The Roles of Hedgehog and Wnt Signaling Pathways in Vertebrate Sex Determination

脊椎動物的性別決定大致可分為：(1)基因、(2)性賀爾蒙、及(3)器官型態三個層次。在基因層面，性別決定基因控制了性腺發育成睪丸或卵巢的方向。整體來說，在不同物種的脊椎動物中，性腺的發育及分化過程極為相似；然而，目前有證據顯示，雖然老鼠和雞其性腺的發育及分化過程雖然相似；但在性別決定上卻可能有截然不同的機制。以實驗用小鼠為例，在胚胎發育過程中，Dhh 僅表現在雄性線的「Sertoli 細胞」中；而且若是移除Dhh 的功能，會造成雄性的「雌性化」與不孕。在研究其分子致病機制時發現：Dhh 突變小鼠的睪丸中缺少負責製造雄性賀爾蒙的「Leydig 細胞」。因此，Dhh 在雄性性別決定及「Leydig 細胞」的分化過程中應該扮演了一個很重要的角色。雞的基因體計畫指出雞的基因中並不包含Dhh 基因；但是雞的雄性腺仍可以正常發育。因此，這個計畫想要探討Hh 訊息傳導途徑在雞的雄性生殖腺中所扮演的角色。相對於Dhh 在雄性腺中的功用，Wnt-4 控制了雌性胚胎中卵巢的分化與發育。在Wnt-4缺陷的雌性小鼠中，卵巢及雌性生殖管的發育都不完全，證明Wnt-4 對卵巢的發育非常重要。而且Wnt-4 在雞生殖腺的功能並沒有被研究過。因此，這個計畫將探討Wnt-4 訊息傳導途徑在雞的卵巢的分化與發育中所扮演的角色。此外，Dax1 也在小鼠胚胎的性別決定中扮演了一個重要的角色；過量或缺少Dax1 皆會造成雌、雄性別間的性別決定錯亂。在小鼠中，Dax1 擷抗雄性性別決定基因Sry 的作用。然而，雞的性染色體為ZW，並不包含Sry 的序列。因此，在這個計畫也將探討Dax1基因在雞的性別決定中所扮演的角色。這個計畫嘗試分別由一個「雄性性別決定基因」、一個「雌性性別決定基因」、和一個「性別決定雙重功能基因」著手，結合生物資訊學和分子生物學的方法，藉由比較小鼠和雞兩種動物雌、雄性腺發育的訊息傳導調控，進一步探討生物的「性別決定機制」。In vertebrates with sex chromosomes, sex determination occurs at three levels: (1) genetic sexdetermination, (2) hormonal sex determination, and (3) phenotypical/morphological sexdetermination. During genetic sex determination, some sex determination genes are expressedexclusively in male or female embryos to direct the two gonad differentiation fates[1]. Generallyspeaking, the processes of gonad development are conserved among all vertebrates; however,current data suggests that the sex determination mechanisms may be very different betweenmammals and avian.In mice, Desert Hedgehog (Dhh) is expressed only in the Sertoli cells of testis. Targeted inactivationof Dhh led to male specific sterility[2]. The testes of Dhh-null mice have abnormal seminiferoustubules, and lack testosterone producing Leydig cells[3-5]. These results implicated the Dhh playsimportant roles in male sex determination. However, there is no evidence that Dhh exist in chickengenome. We hypothesize that either the other members of the Hh family substitute the function ofDhh in chicken testis or the chicken embryos evolved a Dhh-independent mechanism to sustainLeydig cell differentiation. Therefore, I propose to clarify how chicken Leydig cells differentiate inthe absence of Dhh in chicken testis development.On the other hand, Wnt-4 is required for mammalian ovary development, as conditional inactivationof Wnt-4 results in the absence of ovaries, and female reproductive ducts and the ectopic synthesisof testosterone[6]. However, the role of Wnt-4 in avian ovary development has not beencharacterized. Since the conservation of ovarian developmental processes between mouse andchicken, Wnt-4 may also plays important roles in chicken ovary development. Therefore, I intend totest the function of Wnt-4 genes during chicken embryonic gonad development.Dax 1 is another gene involved in sex determination. Transgenic expression of Dax 1 results in maleto female sex reversal[7]. In mice, it was suggested that the expression of Dax 1 is under the controlof Wnt-4 and Dax 1 functions through inhibiting the activity of Sry[7]. Since avian uses ZW sexchromosome and does not contain Sry ortholog in their genomes, I want to understand themechanism of Sry-independent sex determination by Dax 1 in chicken embryo.In this proposal, I plan to characterize the relationship of the sex determination genes, with focus onthe Hh pathway and Wnt pathway to help construct a regulatory hierarchy of sex determinationgenes that will advance our understanding in the evolutionary variations among different species invertebrates

