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Early Mammalian Development Axis determination Neural Development 세포생물학 2 (임현정) June 5, 2009 Axes and planes of section used for describing embryos Axis A-P D-V L-R 11.27 Development of a human embryo from fertilization to implantation Early mammalian embryonic development Development of a human embryo from fertilization. Compaction of the human embryo occurs on day 4, when it is at the 10-cell stage. The embryo hatches from the zona upon reaching the uterus. The zona prevents the embryo from prematurely adhering to the oviduct. Preimplantation development in mice (Gestation length: 20 days) Day 1 Day 2 Day 3 (zona 생략) Day 4 11.29 Cleavage of a single mouse embryo in vitro Cleavage of a single mouse embryo in vitro. 11.31 Hatching from the zona and implantation of the mammalian blastocyst in the uterus Hatching from the zona pellucida and implantation of the mammalian blastocyst in the uterus. (A) Mouse blastocyst hatching from the zona. (B) Mouse blastocysts entering the uterus. (C) Initial implantation of the blastocyst in a rhesus monkey. 11.33 Tissue formation in the human embryo between days 7 and 11 (Part 1) Tissue formation in the human embryo between days 7 and 11. (A,B) Human blastocyst immediately prior to gastrulation. The inner cell mass delaminates hypoblast cells that line the blastoceol, forming the extraembryonic endoderm of the primitive yolk sac and a two-layered (epiblast and hypoblast) blastodisc. The trophoblast divides into the cytotrophoblast, which will form the villi, and the syncytiotrophoblast, which will ingress into the uterine tissue. 11.33 Tissue formation in the human embryo between days 7 and 11 (Part 3) Tissue formation in the human embryo between days 7 and 11. (C) Meanwhile, the epiblast splits into the amniotic ectoderm (which encircles the amniotic cavity-blue) and the embryonic epiblast (purple”embryo proper”). The adult mammal forms from the cells of the embryonic epiblast. (D) The extraembryonic endoderm forms the yolk sac. The actual size of the embryo at this stage is about that of the period at the end of this sentence. Amnion structure and cell movements during human gastrulation. (A,B) Human embryo and uterine connections at day 15 of gestation. (A) Sagittal section through the midline. (B) View looking down on the dorsal surface of the embryo. (C) The movements of the epiblast cells through the primitive streak and Hensen’s node and underneath the epiblast are superimposed on the dorsal surface view. At days 14 and 15, the ingressing epiblasts are thought to replace the hypoblast cells, while at day 16, they fan out to form the mesodermal layer. 11.32 Schematic diagram showing the derivation of tissues in human and rhesus monkey embryos Schematic diagram showing the derivation of tissues in humans and rhesus monkey embryos. The dashed line indicates a possible dual origin of the extraembryonic mesoderm. 11.30 Maintaining lineages in the mouse blastocyst Maintaining lineages in the mouse blastocyst. Proposed functions of the Nanog, Oct4, and Stat3 proteins in retaining the uncommitted pluripotent fate of embryonic cells. Oct4 stimulated the morula cells retaining its expression to become “Inner Cell Mass (ICM)” and not trophoblast. Nanog works at the next differentiation event, preventing the ICM cells to becoming hypoblast, and promoting their becoming the pluripotent embryonic epiblast. Stat3 is probably involved in the self-renewal of these pluripotent cells. Meanwhile, Cdx2 in the trophoblast prevents Oct4 and Nanog expression, thereby stabilizing the trophoblast lineage. 11.35 Human embryo and placenta after 50 days of gestation Human embryo and placenta after 50 days of gestation. The embryo lies within the amnion, and its blood vessels can be seen extending into the chorionic villi. Amnion (양막) Chorion (융모막) Yolk sac (난황막) 11.36 Relationship of the chorionic villi to the maternal blood supply in the uterus Relationship of the chorionic villi to the maternal blood supply in the uterus. 11.37 The timing of human monozygotic twinning with relation to extraembryonic membranes Diagram showing the timing of human monozygotic twinning with relation to extraembryonic membranes. (A) Splitting occurs before the formation of the trophoblast, so each twin has its own chorion and amnion. (B) Splitting occurs after trophoblast formation but before amnion formation, resulting in twins having individual amnionic sacs but sharing one chorion. (C) Splitting after amnion formation leads to twins in one amnionic sac and a single chorion. Axis determination: Anterior-posterior patterning in the mouse embryo Key points to remember in A-P Patterning 1. Signaling centers 2. Gastrulation 3. Morphogen gradient 4. Hox genes Mesodermal induction by the vegetal endoderm in Xenopus Functions of the organizer: 1.The ability to self-differentiate dorsal mesoderm (chordamesoderm) 2.The ability to dorsalize the surrounding mesoderm into paraxial mesoderm (somites, etc.) 3.The ability to dorsalize the ectoderm, inducing the formation of the neural tube 4.The ability to initiate the movements of gastrulation Signaling centers 1. 