Axis Formation and Gastrulation II

The Xenopus Oocyte is Asymmetric “Animal” VegT Vg1 “Vegetal” Maternal Determinants VegT: Transcription factor -Promotes “vegetal” identity (endoderm) -Activates mesoderm inducers (nodals/TGF-ß’s) Vg1: Another nodal/ TGF-ß’s Just like for Drosophila, Xenopus egg is being patterned during oogenesis Animal--Vegetal axis established (not really AP or DV) VegT is transcription factor Maternal VegT RNA localized to vegetal side and promotes vegetal or endoderm identity

Sperm Entry Point Determines D/V Axis in Xenopus “Animal” An Cortical Rotation D VegT Wnt11 Maternal Determinants VegT: Transcription Factor Promotes “vegetal” identity (endoderm) Wnt11: Signaling Ligand Promotes Dorsal identity V “Vegetal” Veg Sperm entry point detemines future ventral side Induces MT driven “cortical rotation” where cortex of zygote rotates relative to rest of cytoplasm Opposite point is dorsal--grey cresent Wnt11 thought to rotate and induce dorsal Wnt11 acts through canonical wnt pathway (Tao Heasman, 2005, Heasman lab 2007) Basically--whole idea is to make a vegetal (VegT) to dorsal (Wnt11) axis orthogonal to An-Veg axis

Specifying the Germ Layers Mesoderm Inducers are TGF-ß Family Members (Vg1, Nodals (Xnr’s), Activin) -higher in dorsal mesoderm Wnt11

Specifying The Spemann-Mangold Organizer V D Wnt11 Nodals (TGF-ß’s)

Transplant Dorsal Blastopore Lip Donor: Pigmented (newt) V D V D Host: Unpigmented (newt) Spemann and Mangold, 1924

V D V D Form Second Body Axis Spemann and Mangold, 1924

Normal Body Axis Second Body Axis Spemann and Mangold, 1924 V D V D Also two sites of gastrulation Second Body Axis: Dorsal Mesoderm (Notocord)--Donor Cells Neural Plate--Host Cells Other mesoderm--mix of Donor and Host Donor tissue “organized” host tissue to take on new cell identities Blastopore lip = an Organizer Spemann and Mangold, 1924

Specifying The Speman-Mangold Organizer V D V D wnt11 nodals The Organizer: -Patterns the mesoderm along the D/V axis -Determines the site of gastrulation -Allows for induction of the nervous system (neuroectoderm)

Fate Map of the Xenopus Blastula An V D Veg

D V Fly Frog sog Hypothesis: Neural Induction The dorsal lip secretes a signaling molecule that patterns the mesoderm and induces neural plate specification Search for molecules that can mimic organizer activity Find secreted INHIBITORS of TGF-ß ligands (Chordin, Noggin, Follistatin) Neural is DEFAULT state and TGF-ß signaling is required to INDUCE ectoderm sog D V Fly TGF-ß (BMP, Dpp) Chordin, Sog Xolloid/Tolloid xolloid Frog Note: BMP4 is different type of TGF-ß than nodals -nodals form early gradient that is high in dorsal regions -BMPs form later gradient that is high in ventral regions

Fish and Frog Embryos Appear Very Different Yolk Cell

Fish and Frogs Do Things Similarly -The oocyte has an Animal-Vegetal Axis -The Wnt pathway initiates the D/V Axis (unclear how--doesn’t seem to be sperm) -BMPs and Wnts pattern the mesoderm V D V D BMPs V D Also get nuclear accumulation of b-catenin in future dorsal region of fish embryo Not known how this is regulated -does not seem to be due to sperm entry point (which is fixed at micropyle) -initial cleavages do not appear to predict D/V axis -but Gore 2005 show localization of squint (nodal) at early cleaveages and eventually do dorsal side -Scheir and Talbot show nodal ligands not important for D/V -BUT this still indicates early cleavages define D/V, in conflict with lineage analysis -also, disruption of microtubules soon after fertilization does disrupt D/V--also in contrast to lineage work -also, localization of germ plasm clearly indicates that machinery for post-fertilization localization of determinants exists MVD: could cytoplasmic bridges affect early fate map so identity is really more restricted early than these studies would suggest?? Organizer (Shield) V nodals D V D Wnts -Nodal (and FGF) signaling specifies the mesoderm (and endoderm) with a dorsal bias -Dorsal mesoderm makes the organizer (shield)

Fate Mapping (Lineage Tracing) to Investigate Cell Identity and Developmental Potential Xenopus An V D Veg Zebrafish

It is not birth, marriage or death, but gastrulation, which is truly the most important time in your life.               - Lewis Wolpert (1986) Gastrulation

Cell Movements Relevant for Gastrulation Moving cells around Getting cells inside Spreading tissues out Making tissues longer Convergence/extension

Xenopus Gastrulation Initiates at the Organizer (aka Dorsal Blastopore Lip)

