骨形態發生蛋白（BMPs）是調節小鼠發育中肢體的頂外胚層脊（AER）功能和趾間程序性細胞死亡（PCD）的分泌信號。然而這些信號的細胞內第二信使者的身份不明。本研究旨在分析Smad蛋白在受骨形態發生蛋白調節的頂外胚層（AER）功能和趾間程序性細胞死亡的功用和相互作用。 基因剔除骨形態發生蛋白信號元件，包括細胞內第二信使者和骨形態發生蛋白，會導致早期胚胎死亡。本研究採用Cre/loxP系統，選擇性地在小鼠發展中肢體的頂外胚層脊和腹側外胚層剔除Smad1和/或Smad5基因。 本研究採用Smad1和/或Smad5 floxed等位基因和En1[superscript Cre/]⁺ 敲等位基因。 / 單一選擇性地在發展中肢體的頂外胚層脊和腹側外胚層剔除Smad1或Smad5不會導致肢體畸形。然而，同時選擇性剔除Smad1/Smad5會導致帶子手指和腳趾(syndactyly)。帶子手指和腳趾的形成是因趾間程序性細胞死亡減少和趾間細胞異常增生。 Smad1/Smad5雙突變體的細胞跟踪實驗顯視腹側外胚層增厚和原在腹側的外胚層En1[superscript Cre/]⁺後裔細胞出現異位轉移到背側趾間位置。 在分子水平上，Fgf8在Smad1/Smad5雙突變體趾間外胚層的表達延長至胚胎發育的第十三天(E13)。這異位Fgf8表達可作為一個趾間的上皮細胞和間質細胞的生存信號。本研究結果表明，Smad1和Smad5在骨形態發生蛋白信號中擔當必需角色，它們充當頂外胚層脊和腹側外胚層細胞內骨形態發生蛋白信號的細胞內第二信使者，並互補對方的功用。頂外胚層脊和腹側外胚層的Smad1/Smad5信號調節趾間組織萎縮。因Smad1/Smad5雙突變體在趾間的程序性細胞死亡出現缺陷，它將會是一個研究程序性細胞死亡調節機制的重要模型。 / Bone morphogenetic proteins (BMPs) are secreted signals that regulate apical ectodermal ridge (AER) functions and interdigital programmed cell death (PCD) of developing mouse limb. However, the identities of the intracellular mediators of these signals are unknown. The present study aims at investigating the role and interaction of Smad proteins in BMPs-regulated AER functions in limb development. Inactivation of BMP signaling components, including intracellular mediators and BMP ligands, will lead to early embryonic lethality. To circumvent the problem, Cre/loxP system was employed to inactivate Smad1 and/or Smad5 selectively in AER and ventral ectoderm of developing mouse limb. Smad1 or/and Smad5 floxed alleles and an En1[superscript Cre/]⁺ knock-in allele was employed for the study. / Single inactivation of either Smad1 or Smad5 did not result in limb abnormalities. However, the Smad1/Smad5 double mutants exhibited syndactyly due to a reduction in interdigital PCD and an increase in interdigital cell proliferation. Cell tracing experiments in the Smad1/Smad5 double mutants showed that ventral ectoderm became thicker and the descendents of ventral En1[superscript Cre/]⁺ expressing ectodermal cells were located at dorsal interdigital regions. At the molecular level, Fgf8 expression was prolonged in the interdigital ectoderm of embryonic day (E) 13 Smad1/Smad5 double mutants, suggesting that the ectopic Fgf8 expression may serve as a survival signal for interdigital epithelial and mesenchymal cells. The result suggests that Smad1 and Smad5 are required and function redundantly as intra-cellular mediators for BMP signaling in the AER and ventral ectoderm. Smad1/Smad5 signaling in the AER and ventral ectoderm regulates interdigital tissue regression of developing limb. The mutants with defects in interdigital PCD could also serve as a valuable model for investigation of PCD regulation machinery. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wong, Yuk Lau. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 121-133). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract of thesis in English --- p.i / Abstract of thesis in Chinese --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Figures --- p.xii / List of Tables --- p.xv / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Limb development: a General Overview --- p.1 / Chapter 1.2 --- Signaling centres and axis determination of developing limb buds --- p.8 / Chapter 1.2.1 --- Apical ectodermal ridge and proximal-distal axis --- p.8 / Chapter 1.2.2 --- The zone of polarizing activity and anterior-posterior axis --- p.8 / Chapter 1.2.3 --- Ectoderm and dorsal-ventral axis --- p.9 / Chapter 1.3 --- Programmed cell death in limb development --- p.10 / Chapter 1.3.1 --- Localization of programmed cell death in developing limb bud --- p.10 / Chapter 1.3.2 --- Functions of programmed cell death in limb development --- p.11 / Chapter 1.4 --- Regulation of programmed cell death in developing mouse limb --- p.12 / Chapter 1.4.1 --- Tissues of limb bud involved in