다른 종에서는 Organizer (양서류) 또는 Hensen’s node (닭) 으로 불리운다. 2. 포유류에는 2개가 있다: the node, the anterior visceral endoderm (AVE). 3. Node는 전체 body patterning을 조절하며 node와 AVE가 협력하여 배아의 머리쪽 구조를 형성한다. 4. Neural tube를 유도하는 notochord는 node의 ciliated cell들이 등쪽으로 들어가면서 형성된다. 5. Chordamesoderm인 notochord는 ectoderm 의 일부를 neural tube로 유도한다. 11.39 Axis and notochord formation in the mouse (Part 1) Day 7 Axis and notochord formation in the mouse. (A) In the 7-day mouse embryo, the dorsal surface of the epiblast (embryonic ectoderm) is in contact with the amnionic cavity. The ventral surface of the epiblast contacts the newly formed mesoderm. In this cuplike arrangement, the endoderm covers the surface of the embryo. The node is at the bottom of the cup. The two signaling centers, the node and the anterior visceral endoderm (AVE), are located on opposite sides of the cup. Eventually, the notochord will link them. The caudal side of the embryo is marked by the presence of the allantois. AVE: Nodal protein의 antagonist인 Lefty1과 Cerberus, Wnt inhibitor인 Dickkopf를 발현한다. 즉 posterior region 형성에 필요한 유전자들을 저해함으로써 anterior 구조형성에 기여한다. 11.39 Axis and notochord formation in the mouse (Part 2) Day 8 Axis and notochord formation in the mouse. (B) By embryonic day 8, the AVE lines the foregut, and the prechordal mesoderm is now in contact with the forebrain ectoderm. The node is now father caudal, due largely due to the rapid growth of the anterior portion of the embryo. 11.39 Axis and notochord formation in the mouse (Part 3) Ventral surface of a 7.5-day mouse embryo. The presumptive notochord cells extend from the node into the endoderm of the primitive gut, converging medially to begin formation of the notochord. Node Embryonic turning. In mice, from about embryonic day 7.5 to 9, the embryo becomes rotated around its long axis leading to ventral closure of the gut. Morphogen gradient A stable concentration gradient: cannot be produced simply by releasing a pulse of the morphogen. 조건: “source” and “sink” gradient 형성 중요개념: Threshold responses Polarity “Mirror symmetry” 그림 설명: Properties of morphogen gradients. (a)Normal development of an animal with a head and three segments. (b)Graft of the posterior source to the anterior causes formation of a U-shaped gradient and produces a double-posterior animal. (c)Insertion of an impermeable barrier causes formation of a large gap in the pattern. The binary codes indicate the activity in each body region of the genes, 3,2, and 1, respectively (1=on, 0=off). Anterior-posterior patterning in the mouse embryo (A) Concentration gradients of BMPs, Wnts, and FGFs in the late gastrula mouse embryo (depicted as a flattened disc). The primitive streak and other posterior tissues are the sources of Wnt and BMP proteins, whereas the organizer and its derivatives produce antagonists. (C) Retinoic acid, Wnt3a, and Fgf8 each contribute to posterior patterning, but they are integrated by the Cdx family of proteins that regulate the activity of the Hox genes. 11.40 Expression of BMP antagonists in the mammalian node Expression of BMP antagonists in the mammalian node. Homeotic genes (A) A head of a wild-type fruit fly. (B) Head of a fly containing the Antennapedia mutation that converts antenna into legs. 3` Evolutionary conservation of homeotic gene organization in fruit flies and mice. 5` * Homeotic genes: mutations of those lead to homeotic transformations of specific segments along the AP body axis 1. 2. 3. 4. 5. 6. Homeobox (183-bp) Transcription factors (61-aa homeodomain) Temporal colinearity Spatial colinearity Chromosomal duplication Autoregulation or cross-regulation Paralogous group paralogous group * Hox gene expression domains in the developing vertebral column vertebrae occipital bone Experimental analysis of the Hox code 1. Gene targeting 2. Retinoic acid teratogenesis 3. Comparative anatomy 11.43 Axial skeletons of mice in gene knockout experiments Gene targeting Lumbar thoracic Sacral lumbar 11.44 The effect of retinoic acid on mouse embryos (Part 1) Retinoic acid teratogenesis 11.44 The effect of retinoic acid on mouse embryos (Part 2) 11.45 Schematic representation of the chick and mouse vertebral pattern along the anteriorComparative anatomy posterior axis (Part 1) Axis determination: Left-right patterning in the mouse embryo Key points to remember 1. Ciliary