Organogenesis MCB141 part III Spring 2012 Lecture 1 Harland Mammalian gastrulation Gilbert 9 305-308; 311-313 Gillbert 8: 354; 358-9; Gilbert 7 371, 375-6 Also references are given below to Gilbert's Online sixth edition. This has to be searched for text, so I will give the links when possible (for mammalian early development it is Gilbert6). I will also reference journal articles, which can be accessed from campus IP addresses, or via the library’s proxy server. Sometimes (especially with Elsevier journals) one has to open the journal article before navigating to the individual figure. For a summary of mouse gastrulation, see Lu and Robertson, figure 1 1.The topology of the mouse gastrula is the same as that of the chick (and human) embryo. Like the chick, the primitive streak forms at the edge of the epiblast, and cells ingress into the space between the epiblast and primitive endoderm (hypoblast). However, the disk of the chick is distorted in the mouse into a cup shape. Instead of sitting on top of the yolk, the embryonic disk develops above the blastocoel, and the edges of the disk are attached to extraembryonic ectoderm. 2.Following the egg cylinder stage, the primitive streak forms at the edge of the cup, and spreads down to the distal tip of the epiblast. Cells ingress from the single cell thick epithelium of the epiblast, outwards so that they form mesoderm between the epiblast and the hypoblast (or primitive endoderm). This primitive endoderm will ultimately be enclosed in the developing gut, so is called the visceral endoderm. 3.The mesoderm migrates out around the epiblast as “wings” of mesoderm, until these wings meet at the other side, under the AVE (Anterior Visceral Endoderm). As in the chick, this migrating mesodermal tissue forms the paraxial mesoderm of the somites, and the lateral plate. Although there are no good movies of this in the mouse embryo, one can infer a lot of these movements from a movie of the simpler chick embryo1 or movie2 (from CD Stern lab) 4.The distal tip of the extending early primitive streak has organizer activity and when the streak is fully extended it forms an anatomically distinct structure, the node. The node, as in the chick, is the mouse “organizer” and precursor of the midline of the embryo, the notochord, and definitive endoderm. Transplantation of the node (marked with a lacZ transgene) showed that it could induce ectopic axial structures. 5.How does the mouse embryo break these symmetries? Unlike other embryos, the mammalian embryo has no prominent axes when shed from the ovary. Breaking symmetries relies on amplification of a series of small differences. 6. First there is the inside outside decision (trophectoderm versus epiblast) . Inside/outside is driven by activation of Cdx2 expression in the outer epithelial cells. The initiating signal from the adhesion to gene expression is still not completely understood, though uses a conserved “hippo” pathway, that is used in flies for size regulation. The inner cells remain totipotent by default. 7. The next decision in the Inner Cell Mass to generate the epiblast (still totipotent) and the primitive endoderm comes fromcell sorting. Stochastic decisions within the ICM turn on Oct4, Nanog, and Sox2 which maintain totipotency, versus the alternative GATA6 which differentiates the primitive endoderm. Each pathway positively feeds back on itself, while repressing the other. So the inner cell mass generates a salt and pepper pattern of GATA6 and Oct4 cells, which then sort out. 8. Inner cell mass cells can be cultured and propagated as Embryonic Stem Cells. These can be cultured and manipulated, and then reintroduced into a blastocyst, where they can take part in normal development,including forming germ cells. Usually genetically marked cells the ES cell contribution to the mosaic embryos can be seen. Thus, ES cells can me manipulated, but then form mice. 9.we’ll leave the summary of nodal signaling to the next lecture- 10. As gastrulation progresses, there is a huge amount of proliferation and size increase. The primitive streak continues to produce mesoderm and endoderm of progressively more posterior identity. Refer to figure 1 of Lu and Robertson. The node produces midline notochord, and definitive endoderm. Somites coalesce on each side of the midline, the neural plate begins to close. Finally, in a triumph of topology, the embryo turns from its backward bent shape at 8.5 days to assume the fetal position, nose to feet. As it turns, it wraps itself in the amniotic membrane. 11.The embryo always turns in the same direction, and also generates clear Left right asymmetry in a variety of internal organs, the heart, liver lungs, gut, brain. During formation of the node, the overlying primitive endoderm forms a gap, and the ciliated cells of the node are exposed. The cilia point posterior, and rotate, inducing a leftward flow. This flow is thought to be causal in setting up the left side. Mutations that eliminate ciliary motility cause randomization of the Left-right axis. The axis can be reversed by flowing fluid in the opposite direction 12.Formation of the mesoderm requires the product of the nodal gene, homologous to the gene you heard about in formation of the mesoderm in Xenopus.. This gene was first identified in the mouse, in one of the early exploitations of Embryonic stem cells. This was a forward genetic screen (as in Patel lecture 3) using retroviruses as mutagens, and screening for interesting phenotypes. The retrovirus-as-mutagen approach has the advantage that the integration can disrupt genes, and provides a DNA marker which can subsequently used to identify and clone the integration site. Retroviruses were used to infect ES cells, where the retroviral RNA genome was reverse transcribed into DNA, and integrated into the genome. ES cells carrying a large number of retroviral insertions (mutations) were then reintroduced in to the blastocyst, the resulting mice would then carry mutations induced by the retrovirus. Germ cells derived from the ES cells propagate the mutations into mice. These heterozygous lines could then be bred to an F2 generation to homozygosity (similar to the screen described for Drosophila), and screened for interesting phenotypes. One such integration completely disrupts mesoderm development, and cloning of the retrovirus tagged locus identified the TGFbeta family member nodal. 13.So to review the origin of the axes: The anterior posterior axis is established by the asymmetry of the AVE, which underlies the future head, and determines the site of the beginning of mesoderm/ primitive streak formation, which in turn is the posterior part of the embryo. the dorsal ventral axis is set up from the topology of epiblast and visceral endoderm. As in Xenopus, an initially small asymmetry is magnified to establish a definitive asymmetry in gastrulation movements, though the timing and mechanism is quite different, as we will see next time.