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成因。作者手稿;在 PMC 2014 年 4 月 23 日推出。
以最终编辑形式发布为:
Identification of adult mineralized tissue zebrafish mutants
成年矿化组织斑马鱼突变体的鉴定
维多利亚·安德烈耶娃、 1 米歇尔·康诺利、 1, 2 凯特琳·斯图尔特-斯威夫特、 1 丹尼尔·弗拉赫、 1 杰弗里·伯特、 1 贾斯汀·卡达雷利 1 和帕梅拉·耶利克 1
Viktoria Andreeva
1Divison of Craniofacial and Molecular Genetics, Department of Oral and Maxillofacial Pathology, Tufts University, 136 Harrison Avenue, Boston, MA 02111
Michelle H. Connolly
1Divison of Craniofacial and Molecular Genetics, Department of Oral and Maxillofacial Pathology, Tufts University, 136 Harrison Avenue, Boston, MA 02111
Caitlin Stewart-Swift
1Divison of Craniofacial and Molecular Genetics, Department of Oral and Maxillofacial Pathology, Tufts University, 136 Harrison Avenue, Boston, MA 02111
Daniel Fraher
1Divison of Craniofacial and Molecular Genetics, Department of Oral and Maxillofacial Pathology, Tufts University, 136 Harrison Avenue, Boston, MA 02111
Jeffrey Burt
1Divison of Craniofacial and Molecular Genetics, Department of Oral and Maxillofacial Pathology, Tufts University, 136 Harrison Avenue, Boston, MA 02111
Justin Cardarelli
1Divison of Craniofacial and Molecular Genetics, Department of Oral and Maxillofacial Pathology, Tufts University, 136 Harrison Avenue, Boston, MA 02111
Pamela C. Yelick
1Divison of Craniofacial and Molecular Genetics, Department of Oral and Maxillofacial Pathology, Tufts University, 136 Harrison Avenue, Boston, MA 02111
本文的最终编辑版本可在 Genesis 上获得
Abstract 抽象
Zebrafish craniofacial, skeletal, and tooth development closely resembles that of higher vertebrates. Our goal is to identify viable adult zebrafish mutants that can be used as models for human mineralized craniofacial, dental, and skeletal system disorders. We utilized a large-scale forward-genetic chemical N-ethyl-nitroso-urea (ENU) mutagenesis screen to identify 17 early lethal homozygous recessive mutants with defects in craniofacial cartilage elements, and 7 adult homozygous recessive mutants with mineralized tissue phenotypes including craniofacial shape defects, fused sutures, dysmorphic or missing skeletal elements, scoliosis, and neural arch defects. One mutant displayed both an early lethal homozygous phenotype and an adult heterozygous phenotype. These results extend the utility of the zebrafish model beyond the embryo, to study human bone and cartilage disorders.
斑马鱼的颅面、骨骼和牙齿发育与高等脊椎动物非常相似。我们的目标是确定可行的成年斑马鱼突变体,这些突变体可用作人类矿化颅面、牙科和骨骼系统疾病的模型。我们利用大规模正向遗传化学 N-乙基亚硝基脲 (ENU) 诱变筛选,鉴定了 17 个具有颅面软骨缺陷的早期致死纯合隐性突变体,以及 7 个具有矿化组织表型的成年纯合隐性突变体,包括颅面形状缺陷、融合缝合线、畸形或缺失的骨骼元素、脊柱侧弯和神经弓缺陷。一个突变体同时表现出早期致死的纯合表型和成年杂合表型。这些结果将斑马鱼模型的效用扩展到胚胎之外,以研究人类骨骼和软骨疾病。
关键词:成人颅面、骨骼、牙齿发育不良
Skeletal malformations, including craniofacial and dental abnormalities, are among the most common birth defects. Orofacial clefting, primarily consisting of cleft lip and/or cleft palate, occurs in about 1 in 700 births, while craniosynostosis, premature fusion of the cranial sutures, occurs in 1 out of 2500 births (Coussens et al., 2007; Juriloff and Harris, 2008). The estimated number of congenital vertebral malformations is approximately 0.5–1 per 1000 births, and skeletal dysplasia, disorders characterized by abnormalities of cartilage and bone growth resulting in abnormal shape and size of the skeleton, occurs in approximately 1 in 4,000 to 5,000 births (Raggio et al., 2009). Normal craniofacial and skeletal development requires precise timing and coordination of proper growth factor signaling pathways, transcription factor activities, and tissue interactions. In the past two decades, several genes implicated in craniofacial and skeletal development have been identified. However, we are still far from fully understanding the complex process of mineralized tissue development, size and shape determination, and homeostasis. Although skeletal malformations can be a part of a genetic syndrome, the majority of cases are non-syndromic, making them even more difficult to study.