regulation of programmed cell death-question to be answered --- p.12 / Chapter 1.4.2 --- Molecular regulation of programmed cell death in developing mouse limb --- p.12 / Chapter 1.4.3 --- Programmed cell death machinery --- p.14 / Chapter 1.5 --- BMPs signaling and limb development --- p.16 / Chapter 1.5.1 --- Functions of BMPs signaling in limb development --- p.16 / Chapter 1.5.2 --- BMPs signaling cascade --- p.17 / Chapter 1.5.3 --- BMPs signaling and programmed cell death --- p.20 / Chapter 1.5.4 --- Intra-cellular mediators of BMPs signaling in limb programmed cell death-question to be answered --- p.21 / Chapter 1.6 --- Scope of the Thesis --- p.22 / Chapter 1.6.1 --- Central aim of the project --- p.22 / Chapter 1.6.2 --- Specific objectives --- p.24 / Chapter Chapter 2 --- Syndactyly in mice lacking Smad1/Smad5 signaling in ventral ectoderm: Implication for its functions in limb development / Chapter 2.1 --- Confirmation of expression of En1[superscript Cre/]⁺ knock-in allele in AER and ventral ectoderm of developing limb bud --- p.27 / Chapter 2.1.1 --- Introduction --- p.27 / Chapter 2.1.2 --- Material --- p.28 / Chapter 2.1.2.1 --- Mouse lines --- p.28 / Chapter 2.1.2.2 --- Chemicals --- p.28 / Chapter 2.1.3 --- Breeding strategy and methodology --- p.28 / Chapter 2.1.4 --- Result --- p.30 / Chapter 2.1.5 --- Discussion --- p.30 / Chapter 2.2 --- En1[supercript Cre/]⁺ inactivation of the Smad1 and Smad5 conditional alleles in the AER and limb ventral ectoderm --- p.33 / Chapter 2.2.1 --- Introduction --- p.33 / Chapter 2.2.2 --- Material --- p.33 / Chapter 2.2.2.1 --- Mouse lines --- p.33 / Chapter 2.2.2.2 --- Antibodies and chemicals --- p.33 / Chapter 2.2.3 --- Methodology --- p.34 / Chapter 2.2.4 --- Results and discussion --- p.36 / Chapter 2.3 --- Single inactivation of Smad1 or Smad5 conditional null allele in the AER and ventral limb ectoderm does not result in observable limb abnormalities --- p.40 / Chapter 2.3.1 --- Introduction --- p.40 / Chapter 2.3.2 --- Material --- p.40 / Chapter 2.3.2.1 --- Mouse lines --- p.40 / Chapter 2.3.2.2 --- Chemicals --- p.41 / Chapter 2.3.3 --- Breeding strategy and methodology --- p.41 / Chapter 2.3.4 --- Results and discussion --- p.41 / Chapter 2.4 --- Inactivation of both Smad1/Smad5 signaling in the limb AER and ventral ectoderm results in interdigital tissue regression defects --- p.45 / Chapter 2.4.1 --- Introduction --- p.45 / Chapter 2.4.2 --- Material --- p.45 / Chapter 2.4.2.1 --- Mouse lines --- p.45 / Chapter 2.4.2.2 --- Chemicals --- p.45 / Chapter 2.4.3 --- Breeding strategies and methodology --- p.45 / Chapter 2.4.4 --- Results --- p.46 / Chapter 2.4.5 --- Discussion --- p.48 / Chapter 2.5 --- Smad1/Smad5 signaling in the limb AER and ventral ectoderm is required for regulating interdigital cell death and cell proliferation --- p.56 / Chapter 2.5.1 --- Introduction --- p.56 / Chapter 2.5.2 --- Material --- p.56 / Chapter 2.5.3 --- Methodology --- p.57 / Chapter 2.5.3.1 --- Cell death assays --- p.57 / Chapter 2.5.3.2 --- Cell proliferation assays --- p.58 / Chapter 2.5.3.3 --- Statistical analysis --- p.59 / Chapter 2.5.4 --- Result --- p.59 / Chapter 2.5.5 --- Discussion --- p.60 / Chapter 2.6 --- Fgf8 is up-regulated at the interdigital distal ectoderm and serves as survival signal for interdigital mesenchyme upon inactivation of the Smad1/Smad5 signaling in AER and ventral ectoderm --- p.66 / Chapter 2.6.1 --- Introduction --- p.66 / Chapter 2.6.2 --- Material --- p.66 / Chapter 2.6.2.1 --- Material for preparation of DIG-labeled RNA probe --- p.66 / Chapter 2.6.2.2 --- Materials for whole-mount and section in-situ hybridization --- p.67 / Chapter 2.6.3 --- Methodology --- p.67 / Chapter 2.6.3.1 --- Preparation of DIG-labeled RNA probe --- p.67 / Chapter 2.6.3.1.1 --- Transformation of DNA into