ITEM METADATA RECORD
Title: Impact of iodothyronine deiodinase deficiency on zebrafish development
Other Titles: Impact van joodthyronine dejodase-deficiëntie op de ontwikkeling van de zebravis
Authors: Heijlen, Marjolein
Issue Date: 16-Apr-2014
Abstract: Normal 0 21 false false false NL-BE X-NONE X-NONE <span style="mso-bidi-font-family:&quot;Times New Roman&quot;;mso-ansi-language:EN-US" lang="EN-US">Thyroid hormones (THs) are important regulatory factors of vertebrategrowth and development. Although the main TH secreted by the thyroid gland is theprohormone T4, the majority of TH actions depend on the binding ofthe active hormone T3 to its nuclear receptors. Appropriate T3availability in developing cells is regulated by iodothyronine deiodinases(D1-D3), the enzymes that activate and inactivate THs. Despite their essentialrole in TH regulation, deiodinases have received little attention in relationto their function during development. In this thesis we have used morpholinoknockdown in the zebrafish as a model to examine the impact of deiodinasedeficiency on early development, with the focus on the TH-inactivatingdeiodinase D3.<span style="mso-bidi-font-family:&quot;Times New Roman&quot;;mso-ansi-language:EN-US" lang="EN-US"> Due to a wholegenome duplication <span style="mso-fareast-font-family:GulliverRM;mso-bidi-font-family:Times;color:black;mso-ansi-language:EN-US" lang="EN-US">in the earlyevolution of ray-finned fishes, zebrafish posses two paralogs for D3, dio3a and <i style="mso-bidi-font-style:normal">dio3b. We showed the expression of both genes during zebrafishembryonic and early larval development. Duringmost of this period, significantly higher levels of <i style="mso-bidi-font-style:normal">dio3b mRNA were detected compared to <i style="mso-bidi-font-style:normal">dio3a mRNA. In line with this, the contribution of dio3b to embryonic D3 activity seemed tobe predominant and the phenotype of dio3bmorphants was more severe and persisted longer compared to <i style="mso-bidi-font-style:normal">dio3a morphants.<span style="mso-bidi-font-family:&quot;Times New Roman&quot;;mso-ansi-language:EN-US" lang="EN-US"> To compare thefunctions of the TH-activating and TH-inactivating pathways in zebrafishdevelopment, we examined the effects following co-knockdown of the activatingdeiodinases (D1 and D2) with those following knockdown of D3. We found that dio1dio2 morphants, as well as dio3b morphants, showed a delayedincrease in body length, eye size and ear size, delayed hatching, and disturbedswim bladder inflation. For all these effects the phenotype was more severe andpersisted longer in dio3b morphants.We further found some phenotypic abnormalities in <i style="mso-bidi-font-style:normal">dio3b morphants that were not present in <i style="mso-bidi-font-style:normal">dio1dio2 morphants. More specifically, <i style="mso-bidi-font-style:normal">dio3b knockdown seemed to cause a disorganized layer formation inthe swim bladder, and caused a reduced size in other endoderm derived organssuch as liver and intestine. dio3bmorphants also showed defects in embryonic and larval motility, and anupregulation of transcription in <span style="mso-ansi-language:EN-US" lang="EN-US">one of the genes involved in fast muscle development (mylpfa).<span style="mso-bidi-font-family:&quot;Times New Roman&quot;;mso-ansi-language:EN-US" lang="EN-US"> To verify whetherthe developmental changes that follow dio3bknockdown are truly caused by excessive T3 availability due todecreased D3 activity, we used two approaches. First, we demonstrated that exposure toexogenous T3 caused similar developmental defects as seen in dio3b morphants. Next, we showed thatinjection of human DIO3 mRNA could substantiallyrescue D3 activity as well as the observed phenotype. Takentogether, we have demonstrated that maintaining a critical balance betweenTH-activation and TH-inactivation is important for development to proceednormally, and showed that zebrafish is an excellent model to further explorethe role of deiodinases during early vertebrate development.