al. Severe nutritional
M AN U
iron-deficiency anaemia has a negative effect on some bone turnover biomarkers in rats. Eur J Nutr. 2012. 51(2): 241-7.
[14]
Witten PE, Huysseune A. A comparative view on mechanisms and functions of
TE D
skeletal remodelling in teleost fish, with special emphasis on osteoclasts and their function. Biol Rev Camb Philos Soc. 2009. 84(2): 315-46.
Andreeva V, Connolly MH, Stewart-Swift C, et al. Identification of adult
EP
[15]
mineralized tissue zebrafish mutants. Genesis. 2011. 49(4): 360-6.
Pasqualetti S, Banfi G, Mariotti M. The zebrafish scale as model to study the
AC C
[16]
bone mineralization process. J Mol Histol. 2012. 43(5): 589-95.
[17]
Witten PE, Hansen A, Hall BK. Features of mono- and multinucleated bone
resorbing cells of the zebrafish Danio rerio and their contribution to skeletal development, remodeling, and growth. J Morphol. 2001. 250(3): 197-207.
[18]
Donovan A, Brownlie A, Zhou Y, et al. Positional cloning of zebrafish
ACCEPTED MANUSCRIPT ferroportin1 identifies a conserved vertebrate iron exporter. Nature. 2000. 403(6771): 776-81.
[19]
Zhang AS, Enns CA. Molecular mechanisms of normal iron homeostasis.
[20]
RI PT
Hematology Am Soc Hematol Educ Program. 2009 : 207-14.
Beaumont C. Molecular mechanisms of iron homeostasis. Med Sci (Paris).
Detrich HW 3rd, Kieran MW, Chan FY, et al. Intraembryonic hematopoietic
M AN U
[21]
SC
2004. 20(1): 68-72.
cell migration during vertebrate development. Proc Natl Acad Sci U S A. 1995. 92(23): 10713-7.
[22]
Walker MB, Kimmel CB. A two-color acid-free cartilage and bone stain for
[23]
TE D
zebrafish larvae. Biotech Histochem. 2007. 82(1): 23-8.
Chen B, Yan YL, Liu C, et al. Therapeutic effect of deferoxamine on iron
EP
overload-induced inhibition of osteogenesis in a zebrafish model. Calcif
AC C
Tissue Int. 2014. 94(3): 353-60.
[24]
Yan YL, Miller CT, Nissen RM, et al. A zebrafish sox9 gene required for
cartilage morphogenesis. Development. 2002. 129(21): 5065-79.
[25]
Fraenkel PG, Traver D, Donovan A, Zahrieh D, Zon LI. Ferroportin1 is required for normal iron cycling in zebrafish. J Clin Invest. 2005. 115(6): 1532-41.
FIGURE LEGENDS
ACCEPTED MANUSCRIPT Fig.1. The O-dianisidine staining of zebrafish embryos at 48hpf. a) wild-type sibling as control; b) wehtp85c homozygotes. The brown coloration was reduced in wehtp85c homozygotes.
RI PT
Fig.2. Wild-type and wehtp85c homozygotes stained with Alizarin Red S and alcian blue. At 5 dpf, the wehtp85c homozygotes displayed an evident decrease in head skeleton formation compared to controls; and there is no vertebrae examined (A, B);
SC
At 7 dpf, calcified vertebrae of wehtp85c homozygotes were less than wild-type (C, D);
M AN U
At 14 dpf , the formation of vertebral spur and coccyx of wehtp85c homozygotes was less and thinner than wild-type (E, F); At 5dpf, no significant differences in cartilage were detected (G, H). WT: A, C, E, G and wehtp85c mutant: B, D, F, H. Fig.3. Relative mRNA expression of alpl, runx2a, col1a1a and sox9b in wild-type
TE D
and wehtp85c homozygotes at 7 dpf. The expression of alpl (A)，runx2a (B)，col1a1a (C) significantly decreased in wehtp85c homozygotes compared to wild-type siblings (p < 0.05). The expression of Sox9b (D) was slightly decreased in wehtp85c homozygotes
EP
compared to wild-type sibling (p = 0.09).