骨骼畸形,包括颅面和牙齿异常,是最常见的出生缺陷之一。口面部裂主要由唇裂和/或腭裂组成,大约每 700 例新生儿中就有 1 例发生,而颅缝早闭(颅缝过早融合)每 2500 例新生儿中就有 1 例发生(Coussens 等人,2007 年;Juriloff 和 Harris,2008 年)。先天性椎体畸形的估计数量约为每 1000 名新生儿中有 0.5-1 名,骨骼发育不良,即以软骨和骨骼生长异常为特征的疾病,导致骨骼形状和大小异常,大约每 4,000 至 5,000 名新生儿中就有 1 名发生(Raggio 等人,2009 年)。正常的颅面和骨骼发育需要精确的时间安排和适当的生长因子信号通路、转录因子活性和组织相互作用的协调。在过去的二十年中,已经确定了几个与颅面和骨骼发育有关的基因。然而,我们仍远未完全了解矿化组织发育、大小和形状确定以及稳态的复杂过程。虽然骨骼畸形可能是遗传综合征的一部分,但大多数病例是非综合征性的,这使得它们更难研究。
The zebrafish has proved to be not only an excellent model to study vertebrate development but also a useful model for human diseases. There are many advantages of using zebrafish as a model, including ease of handling and economical maintenance, short reproductive cycle, external fertilization and development, production of large numbers of synchronous and rapidly developing embryos per mating, and the optical transparency of zebrafish embryos. These advantages have facilitated large-scale forward genetic mutagenesis screens that have yielded the discovery of numerous mutants, and the identification of genes important for the development of many different organs and tissues, including craniofacial and tooth development (Neuhauss et al., 1996; Piotrowski et al., 1996; Schilling et al., 1996). These elegant studies demonstrated that specification and development of craniofacial skeletal elements are governed by similar signaling pathways in humans and zebrafish. However, almost all known mutagenesis screens had several significant limitations. For one, mutants were usually selected based on visible defects, which limited the detection to the most severe phenotypes. Also, the mutants were screened at early embryonic stages (5–6 days post fertilization (dpf)), which limited the screens to the identification of genes important to the early development of predominantly cartilaginous skeletal elements, while the genes controlling later stages of bone development and homeostasis were not studied.
斑马鱼不仅被证明是研究脊椎动物发育的优秀模型,也是人类疾病的有用模型。使用斑马鱼作为模型有很多优点,包括易于处理和经济维护、繁殖周期短、外部受精和发育、每次交配产生大量同步和快速发育的胚胎,以及斑马鱼胚胎的光学透明度。这些优点促进了大规模的正向遗传诱变筛选,从而发现了许多突变体,并鉴定了对许多不同器官和组织发育(包括颅面和牙齿发育)很重要的基因(Neuhauss 等人,1996 年;Piotrowski等人,1996年;Schilling等人,1996)。这些优雅的研究表明,颅面骨骼元素的规格和发育受人类和斑马鱼相似的信号通路的支配。然而,几乎所有已知的诱变筛选都有几个明显的局限性。首先,突变体通常是根据可见缺陷来选择的,这限制了对最严重的表型的检测。此外,突变体在胚胎早期阶段(受精后 5-6 天 (dpf))进行筛选,这限制了筛选对主要软骨骨骼元件的早期发育重要的基因的鉴定,而控制骨骼发育和稳态后期阶段的基因没有被研究。
We have used a forward genetic chemical mutagenesis screen to identify novel adult craniofacial, skeletal and tooth mutants by screening for mineralized tissue defects in viable adult F3 zebrafish. The mutagenesis of adult male zebrafish was performed as previously described (Solnica-Krezel et al., 1994). Ninety two, adult three month old zebrafish males were subjected to three 1 hour treatments with N-ethyl-nitro-urea (ENU). Mutagenized F0 males were crossed to wild type females to generate 1,084 F1 individuals, which were raised to sexual maturity and outcrossed to wild type adults, generating 131 F2 families. Sexually mature F2 family members were then incrossed in pairwise fashion to generate 12–15 F3 clutches for analysis. The F3 embryos were initially screened daily to 5–6 dpf to identify embryonic lethal (EL) phenotypes. When EL phenotypes were detected and confirmed, embryos were fixed at 4–5 dpf depending on severity of the phenotype, and stained with Alcian Blue (Ab) for cartilage and Alizarin Red (AR) for mineralized structures. Viable siblings from EL phenotype clutches were raised to 16–21 mm in body length, stained with Alizarin red (AR), and screened for adult heterozygous phenotypes. When no EL phenotype was observed, all of the embryos of the clutch were grown to juvenile stage (16–21 mm in body length at ~ 6–8 weeks), stained with AR, and analyzed for mineralized tissue defects.
