臺灣博碩士論文加值系統

決定昆蟲胚胎的型態是由一群很保守專一的基因所調控。其中以果蠅 (Drosophila) 前後體軸形成的機制是最為熟知的，主要受到兩個基因 bicoid 和 caudal 他們的蛋白質產物在胚胎兩端所形成的梯度而影響果蠅胚胎的發育。然而，最近的研究顯示，bicoid 是在演化過程中所產生的新的調控蛋白質。因此，在其他非雙翅目昆蟲例如: 寄生蜂 (Nasonia vitripennis) 和擬穀盜 (Tribolium castaneum)，和果蠅不同的是，這兩種昆蟲是利用 hunchback 和 orthodenticle 這兩個基因之間的交互調控來替代果蠅 bicoid 對於前端發育的影響。

這裡的證據顯示在蚜蟲中，其前端的決定與發育主要是藉由 Aphb 在發育時期中不對稱的分布所影響，而並非藉由和寄生蜂與擬穀盜一樣的機制: 利用hunchback 和 orthodenticle 的相互調控來影響前端的發育。此外在蚜蟲和果蠅 caudal 會在早期胚胎發育過程中形成梯度不同的是，蚜蟲 caudal 並沒有發現有梯度的形成，但是在發育的中後期，它的表現則會集中在胚胎的最末端。綜言之，Aphb、Apotd 和 Apcad 在蚜蟲晚期胚胎發育中仍然擁有一些在其他昆蟲也有的保守性特徵，可是在早期的發育中則相對的變異較大。

相較於果蠅會在還是 syncytial blastoderm 的時期就決定體節的形成，大部分的節肢動物會處於在細胞化狀態下去形成他們的體節。在蚜蟲中，我們選殖出三個保守性的體節極化基因 (segment polarity genes)，分別是 engrailed-1、engrailed-2 和 wingless，並且觀察他們三者在胚胎發育中的表現位置。結果發現在蚜蟲中，第一個出現的體節是胸部第三節 (T3)，然後剩餘體節形成的順序則是先頭部然後才是腹部。另外，Apwg 的表現也和 Apen 一樣是一節一節的，並且和 Apcad 一樣都會表現在胚胎的末端。這樣的結果顯示蚜蟲體節生成的機制和其他 short germ band 的昆蟲是相似的。然而在蚜蟲中，體軸形成相關的基因 (Aphb、Apotd 和 Apcad) 是否會調控體節形成基因 (Apen-1、Apen-2 和 Apwg) 仍然需要進一步的實驗以及分析。

Patterning insect embryos is controlled by a spectrum of well-conserved genes. Anteroposterior axis formation is well understood in Drosophila in which bicoid and caudal encode proteins that form morphogenetic gradients from both poles of the embryo and both of them play a key role in anterior and posterior patterning of the fruit fly embryo. However, recent studies suggest bicoid is an invention of new regulatory protein during the evolution. In some non-dipteran insects such as wasp (Nasonia vitripennis) and beetle (Tribolium castaneum), the anterior specification relies on a synergistic interaction between hunchback and orthodenticle.

Evidence shown here indicates it is the asymmetric localization of Aphb rather than the synergistic interaction between Aphb and Apotd, which regulate the anterior development in the asexual pea aphid. Furthermore, unlike Drosophila caudal which forms a linear concentration gradient in early embryogenesis, Apcad does not show a gradient expression in early developmental stages. But, Apcad transcript is expressed in the posteriormost of the germ band in mid- and late- embryogenesis, and might involve in the germ cell formation in the pea aphid. In conclusion, Aphb, Apotd and Apcad exhibit some conserved features in later embryogenesis in the pea aphid as well as in other insects; however, they diverse in early oogenesis and embryogenesis.

Compared to Drosophila which patterns most of its segments in syncytial blastoderm stage, most arthropods generate its segments from a celluarized environment. Here, three conserved segment polarity genes, engrailed-1, engrailed-2 and wingless have been cloned and illustrated the expression of their transcripts. It is shown that the first appearing segment is the third segment of thorax (T3), and then remaining segments add from head to abdomen sequentially. The expression of Apwg is also shown periodically and co-localized with Apcad transcript in the posterior of the germ band. It suggests that the segmentation mechanism in pea aphid might be conserved, similar to other short germ band insects. However, the possible regulation between genes which involve in axis formation (Aphb, Apotd, and Apcad) and genes which involve in segment formation (Apen-1, Apen-2, and Apwg) remains unclear and worthy of further investigation.

