(发育生物学）IV 早期胚胎发育

1、( (发育生物学）发育生物学）IV IV 早期胚胎发育早期胚胎发育Cleavage and formation of the blastula (I)n In animal egg, the first step in embryonic development is the division of the fertilized egg by cleavage into a number of smaller cells (blastomeres), leading in many animals to the formation of a hollow sphere of cellsthe

2、blastula (a polarized epithelium surrounding a fluid-filled blastocoel). n Cleavage involves short cell cycles, in which cell division and mitosis succeed each other repeatedly without any interventing periods of cell growth (that is, the G1 and G2 phases of the cell cycle). During cleavage therefor

3、e, the mass of the zygote does not increase. Thus, the enormous volume of zygote is divided into increasingly smaller cells.n The cleavage of nuclei oocurs at a rapid rate never seen again (not even in tumor cells). Mitosis in cleavage-stage Drosophila embryos occurs every 10 minutes for over 2 hour

4、s and in just 12 hours forms some 50,000 cells.Cleavage and formation of the blastula (II)n The pattern of embryonic cleavage is determined by 2 major parameters: The amount and distributions of yolk protein within the cytoplasm Factors in the egg cytoplasm that influence the angle of the mitotic sp

5、indle and the timing of its formation n The pattern of cleavage can be divided into 2 types, based on the amount and distribution of the yolk protein: Complete cleavage in which the cleavage furrow extends through the entire egg Incomplete cleavage wherein only a portion of the cytoplasm is cleaved

6、The blastocoel and its function (I)n An amphibian embryo containing 16-64 cells is commonly called a morula (桑椹胚桑椹胚). At the 128-cell stage of an amphibian embryo, the blastocoel （囊胚腔囊胚腔）becomes apparent, and the embryo is considered a blastula.The blastocoel and its function (II)n An amphibian embr

7、yo containing 16-64 cells is commonly called a morula (桑椹胚桑椹胚). At the 128-cell stage of an amphibian embryo, the blastocoel （囊胚腔囊胚腔）becomes apparent, and the embryo is considered a blastulan The blastocoel serves 2 major functions in frog embryos:It permits cell migration during gastrulation It pre

8、vents the cells beneath it from interacting prematurely with the cells above it, thus ensuring the proper cell fate determination .n EP-cadherin functions in the formation of the blastocoel Depletion of EP-cadherin mRNA in the Xenopus oocyte results in the loss of adhesion between blastomeres and th

9、e obliteration of the blastocoelWild type Xenopus embryoEP-cadherin-depleted embryo(by injecting antisense mRNA of EP-cadherin gene into the oocyte)Early embryonic development1 Cleavage and formation of the blastula 1.1 The main patterns of cleavage1.2 The blastocoel and its function2 Gastrulation2.

10、1 The concept and morphogenetic movements2.2 The cell/tissue movements involved in gastrulation3 The cell fate specification and determination3.1 The fate map in sea urchin3.2 The Xenopus fate mapGastrulation (I)n At the end of cleavage, the animal embryo is essentially a closed sheet of cells, whic

11、h is often in the form of a sphere enclosing a fluid-filled interior. Gastrulation converts this sheet of cells into a solid three-dimensional embryonic structure. n Gastrulation is the process of highly coordinated cell and tissue movements whereby the cells of the blastula are dramatically rearran

12、ged. Gastrulation (II) During gastrulation, the cells that will form the endodermal and mesodermal organs are brought to the inside of the embryo, while the cells that will form the skin and nervous system are spread over its outside surface. Thus, the 3 germ layers- outer ectoderm, inner endoderm,

13、and interstitial mesoderm- are first produced during gastrulation. Gastrulation (III) During gastrulation, the cells that will form the endodermal and mesodermal organs are brought to the inside of the embryo, while the cells that will form the skin and nervous system are spread over its outside sur

14、face. Thus, the 3 germ layers- outer ectoderm, inner endoderm, and interstitial mesoderm- are first produced during gastrulation. The outcome of gastrulation for almost all animals is the same: the transformation of a two-dimensional sheet of cells into a three-dimensional embryo, with ectoderm, mes

15、oderm, and endoderm in the correct positions for further development of body structure. Although patterns of gastrulation vary enormously throughout the animal kingdom, there are only a few basic types of cell/tissue movements. Gastrulation (IV)Gastrulation usually involves some combination of the f