<span style="mso-bidi-font-family:&quot;Times New Roman&quot;;mso-ansi-language:EN-US" lang="EN-US"><span style="font-size:11.0pt;line-height:115%;font-family:&quot;Calibri&quot;,&quot;sans-serif&quot;;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:&quot;Times New Roman&quot;;mso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA" lang="EN-US"> Normal 0 21 false false false NL-BE X-NONE X-NONE <w:LatentStyles DefLockedState="false" DefUnhideWhenUsed="true" DefSemiHidden="true" DefQFormat="false" DefPriority="99" LatentStyleCount="267"> <w:LsdException Locked="false" Priority="0" 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Table of Contents: List of abbreviations v
Research focus 1
Chapter 1 Introduction 5
1.1 General background on thyroid hormones 6
1.1.1 Thyroid hormone synthesis and secretion 6
1.1.2 Thyroid hormone transport 8
1.1.3 Thyroid hormone deiodination 8
1.1.4 Thyroid hormone receptors 11
1.2 Zebrafish as a model 13
1.2.1 Advantages of zebrafish as a model organism 13
1.2.2 The life cycle of zebrafish 14
1.3 Zebrafish as a model for peripheral thyroid hormone metabolism in vertebrate development 15
1.3.1 Development of the zebrafish thyroid gland 15
1.3.2 Thyroid hormone levels during zebrafish development 18
1.3.3 Expression and distribution of thyroid hormone transporters during zebrafish development 19
1.3.4 Expression and distribution of deiodinases during zebrafish development 20
1.3.5 Expression and distribution of thyroid hormone receptors during zebrafish development 25
1.3.6 Zebrafish as a model for thyroid hormone-related gene silencing 27
Chapter 2 Comparison of the expression and function of dio3a and dio3b during zebrafish development 31
2.1 Experimental setup and methods 32
2.1.1 Zebrafish care 32
2.1.2 RNA isolation and quantitative RT-PCR 32
2.1.3 Whole mount in situ hybridization 34
2.1.4 Microinjections of antisense morpholino oligonucleotides 34
2.1.5 D3 enzyme activity assay 35
2.1.6 Morphometric analysis 36
2.1.7 Statistical analysis 38
2.2 Results 38
2.2.1 Ontogenic expression of dio3a and dio3b 38
2.2.2 D3 activity following dio3aKD or dio3bKD 40
2.2.3 D3KD affects embryonic and early larval zebrafish development 41
2.2.4 Double KD does not exacerbate the phenotype 45
2.3 Discussion 47
Chapter 3 Comparing the function of the TH-activating and TH-inactivating pathways in zebrafish development 53
3.1 Experimental setup and methods 54
3.1.1 Zebrafish care 54
3.1.2 Microinjection of antisense morpholino oligonucleotides 54
3.1.3 Morphometric analysis and scoring of hatching 55
3.1.4 Histology and whole mount in situ hybridization 55
3.1.5 Motility assays 55
3.1.6 RNA isolation and quantitative RT-PCR 56
3.1.7 Statistical analysis 57
3.2 Results 57
3.2.1 Co-KD of dio1 and dio2 affects zebrafish development 57
3.2.2 Co-KD of dio1 and dio2, as well as KD of dio3b delays the time of hatching 59
3.2.3 dio3bKD leads to defects in endoderm development 61
3.2.4 dio3bKD affects embryonic and larval motility 65
3.3 Discussion 68
Chapter 4 T3 supplementation and rescue of the D3 knockdown phenotype with human DIO3 mRNA 77
4.1 Experimental setup and methods 78
4.1.1 Zebrafish care 78
4.1.2 T3 treatment and microinjections 78
4.1.3 D3 enzyme activity assay 79
4.1.4 Morphometric analysis, scoring of hatching and motility assays 79
4.1.5 Statistical analysis 79
4.2 Results 79
4.2.1 T3 supplementation mimics the effects of dio3bKD 79
4.2.2 Co-injection of human DIO3 mRNA partially rescues the effects of dio3bKD 81
4.3 Discussion 84
Chapter 5 General discussion and future perspectives 87
Summary 97
Samenvatting 101
Supplementary material 105
References 109
ISBN: 978-90-8649-704-1
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Department of Biology - miscellaneous
Animal Physiology and Neurobiology Section - miscellaneous
Molecular Biodiscovery (+)

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