发育生物学——体轴与胚层【学习内容】

1、哺乳动物胚轴形成与胚层分化,Vertebrate embryos show differences in form before gastrulation After gastrulation, rather similar at Phylotypic stage (种系型期): head structure, neural tube, notochord, somites,Life cycle of the mouse,9 weeks fertilization in the oviduct, meiois II completed clevage morula,卵裂,蛙的早期卵裂。 A 第

2、一次卵裂， B 第二次卵裂， C 第四次卵裂，动物极和植物极细胞出现差异。,卵裂期是指受精卵开始有丝分裂并产生由较小的细胞构成的囊胚(blastula) 的过程。主要特点包括： 分裂周期短; 分裂球的体积下降：海胆胚胎的质/核比由550降至6; 早期卵裂中合子基因大多处于休眠状态； 卵裂常经历由均等裂向不均等裂变化。,3. hatching,1. compaction,2. blastocyst cavity formation,gene expression starts,128 cell,Day 7-human Day 5.5-mouse,哺乳动物的早期卵裂发生在输卵管中,Mouse pr

3、e-implantation stages: cleavage, compaction, blastocyst cavity formation,Compaction at the late 8-cell stage, With apical-basal polarization of cells,128-cell blastocyst,compaction,128-cell blastocyst,紧密化(Compaction): 8细胞分裂后不久，卵裂球突然挤在一起，卵裂球之间的接触面增大，形成一个紧密的细胞球体。,哺乳动物卵裂球的compaction: 发生第三次卵裂后不久,小鼠8细胞胚胎

4、compaction前后,小鼠桑椹胚的压缩现象 8细胞时期，小鼠细胞表面光滑，微绒毛均匀分布，压缩后微绒毛仅分布于细胞的外表面，细胞之间的联系加强了。,未压缩的和压缩的8细胞小鼠胚胎的比较,Compaction的机制: 8细胞胚胎的外层胞间形成致密连接 (tight junctions)，可将球内部的细胞与外环境隔绝，起稳定细胞球的作用。而内层胞间形成缝隙连接 (gap junction)，可以交换小分子和离子。,注射E-Cadherin抗体的胚胎不能够致密化。 在4细胞期激活蛋白激酶C导致compaction发生。因此，Compaction可能始于蛋白激酶C的活化，它引起细胞骨架的重排，在膜

5、上均匀分布的E-Cadherin重新定位在胞间相交处。,哺乳动物囊胚细胞命运的早期分化位置决定论 16细胞期桑椹胚(morula):位于内部的少数细胞产生的子细胞将组成内细胞团(inner cell mass)，而位于外部的细胞产生的子细胞大多构成滋养外胚层(trophoblast)。 32细胞期胚泡(blastocyst):位于囊胚腔一端的内细胞团(约10个细胞)将发育胚胎的本体及与其相连的卵黄囊、尿囊和羊膜；外层的滋胚层生成绒毛膜，为胎儿从母体摄取营养物质和氧，并产生激素以避免母体的排斥反应。,内细胞团的产生是哺乳动物早期发育的关键步骤之一。通过对活体胚胎的观察研究，发现这种重要决定作用仅

6、仅依赖于细胞于某一正确时间出现在某一正确的位置。压缩后位于外层的细胞将形成滋养层细胞，而内部的细胞将发育成胚胎。 一个细胞是否成为胚胎或滋养层细胞，完全取决于压缩作用后细胞所处的位置是位于外周还是内部。,哺乳动物胚泡的着床 胚泡在向子宫移动过程中体积增大，是由定位于滋胚层细胞膜囊胚腔一侧的NaK泵将外部Na+泵入腔中，最后通过渗透作用吸水使囊胚腔增大。,随着囊胚腔内离子浓度的增加，水分就通过渗透作用进入进入囊胚腔内。囊胚腔内液体的积累对囊胚腔壁造成一个向外的压力，这种静水压是参与形成和维持囊胚为球形的一个重要作用力。,哺乳动物胚泡的着床 胚胎外的透明带阻止了胚泡与输卵管壁的粘连-异位妊娠。 胚