Chapter 1: Formation of anteroposterior axis in insects....1
1.1 Anteroposterior axis formation in D. melanogaster.....2
1.1.1 Genes hierarchy in anteroposterior axis formation
in D. melanogaster..................................2
1.1.2 Roles of bicoid, hunchback, and orthodenticle in
anterior patterning in D. melanogaster..............4
1.1.3 Roles of caudal in posterior patterning in D.
melanogaster........................................8
1.2 Anteroposterior axis formation in other insects......10
1.2.1 Roles of hunchback and orthodenticle in anterior
patterning in other insects........................11
1.2.2 Roles of caudal in posterior patterning in other
insects............................................14
1.3 Segmentation.........................................16
1.3.1 Roles of engrailed and wingless in D. melanogaster.17
1.3.2 Roles of engrailed and wingless in other insects...22
1.4 Studying anteroposterior axis formation and
segmentation in the pea aphid Acyrthosiphon pisum....23
1.4.1 Life cycles of the pea aphid.......................25
1.4.2 Oogenesis and embryogenesis of the pea aphid.......26

Chapter 2: Materials and Methods..........................29
2.1 Pea aphids...........................................30
2.1.1 Culturing pea aphids...............................30
2.2 Synthesis of riboprobes..............................30
2.2.1 Genes cloning......................................30
2.2.2 5’ and 3’ rapid amplification of cDNA ends.......33
2.2.3 In vitro transcription.............................35
2.3 In situ hybridization................................38
2.3.1 Dissection and fixation of the ovarioles...........38
2.3.2 Whole-mount in situ hybridization and microscopy in
the pea aphid......................................39
2.4 Antibodies generation................................41
2.4.1 Overexpression and purification of fusion protein..41
2.4.2 Affinity purification of polyclonal antibodies.....41
2.4.3 Western blotting...................................42
2.5 Immunostaining.......................................43
2.5.1 Dissection and fixation of the ovarioles...........43
2.5.2 Whole-mount immunostaining and microscopy in the pea
aphid..............................................43
2.6 RNA/protein pull down assay..........................45

Chapter 3: Results 47
3.1 Improving in situ hybridization method in the pea
aphid................................................48
3.1.1 Probe penetration capacity testing.................49
3.1.2 Probe length and concentration testing.............50
3.1.3 Combination of different substrates................51
3.2 Sequences characterization...........................52
3.2.1 Anteroposterior genes: Aphb, Apotd, and Apcad......52
3.2.2 Segmentation genes: Apen and Apwg..................55
3.3 Developmental expression of target mRNAs.............56
3.3.1 Developmental expression of Aphb and Apotd mRNA
during oogenesis and embryogenesis.................56
3.3.2 Developmental expression of Apcad mRNA during
oogenesis and embryogenesis........................59
3.3.3 Developmental expression of Apcad and Aphb during
oogenesis and embryogenesis........................60
3.3.4 Developmental expression of Apen-1,Apen-2, and Apwg
during oogenesis and embryogenesis.................61
3.4 Proteins expression of ApHb, ApEn-1, and ApEn-2......63
3.4.1 Overexpression of fusion proteins: ApHb, ApEn-1, and
ApEn-2.............................................63
3.4.2 Characterization of antibodies against ApHb, ApEn-1,
and ApEn-2.........................................64
3.4.3 Developmental expression of ApHb during oogenesis and
embryogenesis......................................65
3.4.4 Developmental expression of ApEn-1 and ApEn-2 during
oogenesis and embryogenesis........................65

Chapter 4: Discussion.....................................67
4.1 Whole-mount in situ hybridization....................68
4.1.1 The effect of probe length and concentration to
signal intensity...................................68
4.1.2 Different combination of substrates in double in situ
hybridization......................................69
4.1.3 Fluorescent in situ hybridization..................70
4.2 Roles of Aphb and Apotd in anteroposterior axis
formation in the pea aphid...........................71
4.2.1 Anterior localization of Aphb transcripts in early
oogenesis and embryogenesis........................71
4.2.2 Conserved developmental expression of Aphb
transcripts in mid- and late-embryogenesis.........73
4.2.3 Dynamic and conserved developmental expression of
Apotd transcripts in oogenesis and embryogenesis...74
4.2.4 ApHb is not related to anterior development in early
oogenesis and embryogenesis........................75
4.3 Roles of Apcad in posterior development in the pea
aphid................................................76
4.3.1 Dynamic and conserved developmental expression of
Apcad transcripts in oogenesis and embryogenesis...76
4.4 Roles of Apen and Apwg in segments formation in the
pea aphid............................................79
4.4.1 Developmental expression of Apen-1 and Apen-2
transcripts in oogenesis and embryogenesis.........79
4.4.2 Developmental expression of Apwg transcripts in
oogenesis and embryogenesis........................81
4.4.3 Dynamic and conserved developmental expression of
ApEn-1 and ApEn-2 in oogenesis and embryogenesis...83

Chapter 5: Summary........................................84
5.1 Summary..............................................85
5.1.1 In situ protocol in the pea aphid..................85
5.1.2 Anteroposterior axis formation in the pea aphid....85
5.1.3 Segments formation in the pea aphid................86
5.2 Sexual versus asexual embryogenesis..................87
5.2.1 Anteroposterior axis formation in sexual
individuals........................................88
5.3 Functional assays in the pea aphid...................89
5.3.1 RNA interferences..................................89
5.3.2 In vitro culture of ovarioles......................91

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