The Roles of Hedgehog and Wnt Signaling Pathways in Vertebrate Sex Determination

脊椎動物的性別決定大致可分為：(1)基因、(2)性賀爾蒙、及(3)器官型態三個層次。在基因層面，性別決定基因控制了性腺發育成睪丸或卵巢的方向。整體來說，在不同物種的脊椎動物中，性腺的發育及分化過程極為相似；然而，目前有證據顯示，雖然老鼠和雞其性腺的發育及分化過程雖然相似；但在性別決定上卻可能有截然不同的機制。以實驗用小鼠為例，在胚胎發育過程中，Dhh 僅表現在雄性線的「Sertoli 細胞」中；而且若是移除Dhh 的功能，會造成雄性的「雌性化」與不孕。在研究其分子致病機制時發現：Dhh 突變小鼠的睪丸中缺少負責製造雄性賀爾蒙的「Leydig 細胞」。因此，Dhh 在雄性性別決定及「Leydig 細胞」的分化過程中應該扮演了一個很重要的角色。雞的基因體計畫指出雞的基因中並不包含Dhh 基因；但是雞的雄性腺仍可以正常發育。因此，這個計畫想要探討Hh 訊息傳導途徑在雞的雄性生殖腺中所扮演的角色。相對於Dhh 在雄性腺中的功用，Wnt-4 控制了雌性胚胎中卵巢的分化與發育。在Wnt-4缺陷的雌性小鼠中，卵巢及雌性生殖管的發育都不完全，證明Wnt-4 對卵巢的發育非常重要。而且Wnt-4 在雞生殖腺的功能並沒有被研究過。因此，這個計畫將探討Wnt-4 訊息傳導途徑在雞的卵巢的分化與發育中所扮演的角色。此外，Dax1 也在小鼠胚胎的性別決定中扮演了一個重要的角色；過量或缺少Dax1 皆會造成雌、雄性別間的性別決定錯亂。在小鼠中，Dax1 擷抗雄性性別決定基因Sry 的作用。然而，雞的性染色體為ZW，並不包含Sry 的序列。因此，在這個計畫也將探討Dax1基因在雞的性別決定中所扮演的角色。這個計畫嘗試分別由一個「雄性性別決定基因」、一個「雌性性別決定基因」、和一個「性別決定雙重功能基因」著手，結合生物資訊學和分子生物學的方法，藉由比較小鼠和雞兩種動物雌、雄性腺發育的訊息傳導調控，進一步探討生物的「性別決定機制」