competent cells --- p.67 / Chapter 2.6.3.1.2 --- Preparation of recombinant plasmid --- p.68 / Chapter 2.6.3.1.2.1 --- Birnboim and Doly method --- p.68 / Chapter 2.6.3.1.2.2 --- QIAGEN column method --- p.68 / Chapter 2.6.3.1.3 --- Preparation of linearized recombinant plasmid for riboprobe preparation --- p.70 / Chapter 2.6.3.1.4 --- Preparation of DIG-labelled riboprobes for in situ hybridization --- p.70 / Chapter 2.6.3.2 --- Whole-mount in situ hybridization --- p.72 / Chapter 2.6.3.3 --- Section in situ hybridization --- p.73 / Chapter 2.6.4 --- Results --- p.74 / Chapter 2.6.5 --- Discussion --- p.75 / Chapter 2.7 --- Mesenchymal BMP signals are not altered in the developing autopod of the Smad1/Smad5 mutants --- p.81 / Chapter 2.7.1 --- Introduction --- p.81 / Chapter 2.7.2 --- Material --- p.81 / Chapter 2.7.3 --- Methodology --- p.81 / Chapter 2.7.4 --- Result and discussion --- p.81 / Chapter 2.8 --- Inactivation of Smad1/Smad5 in AER and ventral ectoderm results in postaxial polydactyly --- p.87 / Chapter 2.8.1 --- Introduction --- p.87 / Chapter 2.8.2 --- Material --- p.87 / Chapter 2.8.3 --- Methodology --- p.87 / Chapter 2.8.4 --- Results and discussion --- p.88 / Chapter 2.9 --- Smad1/Smad5 inactivation in the limb ventral ectoderm resulted in ventral ectoderm thickening and ectopic En1-expressing cells and their descendents in the dorsal interdigital ectoderm --- p.91 / Chapter 2.9.1 --- Introduction --- p.91 / Chapter 2.9.2 --- Material p91 / Chapter 2.9.3 --- Methodology --- p.92 / Chapter 2.9.3.1 --- Breeding scheme --- p.92 / Chapter 2.9.3.2 --- X-gal stainin --- p.92 / Chapter 2.9.3.3 --- Immunofluorescence --- p.93 / Chapter 2.9.4 --- Results --- p.93 / Chapter 2.9.5 --- Discussion --- p.94 / Chapter 2.10 --- Inactivation of both Smad1 and Smad5 in ventral limb ectoderm does not cause defects in dorsal-ventral patterning --- p.99 / Chapter 2.10.1 --- Introduction --- p.99 / Chapter 2.10.2 --- Material --- p.99 / Chapter 2.10.3 --- Methodology --- p.99 / Chapter 2.10.1 --- Whole-mount in situ hybridization --- p.99 / Chapter 2.10.2 --- Histological analysis --- p.99 / Chapter 2.10.4 --- Results --- p.100 / Chapter 2.10.5 --- Discussion --- p.101 / Chapter 2.11 --- Contribution of present study --- p.104 / Chapter Chapter 3 --- Proteomic analysis on developing limbs of Smad1/Smad5 knock-out mutant: potential protein candidates that regulate cell death and cell proliferation / Chapter 3.1 --- Introduction --- p.106 / Chapter 3.2 --- Materials --- p.106 / Chapter 3.3 --- Methodology / Chapter 3.3.1 --- Protein sample preparation of limb --- p.107 / Chapter 3.3.2 --- Protein quantification --- p.107 / Chapter 3.3.3 --- 2D gel electrophoresis --- p.108 / Chapter 3.3.4 --- Image analysis --- p.109 / Chapter 3.3.5 --- In gel digestion and MALDI-TOF MS --- p.109 / Chapter 3.4 --- Results --- p.110 / Chapter 3.5 --- Discussion --- p.111 / Chapter Chapter 4 --- General Discussion and Future Direction / Chapter 4.1 --- The intracellular signaling components of BMP signaling --- p.115 / Chapter 4.2 --- The programmed cell death machinery in BMPs-regulated interdigital programmed cell death --- p.117 / Chapter 4.3 --- The possible involvement of reactive oxygen species in BMPs- regulated interdigital programmed cell death --- p.118 / Chapter 4.4 --- Interaction of BMP signaling and retinoic acid signaling --- p.119 / Chapter 4.5 --- Potential candidate genes regulated by Smad1/Smad5 signaling --- p.119 / Bibliography --- p.121 / Publication --- p.134
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328041 |
| Date | January 2012 |
| Contributors | Wong, Yuk Lau., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xviii, 134 leaves) : ill. (chiefly col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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