16、ollowing types of movements: Invagination（内陷）: The infolding of a region of cells, much like the indenting of a soft rubber ball when it is poked Involution（内卷）: The inturning inward movement of an expanding outer layer so that it spread over the internal surface of the remaining external cells. Inv

17、agination:Involution:Infolding of cell sheet into embryoInturning of cell sheet over the basal surface of an outer layerExample:Sea urchin endodermExample:Amphibian mesodermTypes of cell movements during gastrulation (I) Gastrulation (IV) Delamination（分层或层裂）: The splitting of one cellular sheet into

18、 two more or less parallel sheets. The result is the formation of a new sheet cells. Epiboly（外包）：The expanding of epithelial sheets so that one cell sheet encloses the inner layers of the embryo. （Epiboly can occur by the cells dividing, by the cells changing their shapes, or by several layers inter

19、calating into fewer layers. Often, all 3 mechanisms are used）. Types of cell movements during gastrulation (II) Delamination:Splitting or migration of one sheet into two sheetsEpiboly:The expansion of one cell sheet over other cells Example:Mammalian and bird hypoblast （下胚层）formationExample:Ectoderm

20、 formation in amphibian, sea urchin, and tunicates Convergent extension （汇聚伸展）：Cells intercalate to narrow the tissue and at the same time move it forward along the body axis. Types of cell movements during gastrulation (III) Convergent extension of the mesoderm during Xenopus gastrulationInitially

21、the mesoderm is in an equatorial ring in the blastula, but during gastrulation it converges and extends along the anterior-posterior axisThe mechanisms underlying gastrulationWe will discuss gastrulation mechanisms in sea urchins and insects, in which it is relatively simple process. The mechanisms

22、of gastrulation in chick and mouse are not well understood, and so Xenopus serves as our model for the more complex gastrulation process in vertebrates.Early embryonic development1 Cleavage and formation of the blastula 1.1 The main patterns of cleavage1.2 The blastocoel and its function1.3 Formatio

23、n of the fluid-filled cavity in the blastula results from the programmed cell death 2 Gastrulation2.1 The concept and morphogenetic movements2.2 The cell/tissue movements involved in gastrulation3 The cell fate specification and determination3.1 The fate map in Sea urchin3.2 The Xenopus fate mapThe

24、cell fate specification and determinationCell fates can be specified by cell induction: cell-cell interactions through paracrine or juxtacrine signaling asymmetric distribution of cell fate determinants: these unevenly distributed molecules into particular cells are usually transcription factors tha

25、t regulate the expression of specific genes in those cells that acquire them. Such asymmetric distributions of the cell fate determinants can happen during cleavageFate maps and the determination of sea urchin blastomeres (I)n The fates of blastula cells can be visualized by tracing their movements

26、and development during gastrulation by the technique of vital dye staining such as fluorescent dyes (injecting individual cells with fluorescent dyes that will glow in the injected cells progeny for many cell divisions).n The studies by the vital dye staining have shown that by the 60 cell-stage, mo

27、st of the embryonic cell fates are specified. Fate map and cell lineage of the sea urchin Strongylocentrotus purpuratusThe 60-cell embryoCell lineage map of the embryoThe signaling molecules involved in cell specification are just now being identified. For example, several pieces of evidence suggest

28、 that the signaling molecules responsible for specifying the micromeres appears to be beta-catenin (the key signal transducer of the Wnt signal pathway). Fate maps and the determination of sea urchin blastomeres (II)The Wnt signal transduction pathwayThe role of beta-catenin in specifying the vegeta

29、l micromeres of the sea urchin embryoWild typeGain-of-function (The accumulation of beta-catenin in every cell by treating the embryo with lithium chloride-blocking the GSK-3 of the Wnt pathway )-Loss of ectodermLoss-of-function (inhibiting beta-catenin accumulation in the micromere nuclei)-Loss of

30、mesoderm/endoderm beta-catenin is stained by a fluorescently labeled antibodyThe cell fate specification of the micromeres by beta-catenin is mediated by the Pmar1 gene. Through the “de-repressed” effect , the Pmar1 gene activates several transcription factors (Tbr, Ets) or ligands (Notch) that dire