AC C
Dextran iron was given to wehtp85c homozygotes at 2 dpf, phenol red was given as control. At 7 dpf, the number of calcified vertebrae was 5.96 ±1.52 (n = 23) in mutant injected by dextran iron and 1.00 ±0.69 (n = 18) in mutant injected by phenol red (p < 0.05) (E, F); Alpl and runx2a expression increased significantly (G, H). Fig.4. Relative mRNA expression of catenin, bmp2a and bmp2b in wild-type and wehtp85c homozygotes at 7 dpf. Catenin has no obvious change in wehtp85c homozygotes compared with wild-type (A) (p=0.13) ； bmp2a, bmp2b were
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
significantly lower (p＜0.05) in wehtp85c compared to WT controls.
ACCEPTED MANUSCRIPT Table Primer sequences Forward sequence(5’ to 3’)
Reverse sequence(5’ to 3’)
runx2a
GACTCCGACCTCACGACAA
CGTCCCGTCAGGAACATC
alpl
CAGGCAAATCAGTGGGAATC
TTGGGCATGTCTGCATCA
col1a1a
CAGGAGCCCAGTGTTGAG
AGCCACCAGACATCTGAGGA
sox9b
GATCGGACAGCGAGACCCC
TCGTTCAGCAGTCTCCAGAGTT
catenin
AGGTGTTGTCAGTGTGCTCC
CCATGCCCTCCTGTTTGGTG
bmp2a
CGGCTTCTGAGCATGTTTGG
CGGATCTTCTGTAGATTCATCATGG
bmp2b
GATCTCGCGCTGTCACTTTTG
TGATCAGTCAGTTCCGGAGGA
AC C
EP
TE D
M AN U
SC
RI PT
mRNA
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0006-291X(16)30768-9
DOI:
10.1016/j.bbrc.2016.05.069
Reference:
YBBRC 35819
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 2 May 2016 Accepted Date: 12 May 2016
Please cite this article as: L. Bo, Z. Liu, Y. Zhong, J. Huang, B. Chen, H. Wang, Y. Xu, Iron deficiency anemia's affect on bone formation in zebrafish mutant, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.05.069. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Iron deficiency anemia's affect on bone formation in zebrafish mutant Lin Bo1 MD, Zhichun Liu1 PhD, Yingbin Zhong2 PhD, Jian Huang2 PhD, Bin Chen3 MD, Han Wang2* PhD, Youjia Xu3* PhD Department of Rheumatology, The Second Affiliated Hospital of Soochow University,
RI PT
1
Suzhou 215004, Jiangsu, China; 2 Center for Circadian Clocks, Soochow University, Suzhou 215003, Jiangsu, China; 3 Department of Orthopaedics, The Second Affiliated
SC
Hospital of Soochow University, Suzhou 215004, Jiangsu, China
M AN U
Correspondence Youjia Xu ([email protected])
Department of Orthopaedics, The Second Affiliated Hospital of Soochow University 1055 Sanxiang Road,
TE D
Suzhou, Jiangsu 215123
Phone:+86-512-67783346
EP
Han Wang ([email protected])
AC C
Center for Circadian Clocks, Soochow University 199 Renai Road,
Suzhou, Jiangsu 215123 Phone: +86-512-65882115 This work was supported in part by the Natural Science Foundation of China (81200507), Clinical Medical Science and Technology Fond of Jiangsu Province (BL2014044), the Scientific and Technological Development Project of Suzhou
ACCEPTED MANUSCRIPT (SYS201341), and the Young Teachers’ Natural Science Foundation of Soochow University (SDY2013A34). All institutional and national guidelines for the care and
AC C
EP
TE D
M AN U
SC
RI PT
use of laboratory animals were followed.
ACCEPTED MANUSCRIPT Iron deficiency anemia affect bone formation in zebrafish mutant ABSTRACT Iron is one of the essential elements of life. Iron metabolism is related to bone
RI PT
metabolism. Previous studies have confirmed that iron overload is a risk factor for osteoporosis. But the correlation between iron deficiency and bone metabolism
SC
remains unclear. Ferroportin 1 is identified as a cellular iron exporter and required for normal iron cycling. In human, the mutation in Ferroportin 1 gene caused
M AN U
hemochromatosis (iron overload) syndrome. In zebrafish, the mutant of ferroportin 1 gene (fpn1), Wehtp85c exhibited the defective iron transport, leading to developing severe hypochromic anemia. To understand the mechanism of fpn1 in iron regulation and bone formation, we used Wehtp85c as a model for investigating iron deficiency and
TE D
bone metabolism. In this study, we examined the morphology of the developing cartilage and vertebrae of the Wehtp85 compared to the wild type siblings by staining
EP
the larvae with alcian blue for cartilage and alizarin red for the bone. In addition, we evaluated the expression patterns of the marker genes of bone development and cell
AC C
signaling in bone formation. Our results showed that wehtp85c mutant larvae exhibited the defects in bone formation, revealing by decreases in the number of calcified vertebrae along with decreased expression of osteoblast novel genes: alpl，runx2a and col1a1a and BMPs signaling genes in osteoblast differentiation: bmp2a and bmp2b. Our data suggest that iron deficiency anemia affects bone formation, potentially through the BMPs signaling pathway in zebrafish.
