Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Germ Layer Formation in Chick and Mouse Embryos
Transcript
Early Development in Vertebrates: Germ Layer Formation in Chick and Mouse Embryos Top View Side View Ventral Top View Posterior Caudal Tail Sections : Vertical, Horizontal, Sagittal Lateral / Distal / Peripheral Dorsal Medial / Proximal / Central Geometry in Embryology Lateral / Distal / Peripheral Anterior Rostral Head Advantages of using chick embryos in developmental studies 1. Year-round easy accessibility 2. Easy preparation of synchronized embryos in big quantities 3. Extensive manipulations are possible. (explant, injection & in ovo culture) 4. Human gene compatibility 5. Low cost Blastodisc (Blastoderm) 5-6 layers of cells linked together with tight junctions yolk albumin egg shell Most Embryonic Body; Active Cell Cleavage Marginal Belt; boundary; important roles in cell fate specification 25mV membrane potential pH 9.5 PMZ pH 6.5 primary hypoblasts all three germ layers local thickening of PMZ Extraembryonic membrane Linkage between endoderm & yolk sac secondary hypoblasts : P A extension • posterior epiblast thickening • epiblasts anterior to Koller’s sickle • increase in cell height • more globular shape • higher motility • ECM digestion Primitive Streak • epiblasts in PMZ region P A extension • increase in length, decrease in width • ingression of the cells around the streak (first ingressing epiblasts migrate down to basal lamina to form future endoderm) • simultaneous hypoblast extension underneath Hensen’s Node • primitive knot • anterior thickening of primitive streak • ~ organizer in frog Primitive Pit • funnel shape • major cell passing gate to blastocoel • 70% of area pellucida • extension to future head forming region Primitive Groove • depression within the streak • open path to blastocoel • ~ blastopore in frog future head forming region anterior expansion of the embryonic regions between Hensen’s node and head process midbrain hindbrain border Once Hensen’s node hits the head process… posterior regression of Hensen’s node and primitive streak (i.e., shortening of primitive streak) (PA) (AP) (AP) (AP) (PSM) • Anterior-end passing cells migrate anteriorly to form foregut, head mesoderm, and notochord. • Posterior-end passing cells migrate downward to form endodermal and mesodermal tissues. • Order of Development : Anterior > Posterior, Medial > Lateral • individual cell ingression, migration, EMT, and cell fate specification in chick and mouse embryos • continuous invagination, migration, and cell fate specification in amphibians gradual replacement of basal lamina by future endoderm forming cells Visualizing Ingressing and Migrating Cells during Early Development Formation of Germ Layers 1. First group of cells passing through Hensen’s node forms pharyngeal endoderm of foregut (anterior migration & hypoblast displacement) confines hypoblasts in the anterior portion of area pellucida (germinal crescent); precursors of the germ cells later migrating to the gonads through the blood vessel 2. Second group (between epiblasts and endoderms) head mesenchyme, prechordal plate mesoderm (anterior to notochord forming point) (anterior migration, but short traveling to the ventral side) head process formation anterior to the Hensen’s node 3. Third group chordamesoderm notochord forming cells (from Hensen’s node to rostral up to midbrain) hindbrain and trunk (from Hensen’s node to caudal to the end of streak) 4. Lateral portion of primitive streak ingression (for posterior organs) Deep layer : mid-line hypoblast replacement most endodermal origin organs extraembryonic membranes Middle layer mesodermal origin organs Formation of Germ Layers (continued) 5. Mesodermal cell ingression continues. Hensen’s node disappears Primitive streak regression begins. after generating 17 somites Posterior movement of Hensen’s node Posterior extension of notochord cells ingressed through Hensen’s node (until 17 somites) cells ingressed through primitive streak (after 17 somites) AP gradient in developmental maturity anterior region --> already initiate organogenesis (i.e. brain & heart) posterior region --> still active gastrulation Formation of Germ Layers (continued) midbrain level initial HN level before regression HN position during regression Formation of Germ Layers (continued) 6. Finally, the remaining epiblast cells represent presumptive ectodermal cells. Epiboly of the ectoderm : Ectodermal precursor cells continue to migrate and proliferate to surround the yolk for about 4 days. Outer marginal cells of area opaca firmly attach to vitelline envelope of the yolk via fibronectin interactions and these ECM-membrane interactions provide locomotive forces for epiblast cells to wrap around the entire yolk. Thus, after complete and successful gastrulation, 1) The yolk is surrounded by the ectoderms. 