一．内胚层形成
I: Approaches to study organogenesis
Biochemical and molecular approach: purify organ-enriched proteins or transcripts
Explant tissue culture and transplantation approach: suitable for morphogen and drug assay (chick, quail)
Cell lineage tracing: 用荧光染料或是生物素标记细胞 Over-expression approach:
Gene knock-down approach: dominant negative construct, antisense construct; siRNA, morpholino etc
Genetic approach:
1) target gene knock-out: a) homologous recombination; b) TILLING (Targeting Induced Local Lesions IN Genomes)
2) Screening for morphological mutants: most unbiased to identify factors important for development
II: Importance of using model system
II-a: Mouse
Excellent model system for the study of vertebrate development via reverse genetics: homologus recombination。But not an ideal model for forward genetics:1)Intrauterine embryonic development 2) Few progenies, big space and high cost for maintenance.
II-b: Zebrafish
small size;high fecundity;short generation time;external embryogenesis;translucent embryos; Information obtained generally applicable to other vertebrates: similarity in many developmental processes and similar mechanism in disease occurrence.
Ⅲ 研究内胚层发生相关基因的方法
1． Cell lineage tracing：用荧光染料或是生物素标记细胞，看细
胞的迁移运动。
2． 寻找突变体：用物理、化学、生物等方法来制造突变体，通过
突变表型来研究基因功能。
3． 研究基因：克隆基因方法1) Candidate gene strategy 2)
Map-based cloning。在斑马鱼中与内胚层发生相关的几个基因Oep (Nodal co-receptor)；Cas (Sox-related transcription factor)；Bon (Mix-type HD transcription factor)；Fau (Gata5: Zinc-finger transcription factor)
4． 研究基因表达谱：基因表达的位置，时序
5． 基因间上下游关系：通过rescue 实验会判断基因的上下游关系
6． 比较不同物种间内胚层发生
二．肝脏发生
Ⅰ小鼠肝脏发生的五个步骤
precardiac mesoderm
transversum
mesenchyme
Competency - Definitive endoderm is rendered competent to follow a hepatic fate (Hnf3 and Gatas)
Specification - Commitment of the endoderm to a hepatic fate (TFs unknown)
Liver Bud - ventral endoderm generates liver bud in a process that requires both endothelial cells (Hex )
Growth - Pre-hepatic cells delaminate from foregut and migrate into septum transversum (Prox1, Hlx , c-Jun , Xbp1, and NF-B Differentiation - Concurrent with growth phase (Hnf4)
A：腹部的前肠内胚层获得了发育成多种器官的能力，由相邻的原始横隔间质细胞分泌的Foxa 信号可以诱导器官发生。
B：在器官发生过程中，心脏生成的中胚层分泌的FGF 信号，可能同原始横隔间质细胞分泌的BMP 信号起始了肝脏发生，同时阻滞了胰脏生成。距离较远的前肠内胚层细胞不受BMP 信号抑制，从而起始了胰脏基因的重排。
A：当肝发生内胚层确定以后，肝细胞开始向中肠延伸，同时肝脏内皮
细胞也变成了柱状。这些转变是由原始横隔间质细胞、原始内皮细胞
分泌的BMP,Hgf，Vegfr2蛋白及Hex，Prox1，Hlx，c-Met 等转录因子调控的。
B:肠盲囊小窝形成，肝细胞周围细胞基质重排，E-cadherin 降低等现象标志着肝脏芽体的形成。
Ⅱ 斑马鱼的肝脏发生
斑马鱼肝脏发生与小鼠稍有差别，研究的方法已经在第一部分简述。至于具体的哪个基因发挥什么作用，表达谱是怎么样的，个人觉得不重要也不需要知道。
一．内胚层形成
I: Approaches to study organogenesis
Biochemical and molecular approach: purify organ-enriched proteins or transcripts
Explant tissue culture and transplantation approach: suitable for morphogen and drug assay (chick, quail)
Cell lineage tracing: 用荧光染料或是生物素标记细胞 Over-expression approach:
Gene knock-down approach: dominant negative construct, antisense construct; siRNA, morpholino etc
Genetic approach:
1) target gene knock-out: a) homologous recombination; b) TILLING (Targeting Induced Local Lesions IN Genomes)
2) Screening for morphological mutants: most unbiased to identify factors important for development
II: Importance of using model system
II-a: Mouse
Excellent model system for the study of vertebrate development via reverse genetics: homologus recombination。But not an ideal model for forward genetics:1)Intrauterine embryonic development 2) Few progenies, big space and high cost for maintenance.
II-b: Zebrafish
small size;high fecundity;short generation time;external embryogenesis;translucent embryos; Information obtained generally applicable to other vertebrates: similarity in many developmental processes and similar mechanism in disease occurrence.
Ⅲ 研究内胚层发生相关基因的方法
1． Cell lineage tracing：用荧光染料或是生物素标记细胞，看细
胞的迁移运动。
2． 寻找突变体：用物理、化学、生物等方法来制造突变体，通过
突变表型来研究基因功能。
3． 研究基因：克隆基因方法1) Candidate gene strategy 2)
Map-based cloning。在斑马鱼中与内胚层发生相关的几个基因Oep (Nodal co-receptor)；Cas (Sox-related transcription factor)；Bon (Mix-type HD transcription factor)；Fau (Gata5: Zinc-finger transcription factor)
4． 研究基因表达谱：基因表达的位置，时序
5． 基因间上下游关系：通过rescue 实验会判断基因的上下游关系
6． 比较不同物种间内胚层发生
二．肝脏发生
Ⅰ小鼠肝脏发生的五个步骤
precardiac mesoderm
transversum
mesenchyme
Competency - Definitive endoderm is rendered competent to follow a hepatic fate (Hnf3 and Gatas)
Specification - Commitment of the endoderm to a hepatic fate (TFs unknown)
Liver Bud - ventral endoderm generates liver bud in a process that requires both endothelial cells (Hex )
Growth - Pre-hepatic cells delaminate from foregut and migrate into septum transversum (Prox1, Hlx , c-Jun , Xbp1, and NF-B Differentiation - Concurrent with growth phase (Hnf4)
A：腹部的前肠内胚层获得了发育成多种器官的能力，由相邻的原始横隔间质细胞分泌的Foxa 信号可以诱导器官发生。
B：在器官发生过程中，心脏生成的中胚层分泌的FGF 信号，可能同原始横隔间质细胞分泌的BMP 信号起始了肝脏发生，同时阻滞了胰脏生成。距离较远的前肠内胚层细胞不受BMP 信号抑制，从而起始了胰脏基因的重排。
A：当肝发生内胚层确定以后，肝细胞开始向中肠延伸，同时肝脏内皮
细胞也变成了柱状。这些转变是由原始横隔间质细胞、原始内皮细胞
分泌的BMP,Hgf，Vegfr2蛋白及Hex，Prox1，Hlx，c-Met 等转录因子调控的。
B:肠盲囊小窝形成，肝细胞周围细胞基质重排，E-cadherin 降低等现象标志着肝脏芽体的形成。
Ⅱ 斑马鱼的肝脏发生
斑马鱼肝脏发生与小鼠稍有差别，研究的方法已经在第一部分简述。至于具体的哪个基因发挥什么作用，表达谱是怎么样的，个人觉得不重要也不需要知道。