我们使用正向遗传化学诱变筛选,通过筛选可存活的成年 F3 斑马鱼中的矿化组织缺陷来识别新的成年颅面、骨骼和牙齿突变体。如前所述,成年雄性斑马鱼的诱变作用(Solnica-Krezel等人,1994)。92 只成年 3 个月大的雄性斑马鱼接受了 3 次 1 小时的 N-乙基硝基尿素 (ENU) 治疗。诱变的 F0 雄性与野生型雌性杂交产生 1,084 个 F1 个体,这些个体被培育到性成熟并杂交到野生型成虫,产生 131 个 F2 家族。然后以成对方式将性成熟的 F2 家族成员杂交,以产生 12-15 个 F3 离合器进行分析。F3胚胎最初每天筛选至5-6 dpf,以确定胚胎致死(EL)表型。当检测并确认EL表型时,根据表型的严重程度,将胚胎固定在4-5 dpf,并用阿尔新蓝(Ab)染色软骨,用茜素红(AR)染色矿化结构。将EL表型离合器的可存活兄弟姐妹提高到体长16-21毫米,用茜素红(AR)染色,并筛选成虫杂合表型。当未观察到EL表型时,将离合器的所有胚胎生长到幼年期(~6-8周时体长为16-21毫米),用AR染色,并分析矿化组织缺陷。
Out of the 49 screened F2 families, we isolated 22 mutants which displayed an EL phenotype (Table 1). Two mutants had EL phenotypes but did not display any visible craniofacial defects (83N and 91N, Table1 and data not shown). Three EL mutants (8N, 30N and 85N) exhibited a small head, small eye embryonic phenotype. Ab/AR staining revealed that these mutants did not develop any detectable craniofacial structures (Table1, data not shown). One of the mutants, 93N, appeared to be anemic, and exhibited reduced pigmentation and a curved tail. Interestingly, cartilaginous craniofacial elements were present in the 93N mutant, but stained only faintly with Ab suggesting defects in extracellular matrix formation and cartilage differentiation (Table1, Figure 1). The largest group of EL mutants had several common features, including smaller heads, some with curved bodies, and heart edema (7N, 9N, 12N, 69N, 78N, 100N, 131N, Table1). Ab/AR staining revealed similar skeletal defects, such as reduced or defective anterior arches, as well as absent posterior arches (Figure 1, Figure 3 and data not shown). One mutant, 69N lyon (lyo), displayed a distinctive phenotype (Figure 1) consisting of a shortened Meckel’s cartilage, rostrocaudal elongated and split ethmoid plate resembling cleft palate, and shortened and perhaps duplicated ceratohyal cartilage elements (Figure 1). The surviving putative heterozygous and wild type siblings of all EL phenotype families were also analyzed for adult phenotypes, with the expectation that 2/3 would be heterozygous, and the remaining 1/3 would be wild type. Out of the 22 EL families that were analyzed, we found one family, 78N/knjaz(knz), which also displayed adult skeletal defects. AR staining revealed upper jaw shape defects in knjaz mutants, and defects in tail structures (Figure 2). Pleurostyle and hypural bones were not properly formed and articulated in the tails of knjaz mutants, as compared to wild type siblings (Figure 2).