Sox9與Wnt訊息傳導途徑對鳥類性別決定的影響

Sox9 與Wnt 訊息傳導途徑對鳥類性別決定的影響哺乳動物中除鴨嘴獸外，都是由Y 染色體來決定雄性的發育。自人類XY 女性與XX 男性的染色體變異研究中發現，雄性的發育的分子機制在於Y 染色體上一個約35,000 鹼基小片段，其中包含了一個基因稱之為Sry (Sex-determination Region on Ychromosome)。Sry 透過一系列的機制控制了生殖腺中Leydig cell、Sertoli cell 與細精管等體細胞的形成。然而，其他的脊椎動物並不具有XY 性染色體，也沒有Sry 基因，而是演化出了其他的性別決定機制。在這些非XY 的動物胚胎發育過程中，他們的性別決定時期，雄性生殖腺都有Sox9 的表現，雌性生殖腺都有Wnt4 的表現；因此Sox9 與Wnt4被認為是所有脊椎動物性別決定中最重要的因子。然而，在正常雄性與雌性中，生殖腺的發育是互不相容的；所以我們推論Sox9 與Wnt4 之間很可能有相互拮抗的作用。我們將以雞胚胎及青春期發育前期的小公雞為材料，在其生殖腺中分別加強或去除Sox9 及Wnt 訊息傳導途徑的活性，改變雄性與雌性生殖腺體細胞性別決定的分化，藉此探討Sox9 與Wnt4 之間交互作用的分子機制。The interaction between Sox9 andWnt signaling pathway in avian sex determinationSex determination in mammals is regulated by the presence of Y chromosome in male, exceptPlatypus. Molecular analysis has revealed that a small region on the Y chromosome, Sry,controls the male determining cascade of differentiation, including the formation of Leydigand Sertoli cells in testis, testis cord formation, and synthesis of testosterone. However, manynon-mammal vertebrates do not have XY sex chromosome and have evolved a different sexdetermining mechanism. Here we propose to study two genes, Sox9 and Wnt4 that have beingshown to promote male and female gonad formation in mouse and human. As Sox9 andWnt4antagonize each other's function during chondrogenesis, we hypothesize that the mutualexclusive differentiation of testis and ovary may share a similar regulatory mechanism.Wewill use chicken embryos and pre-pubertal chicken to characterize the interactions betweenSox9 andWnt signaling pathway in somatic gonads. By manipulating the activity of Sox9 andWnt pathway, we expect to alter the normal gonad development

Functional Analysis of Hedgehog Signaling Pathways

在無脊椎動物和脊椎動物的胚胎發育的過程中，Hedgehog 訊息傳導途徑控制了身體中軸前後方向的決定，並參與多種器官的形成[1-3]。近期以哺乳動物為材料的研究發現，Hedgehog 訊息傳導途徑在幹細胞更新、組織修復、及癌症的發生均扮演重要的角色[4-10]。目前利用遺傳學的方法，全世界果蠅的實驗室共同建立了Hedgehog 訊息傳導途徑的基本架構。簡略來說，細胞外的Hedgehog 蛋白質會與受體細胞膜上的受體蛋白質結合，並激活受體細胞內一系列的活化作用，最終傳導至轉錄因子cubitus interuptus[11]。脊椎動物的Hedgehog 訊息傳導途徑，其基本架構雖然和果蠅的系統大同小異，但在活化機制的調控上，則更為複雜。以上述的轉錄因子為例，在果蠅中只有一個基因，但在老鼠的基因內卻有三個同源基因存在，而且它們的功用會互相影嚮；因此脊椎動物的Hedgehog 訊息傳導途徑仍有待大家更深入的研究、探討。因為個人目前的幹細胞研究領域和Hedgehog 訊息傳導途徑有緊密的關聯性；因此，在這個計畫中，我將以雞胚胎為材料，研究脊椎動物的Hedgehog 訊息傳導途徑。其中將專注於cubitus interuptus的三個同源轉錄因子基因Gli1、Gli2 與Gli3 其蛋白質產物的活性調控機制： (1) Gli1受到磷酸脢PKA 磷酸化的活性調控，(2) Gli3 受到蛋白質分解脢(slimb)控制的活性轉化(3) Gli 蛋白質在經過SUMO 修飾後的活性轉化(4) 尋找Hedgehog 訊息傳導途徑中新的因子，並研究其在胚胎發育中的功能