ACCEPTED MANUSCRIPT Keywords Fpn1
RI PT
Iron deficiency anemia Bone formation Zebrafish
SC
INTRODUCTION
M AN U
Bone is a dynamic connective tissue, which constantly remodels itself to accommodate growth and mechanical loads, and to maintain mineral homoeostasis. Bone metabolism is influenced by some trace elements in the body, such as calcium, phosphorus, magnesium, etc.
TE D
Iron is essential for growth and proliferation of cells because it act as a protein cofactor for hemoglobin synthesis, DNA synthesis and mitochondrial respiration. The
EP
regulation of iron content is important since iron overload prompt the generation of reactive oxygen species which damage DNA and proteins, while iron deficiency leads
AC C
to cell cycle arrest even cell death[1, 2]. Dysregulation of iron homeostasis may cause hematological, metabolic and neurological diseases. In recent years, the correlation between bone metabolism and iron is being a hotspot of research. Weinberg
[3]
firstly proposed that iron overload was a risk factor for
osteoporosis in 2006. Kim et al. [4] conducted a 3-year retrospective longitudinal study and demonstrated that iron overload is related to osteoporosis in healthy adults, especially in postmenopausal women. Further more, several previous studies have
ACCEPTED MANUSCRIPT proved that iron overload promoted osteoclast differentiation and bone reabsorption [5, 6]
. Iron overload has been recognized as an independent risk factor for osteoporosis.
However, besides iron overload, abnormal iron homeostasis also includes iron
RI PT
deficiency. Iron deficiency is one of widespread diseases in human beings with major consequences for human health and socioeconomic development. Globally, 1.5-1.7 billion people suffered by iron deficiency anemia, approximately 24.8% of the world
SC
population, while the highest prevalence is in preschool-age children (47.4%,
M AN U
283–303 million children) [7]. Previous studies have proved that iron deficiency, even in absence of anemia, adversely affects the physical growth and cognitive performance of children
[8]
. It can also affect the immune status of human beings
[9]
.
In terms of bone metabolism, it has been reported that iron deficiency diet can affect [10]
. However, the molecular mechanism of the iron deficiency effect
TE D
bone formation
on bone metabolism still remains unclear.
[11-13]
.
EP
Severe iron deficiency anemia has a negative effect on bone metabolism
However, the relationship between iron deficiency and bone loss remains unclear. The
AC C
zebrafish model is rapidly gaining prominence in the study of many aspects of biological and medical research. For example, zebrafish can serve as a good model for studying the development of the skeletal system due to its rapid bone development, simple bone structure, external embryonic development, and body transparence. The latter allows for easy observations of bone morphology in live embryos or larvea [14-16]. In
addition,
in
the
early stage
of
zebrafish
bone
development (<
20 dpf), osteoclast has not yet appeared while only osteoblast and bone formation
ACCEPTED MANUSCRIPT exist[17]. Zebrafish mutant, wehtp85may provide a useful tool for studies of diseases related to pathological bone formation, such as osteoporosis or osteomalacia. Wehtp85c is the autosomal recessive zebrafish mutants of ferroportin 1 gene (fpn1), in
between 10 and 14 days of age
[18]
RI PT
which homozygotes develop severe anemia within 48 hours after fertilization and dies . Fpn1 is the only non-heme iron exporter with
function in iron efflux, which is mainly expressed in duodenal mature enterocytes,
. Dietary iron exists mainly in form of ferric (Fe3+) and is reduced to ferrous (Fe2+)
M AN U
[2]
SC
reticuloendothelial macrophages, placental syncytiotrophoblast cells and hepatocytes
by duodenal cytochrome B (Dcyt B) prior to transport. The transport of non-heme iron across the apical membrane occurs via the divalent metal transporter 1 (DMT1). Cytosolic iron in intestinal enterocytes can be either stored in ferritin or exported into
TE D
plasma by the basolateral iron exporter FPN1
[19]
. In the plasma, iron binds with
transferrin and is imported into erythroid precursors and other cells via transferrinendocytosis.