在筛选的 49 个 F2 家族中,我们分离出 22 个显示 EL 表型的突变体(表 1)。两个突变体具有EL表型,但没有显示任何可见的颅面缺损(83N和91N,表1和数据未显示)。三个EL突变体(8N、30N和85N)表现出小头、小眼胚胎表型。Ab/AR染色显示,这些突变体没有形成任何可检测到的颅面结构(表1,数据未显示)。其中一个突变体93N似乎是贫血的,并表现出色素沉着减少和弯曲的尾巴。有趣的是,93N突变体中存在软骨颅面元件,但仅用Ab微弱染色,表明细胞外基质形成和软骨分化存在缺陷(表1,图1)。最大的一组EL突变体有几个共同特征,包括较小的头部,一些具有弯曲的身体和心脏水肿(7N,9N,12N,69N,78N,100N,131N,表1)。Ab/AR染色显示类似的骨骼缺损,例如前弓减少或有缺陷,以及后弓缺失(图1,图3,数据未显示)。一个突变体,69N lyon(lyo),显示出一种独特的表型(图1),包括缩短的Meckel软骨,类似于腭裂的尾部细长和分裂的筛板,以及缩短和可能重复的角膜软骨元素(图1)。还分析了所有EL表型家族中幸存的假定杂合子和野生型兄弟姐妹的成体表型,预计2/3为杂合子,其余1/3为野生型。在分析的 22 个 EL 家族中,我们发现了一个家族,78N/knjaz(knz),它也显示出成人骨骼缺陷。 AR染色揭示了knjaz突变体的上颌形状缺陷和尾部结构缺陷(图2)。与野生型兄弟姐妹相比,胸膜骨和胸骨在knjaz突变体的尾巴中没有正确形成和铰接(图2)。
早期致死性颅面表型
Examples of EL phenotypes are presented. For each, the left hand panels show lateral views of living embryos, middle panels show lateral views of Alcian blue and Alizarin Red (Ab/AR) stained larvae, and right hand panels show ventral views of Ab/AR stained larvae. Small arrows point to pharyngeal teeth (pt). For the 69N Lyon mutant, the black arrow indicates a smaller lower jaw of 69N lyon (lyo), the red arrow indicates a rostrocaudally extended ethmoid plate, a n d the white arrow indicates a shortened Meckel’s cartilage. The black arrowheads point to rudimentary branchial arch cartilages of 69N lyo embryo, and an asterisk indicates the clefted ethmoid plate. The black arrow in the 93N mutant panel indicates aberrant shape of the lower jaw. The black arrows in the 100N mutant panel indicates a reduced lower jaw, Meckel’s cartilage, and ceratohyal cartilage element. Abbreviations: ch, ceratohyal; eth, ethmoid plate; e, eye; M, Meckel’s cartilage; pt, pharyngeal teeth; wt, wild type; ch, 1–5, branchial arches.
介绍了EL表型的例子。对于每个面板,左侧面板显示活胚胎的侧视图,中间面板显示阿尔新蓝和茜素红 (Ab/AR) 染色幼虫的侧视图,右侧面板显示 Ab/AR 染色幼虫的腹视图。小箭头指向咽齿 (pt)。对于69N Lyon突变体,黑色箭头表示69N Lyon(lyo)的下颌较小,红色箭头表示尾部延伸的筛板,白色箭头表示Meckel软骨缩短。黑色箭头指向69N lyo胚胎的基本分支弓软骨,星号表示裂裂的筛板。93N突变体面板中的黑色箭头表示下颌的异常形状。100N 突变体面板中的黑色箭头表示下颌、Meckel 软骨和角透明软骨元素减少。缩写:ch,ceratohyal;eth, 筛板;e, 眼睛;M: 梅克尔软骨;pt, 咽齿;wt, 野生型;ch,1-5,分支拱。
78N knjaz (knz) 突变体的纯合和杂合表型
(a, b) Lateral view of live 4 dpf wt (a) and 78N knjaz (knz) mutant (b) larvae. (c–f) Lateral (c, d) and (e, f) views of Ab/AR stained 4 dpf wt (c, e) and 78N knjaz (knz) mutant (e, f) embryos. Arrows in (d) and (f) indicate Meckel’s cartilage, and arrowheads in (d) indicate missing branchial arche cartilages. (g–j) Lateral view of AR stained of adult wt (g, i) and 78N knjaz (knz) mutant (h, j) zebrafish. Arrow in (h) points to altered upper jaw morphology in knz mutants. Arrow in (j) points to defects in pleurostyle and hypural bones of knz mutant. Abbreviations: ch, ceratohyal; eth, ethmoid plate; e, eye; M, Meckel’s cartalige; wt, wild type; 1–5, branchial arches.
