Unit 13a Amphibian Axis Specification The African Clawed Frog Xenopus laevis Gilbert 10e Fig 8 1 Frog axis specification Early events Spemann The Organizer The organizer forms on the dorsal side of the embryo Its cells come to lie under the future ectoderm It is capable of inducing cells around it to become neural ectoderm Gilbert 7e Fig 10 21 Spemann The Organizer The organizer is crucial to axis specification Key questions 1 What early events are required 2 What molecular signals are responsible for its activities 3 How do the cells of the organizer contribute to gastrulation Gilbert 7e Fig 10 21 The gray crescent is visible after fertilization in some one cell frog zygotes The gray crescent is visible in some frog species but not Xenopus It forms after fertilization due to reorganization of the one cell zygote Gilbert 10e Fig 8 2 Spemann the gray crescent is associated with the ability to form an axis Tissue incapable of forming axial structures forms ventral mesoderm and endoderm called a belly piece German Bauchst ck Conclusion the gray crescent material or something associated with it seems to be crucial for formation of axial structures Gilbert 10e Fig 8 16 The gray crescent forms via cortical rotation Future dorsal side The sperm can enter anywhere in the animal hemisphere The sperm centrosome organizes microtubules that participate in cortical rotation The cortex rotates 30 The side opposite the site of sperm entry will be the dorsal side of the later embryo It is the side where the gray crescent forms in species in which it is visible Gilbert 10e Fig 8 2 Cortical rotation spot movement predicts where the dorsal side will form In Xenopus which has no visible gray crescent Nile blue can be applied to the yolk beneath the cortex the movement can be used to reliably predict where the dorsal side will later form Courtesy Jean Paul Vincent Cortical rotation can be induced to rescue UVtreated Xenopus embryos UV treatment destroys rotation machinery UV irradiation destroys the rotation machinery embedding zygotes in wax and tipping them allows rotation to be imposed UV Interior rotates Kalthoff 1e Fig 9 10 Egg tipping rescues development Wax holds zygote Future dorsal side Cortical rotation is necessary for axis formation Conclusion cortical rotation is necessary for formation of axial structures Adapted from Browder et al 2e Fig 6 5b Microtubules align at the time of cortical rotation Question how could we test whether microtubules are necessary for cortical rotation Gilbert 10e Fig 8 2 Cortical rotation in Xenopus microtubules Nocodazole belly piece D2O excessive head structures Conclusion microtubules promote cortical rotation but excess stability of MTs leads to an excess of anterior structures Cortical rotation in Xenopus frogs in space Conclusion gravity can be used as a tool to impose rotation but normal rotation is a gravity independent process driven by microtubule based movement Animal and vegetal cells are visibly different animal pole Cells that are obviously different in size often differ in terms of their specification How can we show this vegetal pole From Egg to Tadpole Dorsal vegetal cell transplants can rescue UV irradiated embryos Conclusion Dorsal vegetal cells appear to be capable of inducing a functional organizer in embryos that did not undergo cortical rotation This suggests cortical rotation is important for dorsal vegetal cell differentiation Gilbert 8e Fig 10 21 see 10e Fig 8 20 Dorsal vegetal cell transplants can lead to a second axis Conclusion Dorsal vegetal cells appear to be capable of inducing a second organizer in normal embryos This suggests these cells are normally important for inducing an Organizer Gilbert 8e Fig 10 21 see 10e Fig 8 20 Dorsal endoderm can induce dorsal mesoderm Nieuwkoop marked the dorsal side of the endoderm The D V character of induced mesoderm was determined by D V character of endoderm Gilbert 10e Fig 8 19 The Nieuwkoop Center induces the organizer Gilbert 10e Fig 8 19 Conclusions 1 There is a signaling center in the future dorsal endoderm that can induce an organizer This is called the Nieuwkoop Center in honor of Nieuwkoop 2 Other future endoderm can induce other types of mesoderm Cortical Rotation and Dorsal Specification Gilbert 10e Fig 8 22 Dsh GSK 3 binding protein GBP inhibits GSK 3b a component of the destruction complex and Wnt 11 mRNA are displaced dorsally Cortical Rotation and Dorsal Specification Release of Dsh and GSK 3 binding protein GBP inhibits GSK 3b a component of the destruction complex leads to accumulation of bcatenin dorsally Gilbert 10e Fig 8 22 catenin is enriched dorsally catenin V D D V Conclusion b catenin is more abundant on the dorsal side after cortical rotation Gilbert 10e Fig 8 21 D catenin is in nuclei on the dorsal side Dorsal catenin nuclear Ventral catenin non nuclear Gilbert 10e Fig 8 21 Conclusion b catenin is in nuclei on the dorsal side after cortical rotation where it is in a position to alter gene expression catenin is sufficient to cause a Nieuwkoop Center to form Inject catenin mRNA into ventral vegetal cell Duplicated axes result animal dorsal ventral vegetal Wolpert 2e Fig 3 8 Conclusion b catenin is sufficient for formation of the Niewukoop Center and later an Organizer catenin is sufficient to cause a Nieuwkoop Center to form Conclusion b catenin is sufficient for formation of the Niewukoop Center and later an Organizer Gilbert 9e Fig 7 21 Depleting catenin ventralizes embryos Control MO Conclusion b catenin is ultimately necessary for formation of the Niewukoop Center and Organizer courtesy Chris Wylie Dorsal depletion of catenin ventralizes embryos inject morpholino into two dorsal cells inject morpholino into two ventral cells Conclusion b catenin is needed specifically in dorsal cells for formation of the Niewukoop Center and Organizer courtesy Chris Wylie XWnt11 may be required for dorsal specification Xwnt11 antisense oligo knockdown control Conclusion Wnt11 may be important maternally for formation of dorsal structures Adapted from Tao et al 2005 Cell 120 857 871 catenin leads to formation of the Nieuwkoop Center b catenin Tcf leads to expression of siamois and twin transcription factors expressed by Nieuwkoop Center cells adapted from Gilbert 10e Fig 8 22 siamois twin genes Siamois Twin proteins Siamois overexpression leads to a second axis secondary axis Conclusion either siamois or twin is sufficient to cause cells to differentiate as Nieuwkoop Center cells