Xenopus Gastrulation

Zebrafish Fate Map Schier and Talbot, 2005

Zebrafish Gastrulation

Early Zebrafish Cleavages

Zebrafish Epiboly Transforms the Embryo Into a Hemisphere Solnica-Krezel, 2006

Cells Involute and Ingress to Form the Dosal Shield Cells Migrate Anteriorly After Involution Solnica-Krezel, 2002

Convergence/Extension Requires the PCP Pathway Convergence/Extension Also Contributes to A/P Axis Formation Extension of cells along the A/P axis Convergence of lateral cells toward the midline of the embryo Convergence/Extension Requires the PCP Pathway Solnica-Krezel, 2006

The Anterior-Posterior Axis is Also Coupled to Gastrulation The developmental potential and inducing properties of cells in the dorsal blastopore lip change with time: -Cells in the lip early become anterior mesoderm and induce anterior neural tissue -Latter cells become posterior and induce more posterior neural structures -A gradient of Wnt activity is high in posterior and low in anterior

Chick Embryos Look Different but Act Similarly The Chick Embryo Forms as a Flattened Disc of Cells On the Yolk The primitive streak/node is the organizer and expresses goosecoid and chordin The posterior marginal zone initiates primitive streak/organizer formation and expresses Vg1 and Wnt8 The primitive streak elongates with the node/organizer at the leading edge

Hensen’s Node is the Avian Organizer Similar to Xenopus blastopore and Fish dorsal shield in terms of both patterning and gastrulation movements The node can induce nervous system development and a secondary axis when transplanted The node expresses BMP antagonists, like the Xenopus and Zebrafish organizers

All Vertebrate Embryos Have a Spemann-Mangold Organizer Bird Fish Frog Bird Mouse Henson’s Node Dorsal Shield Blastopore Lip Node

Chick Gastrulation Movements -In the Node and Primitive Streak, cells delaminate from the epiblast and ingress to form the endoderm and mesoderm -Epiblast cells continue to enter the streak from lateral regions -Once they have ingressed in the streak, newly formed endoderm and mesoderm move laterally again

Gastrulation and Patterning Follow the Same Logic as Fish and Frogs As with fish and frogs, the first cells gastrulating through the chick organizer (node) become anterior endoderm and prechordal plate mesoderm The next cells through will form notochord These first cells also induce the nervous system from the overlying ectoderm Cells gastrulating through more posterior primitive streak become other mesoderm and endoderm derivatives BUT, note that the organizer MOVES as the primitive streak first advances and then regresses More posterior regions of notocord are formed from cells migrating through node as it regresses

The A/P Axis is “laid down” During Primitive Steak Regression This is in contrast to the active extension of the A/P axis seen in frog and fish embryos (although some active mechanisms do further elongate the chick A/P axis) Consequently, posterior development is delayed relative to anterior development “Laying down” the notocord during regression of Henson’s node

Human Embryos Gastrulate Like Chick Embryos

Different Embryos, Common Themes

Activin (TGF-ß): A morphogen for the mesoderm Animal cap assay (After Green et al., 1992) (After Asashima, 1994)

Lineage Analysis and Fate Mapping Goal: Identify which cells in the early embryo give rise to particular cells in the later embryo or adult In order to understand how a particular cell type develops, you have to know where it comes from

Lineage Analysis and Fate Mapping Approaches: 1) Just watch closely

Lineage Analysis and Fate Mapping Approaches: Just watch closely Cell transplantation Use a Lineage Tracer -Inject into single cells or few cells -Activate in single cells or few cells

Fate Mapping by Single Cell Injection of Lineage Tracer Single cell ionophoretic injection of fluoroscein dextran e.g. Woo and Fraser, 1995

Laser Activation of Lineage Tracer “Caged” fluorescein: e.g. Jopling and den Hertog, 2005 Photo-activatable GFP Absorbance spectrum before PA Absorbance spectrum after PA Patterson and Lippincott-Schwartz, 2002 Can be hard to inject into single cells Get more control using laser activation Also, with PA-GFP don’t even have to inject PA GFP -wt EGFP actually does some photo conversion but still absorbs at 488 prior to PA (so high background) -found mutant with no absorbance at 488 prior to PA -after PA see 100 fold increase in fluorescence after excitation with 488

Lineage Analysis and Fate Mapping Approaches: Just watch closely Cell transplantation Use a Lineage Tracer -Inject into single cells or few cells -Activate in single cells or few cells 4) Genetic labeling -Random labeling -Labeling a specific lineage

Genetic Mosaics Genetic mosaics can be created by mitotic recombination induced by X-rays or a site-specific recombinase

Genetic Tracking of Specific Lineages Question: What do cells that express YFG at time X develop into? Actin Promoter STOP GFP loxP YFG CRE-ER Add tamoxofen at time X Cells expressing YFG at that time permanently labeled with GFP

Some things to worry about: -Can you identify and label your cells of interest? -Does your labeling technique interfere with normal development? -Is your lineage tracer stable over time? -Is your lineage tracer diluted by cell division?