埃及聖䴉(Threskiornis aethiopicus)是一種非洲撒哈拉沙漠以南以及中東地區的本土朱鷺科鳥類，然而近三十年來已經在台灣成為對生態環境威脅極大的外來物種。鳥喙的形狀大小在物種間有高度多樣性，而擁有細長且微微下彎的喙是埃及聖䴉一個顯著的特徵。雖然對於不同型態鳥喙相關的分子發育機制已經有很多研究，但大部分的研究都是針對比較常被研究的物種，包含雞、鴨以及一些雀形目的鳥類，其他物種則很少被研究。在本研究當中，我們進行了埃及聖䴉以及雞早期胚胎顱面結構的轉錄體比較分析，希望可以找出控制埃及聖䴉長出如此長且大的喙的相關分子發育機制。經由比較兩個物種的喙的基因表現，我們發現埃及聖䴉控制鳥喙形狀的發育的分子發育機制可能和先前的研究有很大的不同。首先，我們發現初級纖毛相關的基因可能和物種間喙的型態差異的發育有關。而一些和骨骼發育相關的基因表現差異顯示在埃及聖䴉中成骨細胞分化以及骨骼礦化的時間可能比雞還要晚，符合先前研究所提出來的假說。最後，經由生物分子路徑分析(Ingenuity Pathway Analysis)以及骨密度的測量，我們認為埃及聖䴉可能藉由抑制骨密度相關基因表現發育出如此巨大且細長的鳥喙。經由對於埃及聖䴉鳥喙的發育研究，我們可以對於擁有類似表徵的涉禽類的發育以及演化機制有更深入的認識。

The African sacred ibis (Threskiornis aethiopicus) is a native wading bird in Sub-Saharan Africa, but has become an invasive species in Taiwan for more than 30 years. The African sacred ibis has a prominent long curved beak that enables them to probe into the water or soil for diet. The molecular mechanisms of species-specific development of beak size and shape have been studied for decades, however, most of them focused on few avian species such as chicken and finches. To identify molecular mechanisms underlying the development of long beak in the sacred ibis, we performed a comparative transcriptomic analysis of craniofacial prominences, which are developmental components of avian beak, in early-stage embryos of chicken and sacred ibis. Our data demonstrated a species-specific genetic pattern in the long and large beak development in sacred ibis. We indicated a possible correlation of cilium formation to species-specific beak morphological difference. Differential expression of several genes of skeleton development were partially matched to the previous hypothesis of delayed bone formation and resorption process in the development of a larger beak. In addition, we speculated that a looser trabecular bone structure enables the development of a larger beak in sacred ibis due to the down-regulation of bone mineral density related pathway. This study provides a better understanding of developmental and evolutionary biology of wading birds that have similar traits of long beaks.

摘要 i
Abstract ii
致謝 iii
Introduction 1
Introduction of African sacred ibis 1
Development of the species-specific beak morphology 2
Transcriptomic analysis of beak of chicken and sacred ibis 6
Method 8
Collection and treatment of embryonic materials of chicken and sacred ibis 8
RNA extraction and RNA sequencing 9
De novo assembly 9
Transcript completeness and filter out redundant transcripts 10
Protein sequence prediction and orthologue prediction 11
Functional annotation 11
Transcript mapping and quantification 12
Differential expression analysis 13
Clustering of differential expression genes by gene function 13
Ingenuity Pathway Analysis 15
Measurement of bone mineral density 15
Result 17
Staging embryonic sample of sacred ibis 17
RNA sequencing 18
De novo transcriptomic assembly 19
Transcript filtering and completeness estimation 19
Orthologue prediction 21
Functional annotation 22
Gene clustering by differential expression 22
Functional enrichment analysis of differential expression transcripts 24
Expression of genes associated with beak morphology 26
Differential expression of genes related to cilium formation 28
Expression of genes of skeleton formation 29
Ingenuity Pathway Analysis (IPA analysis) 32
Measurement of bone mineral density 34
Discussion 35
Embryological staging of the sacred ibis 35
De novo assembly method 37
Genetic mechanism of development of beak morphology is highly species-specific 38
High expression of BMP2 might play a role in the development of the large beak in sacred ibis 41
Craniofacial ciliophathy might be associated with the development of species-specific beak morphology 43
The postponement of osteogenic cell differentiation in larger beak 45
Heterochronic characteristic of expression of genes related to bone resorption 47
Collagen involved in the development of beak in diverse morphology 48
The lower BMD in beak of sacred ibis 49
Conclusion 51
Figures and Tables 53
Supplementary Data 74
Reference 121

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