7、泡到达子宫时，胚胎细胞分泌Strypsin（一种类胰蛋白酶)，它使透明带穿孔，胚泡从孔中“孵化” (hatch) 出与子宫壁接触，通过一系列反应而着床。,人类的同卵双生的发生(占出生总数的0.25%),分离发生在滋胚层形成前(约受精后5天前)的分割，有独立的绒毛膜和羊膜。占同卵双生的33%。,发生在滋胚层形成后但羊膜形成前(约受精后59天)的分割，共用绒毛膜，有独立的羊膜。占同卵双生的66%,发生在羊膜形成后(约受精9天后)的分割，共用绒毛膜和羊膜，易出现连体儿。,人类的连体婴儿,嵌合胚：早期卵裂球有同等的发育潜力。自然人群中也出现过同时有XX型和XY型细胞的人，异卵双生的两个胚胎融合形成的单

8、一混合个体。 胚胎干细胞：保持了分化为胚胎本体的潜能的、可在体外增殖的胚胎细胞。在基因功能研究和疾病治疗方面有重要的作用。,哺乳动物中的嵌合胚、胚胎干细胞,人工受精：如主要用于治疗不育症、保存 和运输优良个体。 胚胎切割：在24细胞期分割胚胎细胞，用于繁殖优良家畜个体。,哺乳动物中的人工受精和胚胎切割,原肠作用 Gastrulation,原肠作用是指囊胚细胞有规则的移动，使细胞重新排列，用来形成内胚层和中胚层器官的细胞迁入胚胎内部，而要形成外胚层的细胞铺展在胚胎表面。原肠作用期的胚胎叫原肠胚(gastrula)。,通过原肠作用，胚胎首先建立起三个胚层，即外胚层、中胚层和内胚层；其次为重新占有新

9、位置的胚胎细胞之间的相互作用奠定了基础。因此，原肠作用是从尚未分化到分化为三个胚层和器官原基决定的关键时期。 动物身体的主轴（包括前后轴、背腹轴和左右轴）也会在卵裂和原肠作用期间建立起来，胚胎各部分的细胞会获得各自的发育潜能。,人类的原肠作用,上胚层和下胚层的形成：在原肠作用开始时，内细胞团分裂为两层。与囊胚腔接触的一层为下胚层，将用于形成yolk sac；另一层为上胚层。上胚层细胞间的缝隙将合并、扩大成为羊膜腔，腔中的液体可防止胚胎脱水和保护胚胎受振荡。上胚层将发育为胚胎的本体。,原肠作用前的胚泡，示内细胞团和滋养层细胞。,下胚层细胞从内细胞团中分离出来形成卵黄囊，滋养层细胞分裂形成细胞滋养

10、层和合胞体滋养层；细胞滋养层形成绒毛，合胞体滋养层融入子宫组织。,上胚层分裂形成羊膜外胚层（包绕羊膜腔）和胚胎上胚层，哺乳类胚胎由胚胎上胚层形成。,胚外内胚层形成卵黄囊,人体胚胎三胚层的建立及其命运,人类原肠作用过程中，羊膜结构的形成和细胞的运动。,Note: the human epiblast is a flat disc. The mouse epiblast is cup-shaped-harder to visualize.,40天妊娠后人类的胚胎被羊膜包裹，外面再被绒毛膜包被。血管已深入到绒毛膜内，绒毛膜上的微绒毛最大限度地与母体血液接触。,人类胚胎与母体间的物质交流,人体胚胎细胞

11、的分化路线,Early post-implantation development of the mouse embryo,Inner cell mass: primitive endoderm (extra-embryonic membrane) and primitive ectoderm or epiblast (embryo proper and some extra-embryonic components) Polar trophectoderm: the ectoplacental cone and the placenta Mural trophectoderm: tropho