(a、b)活的 4 dpf wt (a) 和 78N knjaz (knz) 突变体 (b) 幼虫的侧视图。(中至女)Ab/AR 染色的 4 dpf wt (c, e) 和 78N knjaz (knz) 突变体 (e, f) 胚胎的侧视图 (c, d) 和 (e, f) 视图。(d)和(f)中的箭头表示Meckel软骨,(d)中的箭头表示缺失的分支弓软骨。(克-焦)成年 wt (g, i) 和 78N knjaz (knz) 突变体 (h, j) 斑马鱼 AR 染色的侧视图。(h)中的箭头指向knz突变体中上颌形态的改变。(j)中的箭头指向knz突变体的胸膜骨和胸骨的缺陷。缩写:ch,ceratohyal;eth, 筛板;e, 眼睛;M: 梅克尔的卡塔利格;wt, 野生型;1-5,分支拱。
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(a–h) Alizarin Red (AR) stained adult zebrafish. (a,b,e,f) Lateral views of heads of wt (a,e), and 38N bka (b) and 102N duk (f) mutant siblings. The arrows in (b) and (f) point to reduced upper jaw in both mutants. The arrowhead in (f) points to bent parasphenoid in 102N duk mutant. (c,d) Dorsal view of upper jaw in wt (c) and 38N bka mutant (d) siblings. The asterisks indicate patent suture in wt maxilla (c), and fused suture in 38N bka mutant maxilla (d). Arrows in (d) indicate small maxillary bones in 38N bka mutants. (g,h) Lateral view of axial skeleton in wt (g) and 102N duk mutant (h) siblings. Arrows indicate small and misshapen neural arch cartilages in 102N duk mutants.
Table 1
Summary of early lethal phenotypic ENU families.
Family | Name | Phenotype | Embryonic phenotype | Skeletal phenotype | |
---|---|---|---|---|---|
Onset | Lethal | ||||
3N | 2dpf | 3dpf | Heart edema/curved body | Not determined | |
7N | 2dpf | 7dpf | Small head/curved body | Small pharyngeal arches | |
8N | 3dpf | 7dpf | Small head/ curved body | No defined craniofacial cartilage | |
9N | Fast track (ftt) | 3dpf | 8dpf | Small head/ enlarged gut | Reduced/ missing arches/reduced jaw |
11N | 3dpf | 5dpf | Small head/curved body | Reduced cartilage formation | |
12N | 3dpf | 6dpf | Small head/ heart edema | Absent posterior arches / defective ceratohyal | |
18N | 1dpf | 3dpf | Heart edema/ small eye | Not determined | |
30N | 3dpf | 5dpf | Small head/ small eye | No defined craniofacial cartilage | |
32N | 2dpf | 4dpf | Severe deformity | Variable phenotype | |
48N | 2dpf | 6dpf | Small head/ small eye/ curved body | Not determined | |
56N | 2dpf | 5dpf | Curved body | Variable phenotype | |
60N | 3dpf | 6dpf | Curved body | Small pharyngeal arches, reduced low jaw | |
69N | Lyon (lyo) | 3dpf | 6dpf | Small head/ small eye/ curved body | Inverted ceratohyal/reduced or missing posterior arches |
75N | 3dpf | 6dpf | Small head/curved body/ edema | Reduced cartilage formation | |
78N | Knjaz (knz) | 2dpf | 5dpf | Small low jaw/ curved body/ heart edema | Reduced neurocranium/ reduced anterior arches/ absent posterior arches |
83N | 3dpf | 6dpf | Curved body | No defects found | |
85N | 2dpf | 4dpf | Small head/ small eye/ curved body | No defined craniofacial cartilage | |
91N | 2dpf | 4dpf | Small tail/ large heart/ edema | No defects found | |
93N | 4dpf | 6dpf | Curved tail/ reduced pigment/ anemic | Reduced cartilage staining | |
100N | Vader (vdr) | 3dpf | 5dpf | Small head/small eye/ curved body/heart edema | Reduced anterior arches/ absent posterior arches |
130N | 3dpf | 5dpf | Curved body | Not determined | |
131N | 3dpf | 5dpf | Small head/ small eye/ curved body | Reduced or absent anterior arches/ absent posterior arches |
Early lethal phenotypes were apparent 1–3 days post-fertilization (dpf), and were characterized by distinct features, including reduced pharyngeal arch cartilages, as described.