Macrophages
also
play
an
important
role
EP
receptor-mediated
in iron delivery to plasma transferrin through phagocytosis of senescent red blood cell,
AC C
heme catabolism and recycling of iron [20]. In Wehtp85c mutant the hypochromia is caused by inadequate circulatory iron levels， although the erythroid cells are fully capable of haemoglobinization. Therefore, wehtp85c is one of the suitable models of iron deficiency anemia. In this study, we examined the morphology of developing cartilage and vertebrae of the Wehtp85c larvae by staining the fish with alcian blue and alizarin red, respectively.
ACCEPTED MANUSCRIPT In addition, we evaluated the gene expression patterns of bone development markers (e.g., alpl，runx2a，col1a1a and sox9b) and genes involved in cellular signaling during bone development, such as bmp2a and bmp2b. Our investigation may provide a basis
RI PT
for the research of diseases related to pathological bone metabolism, such as iron deficiency anemia.
SC
MATERIALS AND METHODS Animals
M AN U
Wehtp85c Heterozygous zebrafish used in this study were donated by Professor Zhou Yi (Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute and Harvard Medical School, Howard Hughes Medical Institute, Boston,
TE D
Massachusetts 02115 USA). Breeding colonies were kept in 28.5℃with a 10/14h dark/light cycle and staged by hours post-fertilization (hpf) or days post-fertilization (dpf). Protocols for experimental procedures were according to NIH guidelines.
EP
Mutant homozygotes screening
AC C
Ten female and twenty male (4-6 months old) heterozygotes were used for mating to generate the F1 fish, which contain wehtp85c homozygotes, heterozygotes and wild-type siblings. The wehtp85c homozygotes were identified in a morphologic screen for defects in circulating erythroid cells at 48 hpf [18]. All of the wehtp85c heterozygous zebrafish were identified by gene sequencing. O-dianisidine staining
ACCEPTED MANUSCRIPT The expression of the hemoglobin can be demonstrated by the O-dianisidine staining method. The zebrafish at 48 hpf were dechorionated and stained with O-dianisidine solution (0.6mg/ml), sodium acetate (0.01M, pH4.5), H2O2 (0.65%), ethanol (40%) in
RI PT
the dark for 15 minutes[21]. The O-dianisidine solution was removed and phosphatebuffered saline (PBS) was added to stop the reaction. The embryos were observed under the microscope. Brown precipitate is formed when the heme in the hemoglobin
SC
reacts with the O-dianisidine solution. Changes in the intensity of brown precipitate
M AN U
can be considered as a semi-quantitative estimation of erythroid development in zebrafish embryos.
Alizarin Red S and alcian blue staining
Bone and cartilage were stained by alcian blue (CAS No: 33864-99-2) and alizarin
specimen [22].
TE D
red S (CAS No: 130-22-3, Sigma-Aldrich, Diegem, Belgium) respectively on fixed
EP
Quantitative RT-PCR
AC C
Total RNA of larvae were isolated using Trizol (Ambion, Austin, TX; 15596-018), and cDNA was generated by reverse-transcription using reverse transcriptase (Invitrogen, Carlsbad, CA; C28025-014). Expression of alpl，runx2a，col1a1a, sox9b , catenin, bmp2a and bmp2b was examined by quantitative real-time PCR. Quantitative PCR was conducted on an ABI StepOnePlusTM real-time PCR system with SYBR Premix Ex Taq reagents (TaKaRa, Shiga, Japan): one cycle of 95 ℃for 1 min and 40 cycles of 94 ℃for 10 s, 59 ℃for 30 s, followed by melt curve analysis. Relative
ACCEPTED MANUSCRIPT expression was calculated using the formula 2-△△CT. Primers used for PCR are showed in Table 1.