31、ct the development of mesoderm and endoderm.Fate maps and the determination of sea urchin blastomeres (III)The proposed molecular regulatory network for the micromere fate specificationThe Xenopus fate map (I) Fate mapping has shown that cells of the Xenopus blastula have different fates depending o

32、n whether they are located in the deep or the superficial layers of the embryo. The mesodermal precursors exists mostly in the deep layer of cells, The ectoderm and endoderm arise from the superficial layer on the surface of embryo. Fate map of the Xenopus blastula The Xenopus fate map (II) The germ

33、 layers can be mapped onto the oocyte even before fertilization. The surface of the animal hemisphere will become the cells of the ectoderm (skin and nerves), the vegetal hemisphere surface will form the cells of the gut and associated organs (endoderm), and the mesodermal cells will form from the i

34、nternal cytoplasm around the equator. The Xenopus fate map (III)n The transcription factor VegT and the TGF-beta family paracrine Vg1 paly crucial roles in determining the general fate of the Xenopus blastula. n The mRNAs for these signal proteins are located in the cortex of the vegetal hemisphere

35、of Xenopus oocyte, and they are apportioned to the vegetal cells during cleavage (asymmetric cell division). n Depletion of maternal Vg1 or Veg T protein in early embryo by using antisense oligonucleotide leads to the embryos lacking the normal fate map. Thus, the allocation of cells to the 3 germ l

36、ayers depends on pre-localized cytoplasmic determinants laid down in the egg.u Embryos lacking functional Vg1 lacked endoderm and dorsal mesoderm. VegT in the vegetal hemisphere of Xenopus oocyte is important for fate determination of the germ layers of the blastula No endoderm is produced (treating

37、 the embryo with the antisense oligonucleotide of VegT )Early embryonic development4 The axis formationWe will discuss this part in next chapter in detail5 Early embryonic development in invertebrates5.1 Sea urchin5.2 Drosophila6 Early embryonic development in vertebrates6.1 Amphibians (Xenopus)6.2

38、Mammals (Mouse)The axis formationn While cleavage always precedes gastrulation, axis formation begins as early as oocyte formation. It can be completed during cleavage (as in Drosophila) or extend all the way through gastrulation (as it does in Xenopus). n There are three axes that need to be specif

39、ied: the AP (head-anus) axis, the DV (back-belly) axis, and the left-right axis. n Different species specify these axes at different times, using different mechanisms. We will discuss this part in the next chapters in detailEarly embryonic development4 The axis formationWe will discuss this part in

40、next chapter in detail5 Early embryonic development in invertebrates5.1 Sea urchin5.2 Drosophila6 Early embryonic development in vertebrates6.1 Amphibians (Xenopus)6.2 Mammals (Mouse)Early embryonic development4 The axis formationWe will discuss this part in next chapter in detail5 Early embryonic d

41、evelopment in invertebrates5.1 Sea urchin5.2 Drosophila6 Early embryonic development in vertebrates6.1 Amphibians (Xenopus)6.2 Mammals (Mouse)Cleavage and formation of blastula in sea urchinsn Sea urchins exhibit radial holoblastic (complete) cleavage. n After the first 7 divisions, the embryo becom

42、es a 128-cell blastula which consists of mesomeres at the animal hemisphere, and macromeres/micromeres at the vegetal hemisphere. At this time, the cell fate of these blastomeres has been determined.n The blastula stage of sea urchin development begins at the 128-cell stage. Here the cells form a ho

43、llow sphere surrounding the blastocoel.Cleavage and formation of blastula in the sea urchins From the 4th division, unequal cleavages occur in the vegetal tiers, producing macromeres/micromeres Planes of cleavage in the first three divisions and the formation of tiers of cells in divisions 3-63rd4th

44、5th6thGastrulation in sea urchinsn Gastrulation begins with the transition of the vegetal mesoderm from the epithelial to motile mesenchymal cells (间充质细胞间充质细胞) and the migration of the mesenchyme cells into the blastocoel as individual cells. The primary mesenchymal cells later lay down the skeletal

45、 rods of the sea urchin endoskeleton by secretion.n This is followed by an invagination of the endoderm, which extends inside the blastocoel toward the animal pole through convergent extension of endoderm and contraction of filopodia from the secondary mesenchymal cells at the tip of the invaginatin