2) Hypoblasts are replaced by the endoderms. 3) Various mesoderms are appropriately positioned between endoderm and ectoderm. AP Axis (Asymmetry) Formation due to Gravity Caused by Egg Rotation heavy light Hensen’s Node Is the Chick “Organizer” [hoxb1] [otx2] [otx2] [hoxb1] 2O Hensen’s node periphery transplantation Induction of a New Embryo by Transplantation of Hensen’s Node Genes expressed in PMZ entire primitive streak Koller’s sickle Hensen’s node Vg1 Nodal Chordin Shh During Early Gastrulation Medial-Anterior Region BMP FGF from Hensen’s Node Precursor Cells Lateral-Posterior Region BMP o 1 o BMP Signaling 2 Chordin, Noggin, Nodal (BMP antagonists) from Hensen’s Node Inhibition of BMP signaling is critical for early gastrulation BMP (Bone Morphogenetic Protein) Pathway Participating R-Smads for each pathway BMP : Smad 1, 5, 8 TGFb : Smad 2, 3 R-Smad I-Smad Noggin Chordin Gremlin Folistatin Nodal BMP Antagonists Co-Smad Generation of Mesodermal Cells from the Epiblast Mesoderm Epiblast Mesoderm Marker (-) ERNI Sox3 ERNI/Sox3 Brachyury Tbx6L Streak FGF8 Cerberus Hypoblasts (No Cerberus) Nodal Hensen’s Node Retension of Neural Ectoderm in the Epiblast FGF8 slower induction than Brachyury/Tbx6L SIP1 BMP Signaling Neural Tissue Formation Churchill Brachyury Tbx6 positive cell ingression to form mesoderm ERNI/Sox3/Churchill/SIP1 positive cells Neural Ectoderm During Early Gastrulation Anterior Retinoic Acid Posterior Retinoic Acid Retinoic Acid Degrading Enzyme Retinoic Acid Synthesizing Enzyme early stage embryo posterior marker (Hoxb1) Cerberus Left-Right Asymmetry in Chick RIGHT LEFT Ventral View Activin Nodal Cerberus Shh BMP4 BMP Snail Shh Lefty-2 Nodal FGF8 Dorsal View Lefty-2 Nodal Pitx2 Pitx2 Snail e.g. Heart, Spleen Pitx2 e.g. Liver Ventral View Difficulties in Studying Mammalian Embryos 1. Smallest eggs among animal kingdom (100 um in diameter, 1/100 size to Xenopus egg) 2. Too fragile (manipulations almost impossible) 3. Material availability 4. Internal development Key Features in Mammalian Embryogenesis I. Rotational Cell Cleavage Rotational Cleavage Multiple One Cells Cell Repeat Meridonial Cleavage Meridonial Cleavage Meridonial Cleavage Equatorial Cleavage Meridonial ……. Cleavage Equatorial ……. Cleavage Meridonial ……. Cleavage Equatorial ……. Cleavage Key Features in Mammalian Embryogenesis II. Compaction, ICM, Trophoblast & Blastocyst 2 cell stage 4 cell stage uncompacted 8 cell stage compacted 8 cell stage compaction compacted 16 cell stage morula (embryo proper) ICM + Trophoblast (chorion) Blastocyst Key Features in Mammalian Embryogenesis III. ICM vs. Trophoblast Key Features in Syncitio-Trophoblast Mammalian Embryogenesis Amniotic Ectoderm IV. Blastocyst Penetration, Imaginary Yolk Sac, & Amniotic Cavity Epiblast Amniotic Structures Imaginary Yolk Sac Uterine Tissue Hypoblast Uterine Epithelium Extraembryonic Endoderm Extraembryonic Mesoderm Epiblast Hypoblast Uterus Lumen Blastocoel Trophoblast Blastocyst Key Features in Mammalian Embryogenesis V. Gastrulation & Cell Migration from Bilaminar Germ Disc Key Features in Mammalian Embryogenesis VI. Chorionic Villi Circulation (Exchange) • Small molecule (nutrients, O2) supply from mother to fetus via capillary vessel diffusion • Fetus sends metabolic toxic wastes to maternal circulation system. • BUT, no merge or fusion between maternal and zygotic blood vessels Production of Chimeric Mice Zona Pellucida : extracellular matrix of egg, essential for sperm binding, prevent attachment of blastocyst to oviduct wall (if fails, ectopic tubal pregnancy hemorrhage) AP Patterning by Hox Gene Expression • 4 clusters of Hox gene per haploid set • located on 4 different chromosomes (mouse : Hox a to d, human : Hox A to D • gene composition and relative location order are conserved from fly to human • Hox positive tissues - anterior boundary of the hindbrain to tail - tissues along the dorsal axis (neural tube, neural crest, paraxial mesoderm, medial surface ectoderm • a given combination of Hox gene expression dictates a specific level in the A-P axis • anterior to posterior retinoic acid gradient regulates Hox gene expression • anterior Hox gene repression by RA • RA-indep posterior Hox gene expression • Hox gene mutation more severe effects in anterior patterning • RA treatment alters Hox gene expression. Experimental Confirmation of Hox Code Hypothesis Hox10 K.O. lumbar to thoracic Hox11 K.O. sacral to lumbar more RA anterior reduction posterior expansion less RA anterior expansion posterior reduction Comparative Anatomy Supports Hox Code Hypothesis # of vertebrae for each level Chick 14 7 4 8 3 Mouse 7 13 6 4 6