Defect in hemoglobin synthesis of wehtp85c homozygotes
RI PT
RESULTS
Wehtp85c homozygotes were screened and identified by gene sequencing. Wild-type
SC
siblings were selected as control. Use O-dianisidine staining to detect the hemoglobincontaining cells. The mutation of the fpn1 gene in wehtp85c homozygotes
displayed
M AN U
the synthesis of hemoglobin reduced significantly, resulting in dysfunction of iron absorption, at 2 dpf (Fig.1). These results confirmed that wehtp85c homozygotes suffered from iron deficiency anemia.
TE D
Decreased bone calcification in wehtp85c homozygotes
Alizarin red is a dye that specifically binds to calcium salts. So it is widely used to
EP
detect and quantify mineralization of the bone. To examine skeletal development in wehtp85c homozygotes, we applied Alizarin Red S staining compared to wild-type
AC C
siblings. As shown in Figure 2, the defects in bone development of wehtp85c homozygotes at 5 dpf, 7 dpf and 14 dpf were detected. In wild-type larvae, mineralization of head skeleton occurs at 5 dpf, the axial skeleton at 7dpf and caudal fin rays at day 12. At 5 dpf, alizarin red stains in head skeleton both in wild-type and wehtp85c homozygotes, but the mutant fish displayed an evident decrease in staining area in head skeleton compared to controls (Fig.2 A and B). Morevoer, alizarin red stained vertebrae were observed in 75% of wild-type larvae
ACCEPTED MANUSCRIPT examined (n=24), but none of wehtp85c homozygotes examined (n = 21). At 7 dpf, in wild-type fish, 5.35±1.23 calcified vertebrae were detected (n=20), whereas in wehtp85c homozygotes, only 1.05±1.00 calcified vertebrae were detected (n=20) (Fig.2
RI PT
C and D). During development, the number of calcified vertebrae increased and the vertebral spur and coccyx appeared. But the formation of vertebral spur and coccyx of wehtp85c homozygotes was less and thinner than wild-type (Fig.2 E and F). These
SC
results indicated that wehtp85c homozygotes displayed defects in bone calcification.
M AN U
Normal cartilage development in wehtp85c homozygotes
The development of cartilages was evaluated by staining the larvae with alcian blue. Alcian blue is a cationic dye that binds to mucopolysaccharide under acidic conditions [22]
.
Cartilage
is
a
strongly
sulfated
connective
tissue,
which
contains
TE D
mucopolysaccharide, and thus can be stained with alcian blue. The wild-type and wehtp85c homozygotes at 5dpf were stained. No obvious differences in alcian blue
EP
staining were observed between wild type and wehtp85c homozygotes larvae (Fig.2 G
AC C
and H), suggesting that the development of cartilage in the mutants was not affected. Gene expression of bone markers in wild-type and wehtp85c homozygotes To investigate the bone development, we examined the expression of alpl, runx2a, col1a1a and sox9b in developing wild-type and mutant larvae. Alpl, runx2a, col1a1a are involved in different stages of bone development[23]. In the early stages of bone development, sox9b plays an important role in chondrogenic differentiation[24].We performed qRT-PCR to examine the expression of alpl，runx2a，col1a1a and sox9b
ACCEPTED MANUSCRIPT using total RNA isolated from 7 dpf wild-type and wehtp85c homozygotes larvae (Fig.3 A-D; n=6 for each qRT-PCR).The expression of alpl，runx2a，col1a1a significantly decreased in wehtp85c homozygotes than in wild-type fish (p < 0.05). However, the
RI PT
expression of Sox9b in wehtp85c homozygotes slightly decreased in comparison to the expression in wild-type, with the difference being statistically insignificant (p = 0.09).
SC
Dextran iron rescued bone formation in wehtp85c homozygotes
To confirm the effect of iron deficiency on bone formation, dextran iron was given to
M AN U
wehtp85c homozygotes at 2 dpf, while phenol red was given as control. Due to the defect of iron absorption in intestine, dextran iron was given intravenously rather than by feeding. At 7 dpf, the number of calcified vertebrae was 5.96 ±1.52 (n = 23) in mutant injected with dextran iron and 1.00 ±0.69 (n = 18) in mutant injected with
TE D
phenol red (p < 0.05) (Fig.3 E, F). Furthermore, alpl and runx2a expression increased significantly (Fig.3 G, H). The results suggested that injection of dextran iron can
EP
rescue the bone formation defect of wehtp85c homozygotes.