46、g endoderm. This process will eventually form the gut.Apical constrictionEarly embryonic development4 The axis formationWe will discuss this part in next chapter in detail5 Early embryonic development in invertebrates5.1 Sea urchin5.2 Drosophila6 Early embryonic development in vertebrates6.1 Amphibi

47、ans (Xenopus)6.2 Mammals (Mouse)Cleavage and formation of blastula in Drosophila n Most insect eggs undergo superficial cleavage (incomplete), wherein a large mass of centrally located yolk confines cleavage to the cytoplasmic rim of the egg.n One of the fascinating features of this cleavage pattern

48、 is that cells do not form until after the zygote nucleus undergoes several mitotic divisions: Syncytial blastoderm: prior to the 13th division cycle Cellular blastoderm: after the 13th division cycle, approximately 6000 cellsn During the 9th division cycle, above 15 nuclei reach the surface of the

49、posterior pole of the embryo. These nuclei become enclosed by cell membrane and generate the pole cells (PGCs).Laser confocal micrographs of stained chromatin showing superficial cleavage in a Drosophila embryoAt division cycle 10: the syncytial blastoderm with pole cells at the posteriorAfter cycle

50、 13: the cellular blastoderm.Formation of the cellular blastoderm in DrosophilaA: the cellularization processB: the dividing nuclei during the cellularization of the blastodermThe mitotic spindles stained with Abs for tubulinThe cell boundaries labelled with actinC: cross section during cellularizat

51、ionAs the cells form, the domain of the actin expands into the embryo Mesoderm: invagination forming muscle and other connective tissuesEndoderm: invagination forming mid-gutEctoderm: delamination in ventral ectoderm forming the nervous system Genetic control of mesoderm invagination in gastrulation

52、 of the Drosophila embryosn Mesoderm invagination is affected by mutations in the gene twist and snail, which is expressed in the prospective mesoderm before gastrulation. In mutants that lack twist function, a small transient furrow is formed In snail mutants the prospective mesoderm cells flatten,

53、 but no other change occurs Double mutants of twist and snail genes show no cell-shape changes and no invagination.In twist mutant embryos, while no apical contraction is visible, only transient ventral furrow is formed during gastrulationGenetic control of mesoderm invagination in gastrulation of D

54、rosophilan Mesoderm invagination is affected by mutations in the gene twist and snail, which is expressed in the prospective mesoderm before gastrulation. In mutants that lack twist function, a small transient furrow is formed In snail mutants the prospective mesoderm cells flatten, but no other cha

55、nge occurs Double mutants of these two genes show no cell-shape changes and no invagination.n These two genes encode the transcription factors that may therefore be controlling, either directly or indirectly, the expression of cell components such as cytoskeletal proteins, which are required for the

56、 shape changes to oocur.The molecular pathway underlying regulation of Twist in the mesoderm morphogenesisTwistFogT48RhoGEF2 (a guanine exchange factor)Rho (small GTP-binding protein, small GTPase)MyosinIIApical constrictionActin filamentsThe molecular pathway underlying regulation of Twist in the m

57、esoderm morphogenesisTwistFogT48RhoGEF2 (a guanine exchange factor)Rho (small GTP-binding protein)MyosinIIApical constrictionActin filamentsSignal/regulator ?Early embryonic development4 The axis formationWe will discuss this part in next chapter in detail5 Early embryonic development in invertebrat

58、es5.1 Sea urchin5.2 Drosophila6 Early embryonic development in vertebrates6.1 Amphibians (Xenopus)6.2 Mammals (Mouse)Cleavage and formation of blastula in Xenopusn The amphibian egg contains much more yolk, which is concentrated in the vegetal hemisphere. This localized yolk is an impediment to clea

59、vage. So, cleavage in most frog and salamander embryos is a unequal holoblastic (complete) cleavage which establishes two major embryonic regions: a rapidly dividing region of micromeres near the animal pole and a more slowly dividing vegetal macromere area. n As cleavage progresses, the animal regi

60、on becomes packed with numerous small cells, while the vegetal region contains only a relatively small number of large, yolk-laden macromeres. n An amphibian embryo containing 16-64 cells is commonly called a morula (桑椹胚桑椹胚). At the 128-cell stage, the blastocoel becomes apparent, and the embryo is