cells in the node 2. NVPs 3. Mutations 11.47 Left-right asymmetry in the developing human Left-right asymmetry in the developing human. (A) Abdominal cross sections show that the originally symmetrical organ rudiments acquire asymmetric positions by week 11. The liver moves to the right and the spleen moves to the left. (B) Not only does the heart move to the left side of the body, but the originally symmetrical veins of the heart regress differentially to form the superior and inferior venae cavae, which connect only to the right side of the heart. (C) The right lung branches into three lobes, while the left lung (near the heart) forms only two lobes. In human males, the scrotum also asymmetrically. Normal Polysplenia syndrome Asplenia syndrome (no spleen) Complete mirror-image reversal The left-right axis 1. The mammalian body is not symmetrical. 2. situs inversus viscerum (iv): randomizes the left-right axis for each asymmetrical organ independently (Hummel & Chapman, 1959; Layton, 1976) 3. Lack of coordination among visceral organs can cause serious problems, even death. 4. inversion of embryonic turning (inv): Mice homozygous for an insertional mutation at this locus had all their asymmetrical organs on the wrong side of the body. Since all the organs were reversed, this asymmetry did not have dire consequences for the mice. Ciliary cells in of the node 1. The cilia cause fluid in the node to flow from right to left. 2. KIP3B knockout mice: Nodal cilia did not move, and the situs (lateral position) of each asymmetric organ was randomized. 11.48 Situs formation in mammals (Part 1) Situs formation in mammals. (A)Ciliated cells of the mammalian node. (B)Schematic drawing showing the FGF-induced secretion of nodal vesicular parcels from the cells of the node, the movement of the NVPs to the left side, motivated by the ciliary currents, and the rise in Ca2+ concentration on the left side of the node. NVP: small membrane-bound vesicles of 1 um. They contain Sonic hedgehog protein and retinoic acid, etc. 11.48 Situs formation in mammals (Part 3) Situs formation in mammals. (C) Calcium ions (red, green) concentrated on the left side of the node in mice. 12.1 Major derivatives of the ectoderm germ layer The emergence of ectoderm: Central Nervous System (CNS) Major derivatives of the ectoderm germ layer. Head fold stage Ectoderm 1.Neural ectoderm 2.Neural crest cells 3.Epidermal ectoderm Neural ectoderm: neural tube라는 rudiment를 만들어서 분화되는데, 이 과정을 neurulation이라 한다. Neurulation in a chick embryo (dorsal view). The cephalic (head) region has undergone neurulation, while the caudal (tail) regions are still undergoing gastrulation. 12.3 Primary neurulation: neural tube formation in the chick embryo (Part 1) Primary neurulation: neural tube formation in the chick embryo. (A,1) Cells of the neural plate can be distinguished as elongated cells in the dorsal region of the ectoderm. Folding begins as the medial neural hinge point (MHP) cells anchor to the notochord and change their shape, while the presumptive epidermal cells move toward the dorsal midline. (B,2) The neural folds are elevated as the presumptive epidermis continues to move toward the dorsal midline. 12.3 Primary neurulation: neural tube formation in the chick embryo (Part 2) Primary neurulation: neural tube formation in the chick embryo (II). (C,3) Convergence of the neural folds occurs as the dorsolateral hinge point (DLHP) cells become wedge-shaped and the epidermal cells push toward the center. (D,4) The neural folds are brought into contact with one another, and the neural crest cells link the neural tube with the epidermis. The neural crest cells then disperse, leaving the neural tube separate from the epidermis. 12.4 Three views of neurulation in an amphibian embryo (Part 1) Three views of neurulation in an amphibian embryo, showing early (left), middle (center), and late (right) neurulae in each case. (A) Looking down on the dorsal surface of the whole embryo 12.4 Three views of neurulation in an amphibian embryo (Part 3) Three views of neurulation in an amphibian embryo, showing early (left), middle (center), and late (right) neurulae in each case. (B) Sagittal section through the medial plane of the embryo. (C) Transverse section through the center of the embryo. 12.5 Neurulation in the human embryo (Part 1) Neurulation in the human embryo. (A)Dorsal view of a 22-day (8-somite) human embryo initiating neurulation. Both anterior and posterior neuropores are open to the amniotic fluid. (B)10-somite human embryo showing the three major sites of neural tube closure (arrows). (C)Dorsal view of a neurulating human embryo with only its neuropores open. (D)A stillborn infant with ancephaly. 