Characterization of the testicular regeneration potential in premature cockerels

Previous studies have shown that grafted neonatal chicken testicular tissue can develop and produce functional sperm; however, it was unclear whether regenerative processes or proportional growth caused the re-appearance of spermatogenic tissue. We dissociated testicular tissues, performed subcutaneous auto-transplantation of the re-aggregated cells to castrated cockerels, and monitored the post-surgery development of these transplanted aggregates. We found that these transplanted cell aggregates experienced compensatory growth in the form of a 300-fold increase in size, rather than the 30-fold increase observed in normal testis development. Further, these dissociated testicular cell aggregates restored seminiferous tubule structure and were able to produce testosterone and motile sperm. Therefore, we concluded that the dissociated testicular cells from 11-week-old cockerels retained a strong regenerative potential, as they exhibited compensatory growth, restored destroyed structure, and sustained spermatogenesis

TRANSGENIC POULTRY PRODUCTION AND USES THEREOF

本發明所揭一種基因轉殖禽類產製系統，係選取適當週齡之雄性禽類，對其進行不完全閹割，即將該雄性禽類之睪丸組織切除至少50%，再將構築好之病毒載體以注射之方式打入該雄性禽類剩餘之睪丸組織中，而後再以凝膠封住經注射病毒所產生之傷口，促進組織再生而將病毒載體帶入睪丸生殖細胞。藉由本發明所提供之基因轉殖禽類產製系統，可利用經不完全閹割之雄性禽類，直接將外源蛋白基因注入其剩餘睪丸細胞內，使病毒感染睪丸細胞，將外源蛋白基因帶入細胞中，成功地產製基因轉殖禽類，用以表現外源蛋白

Deferasirox has strong anti-leukemia activity but may antagonize theanti-leukemia effect of doxorubicin

Deferasirox (DFX), in addition to its iron-chelation property, has marked anti-proliferative effects on cancer cells. However, the activity and mechanism by which DFX inhibits acute myeloid leukemia (AML) cells remain to be elucidated. Furthermore, the anti-leukemia effect of combining DFX with currently recommended agents doxorubicin (DOX) and cytosine arabinoside (Ara-C) has not been studied. In this study, we show that DFX significantly reduces the viability of three AML cell lines, HL60, THP1, and WEHI3 and two primary leukemic cells harvested from AML patients. DFX induces cell cycle arrest at G1 phase and apoptosis and inhibits phosphorylation of ERK. We also showed that DFX antagonizes the anti-leukemic effect of DOX. On the contrary, combining DFX with Ara-C created a synergistic effect. Our study confirms the anti-leukemia activity of DFX and provides important information on how to select a partner drug for DFX for the treatment of AML in future clinical trials

Activation of liver X receptor suppresses angiogenesis via induction of ApoD

Liver X receptors (LXRs) are important sensors and regulators for cholesterol, fatty acid, and glucose. LXRs play essential roles in the development and progression of cardiovascular diseases. We examined the effects of T0901317, a potent LXR agonist, on angiogenesis of human umbilical vein endothelial cells (HUVECs). Treatment with T0901317 inhibited the tube formation and migration of HUVECs and reduced the in vivo angiogenesis, as determined by chorioallantoic membrane assay. T0901317 stimulated gene and protein expression of LXR target gene apolipoprotein D (ApoD). Overexpression of ApoD suppressed the tube formation of HUVECs. ApoD interacted with scavenger receptor class B member 1 (SR-B1), while knockdown of SR-B1 blocked suppressive effects of T0901317 on HUVEC migration. T0901317 treatment or overexpression of ApoD lessened expression of proteins regulating angiogenesis, including phospho-eNOS S1177, phospho-Akt T308, phospho-Akt S473, eNOS, mammalian target of rapamycin, VEGF-A, VEGF-C, IL-8, RhoB, matrix metalloproteinase (MMP)-8, -9, and monocyte chemoattractant protein 1. Our study suggested that activation of LXR interferes with angiogenesis through induction of LXR target gene ApoD, which in turn suppresses PI3K-Akt-eNOS signaling, an essential pathway regulating angiogenesis. ApoD may be a potential therapeutic target for tumor angiogenesis.-Lai, C.-J., Cheng, H.-C., Lin, C.-Y., Huang, S.-H., Chen, T.-H., Chung, C.-J., Chang, C.-H., Wang, H.-D., Chuu, C.-P. Activation of liver X receptor suppresses angiogenesis via induction of ApoD