AC C
Decreased BMPs signaling in wehtp85c homozygotes In order to explore the mechanism of iron deficiency affect on bone formation, we examined the expressions of the genes involved in bone formation related signal pathways, such as Wnt signaling and BMPs signalling pathways by qRT-PCR , using total RNA isolated from 7 dpf wild-type and wehtp85c homozygotes larvae. Results indicated that the expression of catenin, the key factor of the Wnt signaling pathways, had no obvious change in wehtp85c homozygotes compared with wild-type, while the
ACCEPTED MANUSCRIPT expressions of bmp2a, bmp2b which are the key factor genes of the BMPs signaling pathways were significantly lower than WT controls (p < 0.05). Hence, these results indicated that iron deficiency might down-regulate BMPs signaling pathways and
RI PT
affect bone formation in zebrafish (Fig.4). DISCUSSION
SC
The zebrafish mutant weh is known as the first animal model of fpn1 deficiency. Previous studies proved that this mutant exhibited severe embryonic anemia and
M AN U
would die between 10 and 14 days of age, due to a defect in iron transfer across the yolk syncytial layer, consistent with a block in intestinal iron export, which also caused nonheme iron retention in the intestinal epithelium
[18]
. The presence of
osteoclasts at endosteal surfaces of growing bones was observed only in zebrafish [17]
. So during the survival time of wehtp85c homozygotes, only
TE D
older than 20 days
osteoblast plays a role in bone formation, while osteoclasts have not yet appeared.
EP
Therefore, we used wehtp85c mutant zebrafish as the animal model to study the effect
AC C
of iron deficiency on bone formation in vivo. The defect in fpn1 function in wehtp85c homozygotes blocks iron export, which resulting in accumulation of iron in the reticuloendothelial system and the onset of hypochromic anemia
[25]
. Using O-dianisidine staining, we proved that the synthesis
of hemoglobin in the mutant was decreased significantly. Alizarin red staining was used to monitor the development of skeleton. Results indicated that bone formation decreased significantly in the development of wehtp85c homozygotes zebrafish,
ACCEPTED MANUSCRIPT suggesting that iron deficiency could affect bone metabolism. With osteogenesis being the generic term for bone formation, there are two basic types: intramembranous ossification and endochondral ossification.
Chondrocytes are
RI PT
involved in the process of endochondral ossification, while osteoblasts crucial role in both processes. We examined the expression of genes related to differentiation and mineralization of osteoblasts such as alpl, runx2a and col1a1a, and found all of these
SC
genes were down regulated. On the other hand, sox9b involved in chondrogenesis has
M AN U
no remarkable change in wehtp85c homozygotes. So we conclude that iron deficiency may affect bone formation through differentiation and mineralization of osteoblasts. Furthermore, our study suggested that the defect of bone formation of wehtp85c homozygotes could be rescued by injection with iron dextran. We propose that
TE D
injecting wehtp85c homozygotes with iron dextran rescues the mutants by bypassing the block in intestinal iron absorption.
EP
To further explore the probable mechanism involved in the correlation between iron deficiency and bone formation, we tested the expression of genes of BMPs and Wnt
AC C
signaling pathway, which are the main signaling pathways related to bone formation. The qPCR results suggested genes involved in BMPs signaling pathway were significantly down-regulated, while the expression of key factor genes of Wnt signaling pathway did not change obviously. So we inferred that iron deficiency may affect bone formation through the BMPs signaling pathways. In this work, we have presented evidences implicating iron deficiency may affect bone formation in BMPs signaling pathway that regulates differentiation and
ACCEPTED MANUSCRIPT mineralization of osteoblasts. Our study laid a foundation of studying the effects of iron deficiency on bone development, osteoporosis and bone metabolic disease. In addition, our research provides the experimental data and theoretical basis to seek
RI PT
new targets for the prevention and treatment of osteoporosis, and helps to find the treatment of the osteoporosis caused by iron deficiency. Future investigation will be
relationship to metabolic bone diseases.
M AN U
ACKNOWLEDGEMENTS
SC
needed to further understand the mechanisms underlying bone remodeling and its
This work was supported in part by the Natural Science Foundation of China (81200507), Clinical Medical Science and Technology Fond of Jiangsu Province (BL2014044), the Scientific and Technological Development Project of Suzhou
TE D
(SYS201341), and the Young Teachers’ Natural Science Foundation of Soochow University (SDY2013A34). All institutional and national guidelines for the care and
AC C
References
EP
use of laboratory animals were followed.