12、blast giant cells,Early post-implantation development of the mouse embryo,Egg cylinder: an internal cavity (proaminotic cavity) forms, U-shaped Onset of gastrulation: the appearance of primitive streak,小鼠胚胎上胚层细胞的程序性死亡导致囊胚腔的形成。,Gastrulation in the mouse embryo,6.5 d: beginning of gastrulation, the ap

13、pearance of primitive streak; antero-posterior axis 7.5 d: primitive streak elongates until the bottom of the cup, forming notochord (head process),Gastrulation and neurulation in the mouse embryo,7d: the node formation, anterior ectodermneurectoderm (brain and spinal cord) The anterior part of the

14、embryo grows in size and head fold appears, definitive endoderm replace visceral endoderm to form an outer layer on the ventral surface of the embryo. The notochord begins to form.,Gastrulation and neurulation in the mouse embryo,8d: head is distinct, neural folds have formed, gut have closed, somit

15、es begins to form on either side of the notochord. 10.5d: the complete of gastrulation and neurulation after turning.,Tuning in the mouse embryo,Turning: a developmental quirk peculiar to rodents: human embryos are surrounded by their extra-embryonic membranes from the beginning. 8.5-9.5d: the mouse

16、 embryo becomes entirely enclosed in the protective amnion and amniotic fluid. The visceral yolk sac, a major source of nutrition, surrounds the aminon, the umbilical cord connects to the placenta.,胚轴形成,胚胎细胞形成不同组织、器官，构成有序空间结构的过程称为图式形成（pattern formation）。 在动物胚胎发育中，最初的图式形成主要涉及胚轴（embryonic axes）形成及其一系列

17、相关的细胞分化过程。胚轴指胚胎的前后轴（anterior -posterior axis）和背 腹轴（dorsal -ventral axis）。胚轴的形成是在一系列基因的多层次、网络性调控下完成的。,Body axes formation：小鼠胚胎发育的早期无法辨认体轴,The specification of the inner cell mass of a mouse embryo depends on the relative position of the cells with respect to the inside and outside of the embryos. Tra

18、nscription factors: Cdx2 (滋养层细胞的分化) and Oct/4 (内细胞团的分化),The axes of the early mouse embryo,Blastocyst stage: 4d: inner cell mass is confined to embryonic region and this defines an embryonic-abembryonic axis (geometrically, not cell fate) 5.5d: the antero-posterior axis visible, with the formation o

19、f primitive streak at the posterior end.,The symmetry-breaking event is the specification of the anterior visceral endoderm (前端内脏内胚层, AVE),5.5d: A small region of visceral endoderm at the distal-most end of the cup is specifies as AVE induced by Nodal signaling from the epiblast and begins to prolif

20、erate and extend to one side of epiblast. The anterior fate is restricted to the distal region by inhibitory signals from the extra-embryonic ectoderm to the proximal visceral endoderm; induces anterior ectoderm,The symmetry-breaking event is the specification of the anterior visceral endoderm (前端内脏

21、内胚层, AVE),6.5d: primitive streak begins to form on the opposite side, marking the posterior end of the axis,Role of the AVE in establishing the anterior-posterior axis of the mouse embryo,Cylindrical Symmetry,Bilateral Symmetry,A,P,Cerberus (anti-Nodal) Dkk (anti-Wnt) Lefty (anti-Nodal),Otx2 Anterio

22、r neural ectoderm,Nodal, Cripto (co-receptor For Nodal) Endo-mesoderm induction,Day 5.25 Day 5.5 Day 6.25,or hypoblast,Endo-mesoderm induction, Primitive streak wnt, brachyury, fgf expressed,Epiblast ectoderm,The left-right asymmetry,While the vertebrate body is outwardly symmetric, most internal or

23、gans are in fact asymmetric with respect to the left and right sides, called left-right asymmetry, e.g., the heart, the right lung has more lobes than the left. 1/10,000 in humans, situs inversus mutation in the iv gene causes 50% of mice have hearts that loop to the right. Key proteins in establish

24、ing leftness: extracellelar signal protein Nodal and transcription factor Pitx2,Determination of left-right asymmetry in the chick,left-right symmetry is broken at an early stage by the asymmetric activity of a proton-potassium pump (H+/K+-ATPase) The release of Ca2+ from cells in the left side as p