12.6 Expression of N- and E-cadherin adhesion proteins during neurulation in Xenopus Expression of N- and E-cadherin adhesion proteins during neurulation in Xenopus. (A)Normal development. In the neural plate stage, N-cadherin is seen in the neural plate, while E-cadherin is seen on the presumptive epidermis. Eventually, the N-cadherin-bearing neural cells separate from the E-cadherin-containing epidermal cells. (Neural crest cells express neither of them and they disperse.) (B)No separation of the neural tube occurs when one side of the frog embryo is injected with N-cadherin mRNA, so that N-cadherin is expressed in the epidermal cells as well as in the presumptive neural tube. 12.7 Folate-binding protein in the neural folds as neural tube closure occurs Folate-binding protein in the neural folds as neural tube closure occurs. (A)10-somite mouse embryo stained for folate-binding protein mRNA. (B,C) Sections through embryo (A) at the two arrows, showing the folate-binding protein (dark blue) at the points of neural tube closure. Secondary neurulation in the caudal region of a 25-somite chick embryo. (A)Mesenchymal cells condense to form the medullary cord at the most caudal end of the chick tailbud. (B)The medullary cord at a slightly more anterior position in the tailbud. (C)The neural tube is cavitating and the notochord forming; note the presence of separate lumens. (D)The lumens coalesce to form the central canal of the neural tube. Differentiation of the Neural Tube Early human brain development. The three primary brain vesicles are subdivided as development continues. At the right is a list of the adult derivatives formed by the walls and cavities of the brain. 12.10 Rhombomeres of the chick hindbrain Rhombomeres of the chick hindbrain. 12.12 Occlusion of the neural tube allows expansion of the future brain region Occlusion of the neural tube allows expansion of the future brain region. (A)Dye injected into the anterior portion of a 3-day chick neural tube fills the brain region, but does not pass into the spinal region. (B,C) Sections of the chick neural tube at the base of the brain (B, before occlusion, C, during occlusion). (D) Reopening of the occlusion after initial brain enlargement allows dye to pass from the brain region into the spinal cord region. 12.13 Dorsal-ventral specification of the neural tube (Part 1) Dorsal-ventral specification of the neural tube. Two signaling centers: ectoderm roof plate (BMP) notochord floor plate (Shh) 12.14 Cascade of inductions initiated by the notochord in the ventral neural tube (Part 2) (B) Two cell types in the newly formed neural tube. Those closest to the notochord become the floor plate neurons; motor neurons emerge on the ventrolateral sides. (C) If a second notochord, floor plate, or any other Sonic Hedgehog-secreting cell is placed adjacent to the neural tube, it induces a second set of floor plate neurons, as well as two other sets of motor neurons. Schematic representation of neural crest formation in a chick embryo, shown in cross section. Neural crest cells form at the junction between the Wnt6-expressing epidermal ectoderm and the BMPs produced by the presumptive neural ectoderm. 13.2 Regions of the chick neural crest Regions of the chick neural crest. The cranial neural crest migrates into the pharyngeal arches and the face to form the bones and cartilage of the face and neck. It also produces cranial nerves. The vagal neural crest (near somites 1-7) and the sacral neural crest (posterior to somite 28) form the parasympathetic nerves of the gut. The cardiac neural crest cells arise near somites 1-3; they are critical in making the division between the aorta and the pulmonary artery. Neural crest cells of the trunk (about somite 6 through the tail) make sympathetic neurons and pigment cells (melanocytes), and a subset of these (at the level of somites 18-24) form the medulla portion of the adrenal gland. Pluripotency of trunk neural crest cells. (A) A single neural crest cell is injected with highly fluorescent dextran shortly before migration of the neural crest cells is initiated. The progeny of this cell each receive some of these fluorescent molecules. Two days later, neural crest-derived tissues contain dextran-labeled cells that are descended from the injected precursor. The figure summarizes data from two different experiments. 13.7 Pluripotency of trunk neural crest cells (Part 2) Pluripotency of trunk neural crest cells. (B) Model for neural crest lineage segregation and the heterogeneity of neural crest cells. The committed precursors of neurons (N), glias (G), melanocytes (M), and so forth would be derived from intermediate progenitors, some of which could act as stem cells. The multipotent neural crest stem cell (top) is hypothetical and need not exist.