Among the 27 F2 families that were not embryonically lethal, we identified 4 families with adult homozygous recessive craniofacial phenotypes (Table 2). Several mutants exhibited varying degrees of midfacial hypoplasia. AR staining for mineralized tissues revealed that the 38N belka (bka) mutant exhibited a severe reduction in size of the upper jaw (Figure3). Belka mutants also exhibited missing kinethmoid and fused maxilla (Figure 3). In addition, approximately 50% of belka mutants exhibited scoliosis in the tail region. Similarly to belka, 102N or dushka (duk) mutants exhibited severely reduced upper jaws (Figure 3), however their maxillas were not fused. Dushka mutants also exhibited bent parasphenoid bones and branched neural arches, which are normally straight in wild type zebrafish (Figure3). The least affected mutant, 72N, exhibited only slight midfacial hypoplasia, reduced kinethmoid, and asymmetric maxilla (data not shown). The 72N mutant also exhibited defects in tail structures similar to that observed in knjaz heterozygous mutants (data not shown).The only craniofacial defect we could detect in the fourth mutant, 17N, was a bent basihyal in the lower jaw, as compared to wild type siblings (data not shown). Among the F2 families that did not exhibit EL phenotypes, we found 3 mutants exhibiting axial skeletal defects, but which did not display detectable craniofacial abnormalities (Table 2). In two of these families, 92N/droog (dro) and 99N, we observed defects in neural arch elements, however this phenotype was more dramatic in dro mutants (Figure 4). Mutants in 74N families displayed scoliosis in the tail region (Figure 4). The ratio of mutants to wild type siblings in F3s generated from several pairs of F2 74Ns ranged from 33% to 67%, suggesting a possible dominant phenotype.
Lateral views of wt AR stained axial skeleton (a). Lateral view of AR stained axial skeletons of 92N dro (b–d) and 99N (e–f) mutants. Arrows in (b–f) point to missing neural arches. (g) Lateral view of AR stained wt (g), and 74N mutant (h,i) tails. Arrows point to misshaped tail structures in 74N mutant. Dorsal view of AR stained wt (j) and 74N mutant (k, l) tails. Arrows point to scoliosis in 74N mutant tails.
Table 2
Summary of adult mineralized tissue phenotypic ENU families.
Family | Name | Craniofacial phenotype | Axial skeleton phenotype |
---|---|---|---|
17N | Bent basihyal | Not detected | |
38N | Belka (bka) | Reduced upper jaw, mused maxilla, absent kinethmoid | 50% scoliosis in tail region |
72N | Reduced kinethmoid, asymmetric maxilla | Not detected | |
74N | Not detected | Scoliosis in tail region | |
92N | Droog (dro) | Not detected | Missing neural arches |
99N | Not detected | Missing neural arches | |
102N | Dushka (duk) | Reduced upper jaw, bent parasphenoid bone | Curved neural arches |
Adult phenotypes were identified in 16–21 mm fish (6–8 weeks post fertilization). Craniofacial and skeletal defects were revealed by Alizarin Red stain for mineralized tissues.
Overall, our screen represents one of the few systematic genetic screens focusing on viable adult skeletal defects. We are currently working to map the mutations with assigned names (See Tables 1 and and2)2) to identify and test candidate genes. We anticipate that these studies will contribute to the generation of a more comprehensive collection of mineralized tissue zebrafish mutants relevant to human skeletal, craniofacial, and dental disease phenotypes, and which will become valuable tools for elucidating novel genes and signaling pathways regulating mineralized tissue development, remodeling, and homeostasis.
Materials and Methods
Zebrafish husbandry
Wild type (AB) zebrafish were bred and raised at 28.5°C at Tufts Zebrafish Facility, in a controlled environment with 14/10 h light/dark cycle, as previously described (Westerfield, 1995).
ENU mutagenesis and breeding
ENU mutagenesis was performed as previously described (Solnica-Krezel and Driever, 1994). Briefly, young adult male zebrafish (F0) were mutagenized with ENU and out-crossed to wild type females. The resulting F1 fish were raised to sexual maturity and outcrossed to wild type fish, to generate F2 families. Adult F2 family members were then incrossed in pair-wise fashion to generate F3 fish, which were raised to 16–21 mm (6–8 wpf), and analyzed.
Alcian Blue (Ab) and Alizarin Red (AR) staining
Four to seven dpf zebrafish were stained for cartilage and bone with Ab/AR, as previously described (Yelick and Connolly, 2010). Juvenile stage zebrafish, ~16–21 mm in length, were stained with AR for mineralized tissues using modified versions of previously published methods (Connolly and Yelick, 2010).
Acknowledgements
We would like to acknowledge the expertise and input of all members of the Yelick Laboratory, and in particular, that of Christopher Ban, Evan Conaway, Jose A. Gil, Min Ji, Christopher Rud, Jamie Singh, and Thao Tran, for expert zebrafish husbandry and care. We would also like to acknowledge the support of NIH/NIDCR grant DE018043.
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