[1]
Pietrangelo A. Hepcidin in human iron disorders: therapeutic implications. J
Hepatol. 2011. 54(1): 173-81.
[2]
Zhang Z, Zhang F, An P, et al. Ferroportin1 deficiency in mouse macrophages impairs iron homeostasis and inflammatory responses. Blood. 2011. 118(7): 1912-22.
ACCEPTED MANUSCRIPT [3]
Weinberg ED. Iron loading: a risk factor for osteoporosis. Biometals. 2006. 19(6): 633-5.
[4]
Kim BJ, Ahn SH, Bae SJ, et al. Iron overload accelerates bone loss in healthy
RI PT
postmenopausal women and middle-aged men: a 3-year retrospective longitudinal study. J Bone Miner Res. 2012. 27(11): 2279-90.
Tsay J, Yang Z, Ross FP, et al. Bone loss caused by iron overload in a murine
SC
[5]
[6]
M AN U
model: importance of oxidative stress. Blood. 2010. 116(14): 2582-9.
Jia P, Xu YJ, Zhang ZL, et al. Ferric ion could facilitate osteoclast differentiation and bone resorption through the production of reactive oxygen species. J Orthop Res. 2012. 30(11): 1843-52.
Worldwide Prevalence of Anaemia 1993???¡ì?C2005. WHO Global Database
TE D
[7]
on Anaemia. WHO Library Cataloguing-in-Publication Data_ 2008 .
Iron Deficiency Anaemia Assessment, Prevention, and Control. A Guide for
EP
[8]
Programme Managers; United Nations Children??s Fund; United Nations
AC C
University; World Health Organization .
[9]
Jonker FA, van Hensbroek M B. Anaemia, iron deficiency and susceptibility to
infections. J Infect. 2014. 69 Suppl 1: S23-7.
[10]
Katsumata S, Tsuboi R, Uehara M, Suzuki K. Dietary iron deficiency decreases serum osteocalcin concentration and bone mineral density in rats. Biosci Biotechnol Biochem. 2006. 70(10): 2547-50.
ACCEPTED MANUSCRIPT [11]
Medeiros DM, Stoecker B, Plattner A, Jennings D, Haub M. Iron deficiency negatively affects vertebrae and femurs of rats independently of energy intake and body weight. J Nutr. 2004. 134(11): 3061-7.
Katsumata S, Katsumata-Tsuboi R, Uehara M, Suzuki K. Severe iron
RI PT
[12]
deficiency decreases both bone formation and bone resorption in rats. J Nutr.
[13]
SC
2009. 139(2): 238-43.
Diaz-Castro J, Lopez-Frias MR, Campos MS, et al. Severe nutritional
M AN U
iron-deficiency anaemia has a negative effect on some bone turnover biomarkers in rats. Eur J Nutr. 2012. 51(2): 241-7.
[14]
Witten PE, Huysseune A. A comparative view on mechanisms and functions of
TE D
skeletal remodelling in teleost fish, with special emphasis on osteoclasts and their function. Biol Rev Camb Philos Soc. 2009. 84(2): 315-46.
Andreeva V, Connolly MH, Stewart-Swift C, et al. Identification of adult
EP
[15]
mineralized tissue zebrafish mutants. Genesis. 2011. 49(4): 360-6.
Pasqualetti S, Banfi G, Mariotti M. The zebrafish scale as model to study the
AC C
[16]
bone mineralization process. J Mol Histol. 2012. 43(5): 589-95.
[17]
Witten PE, Hansen A, Hall BK. Features of mono- and multinucleated bone
resorbing cells of the zebrafish Danio rerio and their contribution to skeletal development, remodeling, and growth. J Morphol. 2001. 250(3): 197-207.
[18]
Donovan A, Brownlie A, Zhou Y, et al. Positional cloning of zebrafish
ACCEPTED MANUSCRIPT ferroportin1 identifies a conserved vertebrate iron exporter. Nature. 2000. 403(6771): 776-81.
[19]
Zhang AS, Enns CA. Molecular mechanisms of normal iron homeostasis.
[20]
RI PT
Hematology Am Soc Hematol Educ Program. 2009 : 207-14.