25、art of the symmetry-breaking process. Pitx2: leftness; Lefty (Nodal antagonist) provide midline barrier to prevent Nodal crossing to the right-hand side; Activin inhibit Shh activity,Left-right asymmetry in the mouse embryo,Cilia(纤毛)-directed flow generates left-right asymmetry Directed leftward flo

26、w of extracellular fluid at the node at around the late head-fold stage Leftward flow of fluid propagates the release of intracellular Ca2+ to upregulate Nodal expression on the left side,胚层的来源和特化 (specification),Fate map: tell us which tissues the different regions of the embryo give rise to The fa

27、te maps and the genes involved in germ-layer specification are more similar in all vertebrate models than are in early axis specification 根据被标记细胞的命运，构建两栖类早期囊胚的命运图：labeled by injection of high-molecular-weight molecules such as rhodamine (cannot pass through cell membranes), as rhodamine fluoresces r

28、ed in UV light, so detected under a UV microscope. The fluorescent protein green fluorescent protein (GFP) is now widely used.,胚层的来源和特化 (specification),Fate mapping of the early Xenopus embryo: C3 is labeled with fluorescein (green)- tailbud stage, the cells give rise to mesoderm cells,胚层的来源和特化 (spe

29、cification),Fate map of a late Xenopus blastula Ectoderm: epidermis, nervous system Along the dorso-ventral axis the mesoderm: notochord, somites, heart, kidney and blood Endoderm overlying the mesoderm in the marginal zone,Fate map of a mouse at the late gastrula stage,Node: exclusively notochord T

30、he middle part of the streak: mainly lateral-plate mesoderm The posterior part of the streak: tailbud, extra-embryonic mesoderm that forms the extra-embryonic membranes-the amnion, visceral yolk sac, allantois.,不同脊椎动物的 fate map 基于一个图式的改变,Similarities: the future notochord mesoderm occupies a central

31、 dorsal position; the neural ectoderm lies adjacent to the notochord, with the rest of the ectoderm anterior to it. Differences: mainly to the yolkiness of the different eggs, which determines the patterns of cleavage and influences the shape of the early embryo. Endoderm overlying the mesoderm in t

32、he marginal zone,Fate maps of vertebrate embryos at a late blastula or early gastrula,早期脊椎动物的胚胎细胞的命运并未决定，是可以调整的,Fusion of mouse embryos give rise to a chimera (mice that are mosaics of cells with 2 different genetic constitutions-by fusing 2 embryos): 8-cell stage embryo of an unpigmented strain of

33、mouse is fused with a pigmented strain will give rise to a chimeric animal,Grafting experiments: transplanting the cells or regions to a host embryo, if determined, they will develop according to their original position; if not yet determined, develop in line with the new position Early mouse embryo

34、s can regulate to achieve the correct size: retains considerable capacity to regulate until late in gastrulation. Inner cell mass remain pluripotent up to 4.5 days after fertilization-they can give rise to many cell types. Cells in ICM can be cultured to produce embryonic stem cells (ES cells),小鼠发育相

35、关突变的产生,Studying the role of a particular gene in development, to study the effects of a mutation in that gene Mice: transgenic techniques, overexpress or knock-out a given gene Introducing embryonic stem cells (ES cells) carrying the mutation into the blastocyst,小鼠的中胚层诱导和模式建成发生在原条形成时期,6 days: the AV

36、E induce anterior character in the underlying epiblast and early markers of the future primitive streak (PS) are restricted to the proximal epiblast 6.5 days: BMP activates the expression of mesoderm markers and the posterior movement of cells expressing PS markers results in PS formation,小鼠的中胚层诱导和模式建成发生在原条形成时期,7 days: extra-embryonic mesoderm is produced from the posterior end of PS while the node forms at the anterior end, which will give rise to axial mesendoderm (form doral mesoderm and gut endoderm),小鼠的中胚层诱导和模式建成发生在原条形成时期,The molecular interactio