Beaumont C. Molecular mechanisms of iron homeostasis. Med Sci (Paris).
Detrich HW 3rd, Kieran MW, Chan FY, et al. Intraembryonic hematopoietic
M AN U
[21]
SC
2004. 20(1): 68-72.
cell migration during vertebrate development. Proc Natl Acad Sci U S A. 1995. 92(23): 10713-7.
[22]
Walker MB, Kimmel CB. A two-color acid-free cartilage and bone stain for
[23]
TE D
zebrafish larvae. Biotech Histochem. 2007. 82(1): 23-8.
Chen B, Yan YL, Liu C, et al. Therapeutic effect of deferoxamine on iron
EP
overload-induced inhibition of osteogenesis in a zebrafish model. Calcif
AC C
Tissue Int. 2014. 94(3): 353-60.
[24]
Yan YL, Miller CT, Nissen RM, et al. A zebrafish sox9 gene required for
cartilage morphogenesis. Development. 2002. 129(21): 5065-79.
[25]
Fraenkel PG, Traver D, Donovan A, Zahrieh D, Zon LI. Ferroportin1 is required for normal iron cycling in zebrafish. J Clin Invest. 2005. 115(6): 1532-41.
FIGURE LEGENDS
ACCEPTED MANUSCRIPT Fig.1. The O-dianisidine staining of zebrafish embryos at 48hpf. a) wild-type sibling as control; b) wehtp85c homozygotes. The brown coloration was reduced in wehtp85c homozygotes.
RI PT
Fig.2. Wild-type and wehtp85c homozygotes stained with Alizarin Red S and alcian blue. At 5 dpf, the wehtp85c homozygotes displayed an evident decrease in head skeleton formation compared to controls; and there is no vertebrae examined (A, B);
SC
At 7 dpf, calcified vertebrae of wehtp85c homozygotes were less than wild-type (C, D);
M AN U
At 14 dpf , the formation of vertebral spur and coccyx of wehtp85c homozygotes was less and thinner than wild-type (E, F); At 5dpf, no significant differences in cartilage were detected (G, H). WT: A, C, E, G and wehtp85c mutant: B, D, F, H. Fig.3. Relative mRNA expression of alpl, runx2a, col1a1a and sox9b in wild-type
TE D
and wehtp85c homozygotes at 7 dpf. The expression of alpl (A)，runx2a (B)，col1a1a (C) significantly decreased in wehtp85c homozygotes compared to wild-type siblings (p < 0.05). The expression of Sox9b (D) was slightly decreased in wehtp85c homozygotes
EP
compared to wild-type sibling (p = 0.09).
AC C
Dextran iron was given to wehtp85c homozygotes at 2 dpf, phenol red was given as control. At 7 dpf, the number of calcified vertebrae was 5.96 ±1.52 (n = 23) in mutant injected by dextran iron and 1.00 ±0.69 (n = 18) in mutant injected by phenol red (p < 0.05) (E, F); Alpl and runx2a expression increased significantly (G, H). Fig.4. Relative mRNA expression of catenin, bmp2a and bmp2b in wild-type and wehtp85c homozygotes at 7 dpf. Catenin has no obvious change in wehtp85c homozygotes compared with wild-type (A) (p=0.13) ； bmp2a, bmp2b were
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
significantly lower (p＜0.05) in wehtp85c compared to WT controls.
ACCEPTED MANUSCRIPT Table Primer sequences Forward sequence(5’ to 3’)
Reverse sequence(5’ to 3’)
runx2a
GACTCCGACCTCACGACAA
CGTCCCGTCAGGAACATC
alpl
CAGGCAAATCAGTGGGAATC
TTGGGCATGTCTGCATCA
col1a1a
CAGGAGCCCAGTGTTGAG
AGCCACCAGACATCTGAGGA
sox9b
GATCGGACAGCGAGACCCC
TCGTTCAGCAGTCTCCAGAGTT
catenin
AGGTGTTGTCAGTGTGCTCC
CCATGCCCTCCTGTTTGGTG
bmp2a
CGGCTTCTGAGCATGTTTGG
CGGATCTTCTGTAGATTCATCATGG
bmp2b
GATCTCGCGCTGTCACTTTTG
TGATCAGTCAGTTCCGGAGGA
AC C
EP
TE D
M AN U
SC
RI PT
mRNA
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT