Axis Formation and Mesoderm Induction

Transcription

1 Developmental Biology Biology 4361 Axis Formation and Mesoderm Induction October 27, 2005

2 Amphibian anteroposterior specification polarized eggs animal/vegetal pigment yolk v. clear cytoplasm mitochondrial cloud germinal vesicle localized cytoplasmic components

3 RNA localization Xenopus oocytes Figure 8.25

4 Anteroposterior axis VegT depletion normal Figure 9.7

5 Anteroposterior axis VegT depletion normal VegT depleted Figure 9.7 depletion of VegT = shift from endoderm to mesoderm and ectoderm mesoderm replaced with ectoderm animal region forms only epidermis and no nervous system

6 Dorsalization Xenopus UV = ventralized Figure 9.18

7 Transplantation of dorsalizing activity Figure 9.15

8 Figure 9.19 Early Dorsoventral Determination

9 Gray crescent formation

10 Cortical rotation and Disheveled 1. Fertilization sperm Disheveled protein (Dsh) 2. Cortical rotation Dsh 3. Dorsal enrichment of Dsh

11 Disheveled activity gylcogen synthase kinase 3 Figure 9.21

12 Disheveled activity gylcogen synthase kinase 3 Disheveled protein Transcription factor blocks GSK 3 phosphorylation of β catenin Figure 9.21

13 Molecular basis of dorsoventral axis β catenin degraded β catenin stabilized Tcf 3 proteins siamois gene β catenin proteins siamois gene Repressed TGF b signaling pathway Activated transcription Siamois protein goosecoid gene transcription Goosecoid protein

14 Organizer transplant Spemann s organizer dorsal lip of the blastopore

15 Organizer transplant

16 Organizer proteins expressed almost exclusively in the organizer Nuclear Proteins XLim1 Xnot Otx2 XFD1 XANF1 Goosecoid Secreted Proteins chordin noggin nodal related proteins (several) Cerberus Follistatin Frzb Gilbert: Developmental Biology, 7 th ed (2003) Table 10.2.

17 Organizer gene activity goosecoid mrna can induce a second dorsal axis: goosecoid mrna injection causes formation of a second dorsal blastopore lip produces embryo with two dorsal axes and two sets of head structures Gilbert: Developmental Biology, 7 th ed (2003) Fig

18 Organizer gene activity Rescue of dorsal structures by noggin protein: ventralized embryo without dorsal structures (UV irradiated) dose dependent induction of dorsal structures by injection of noggin mrna overdose of noggin mrna causes formation of dorsal structures at the expense of ventral structures noggin binds to bone morphogenic proteins (BMP2 & BMP4) inhibits binding BMP receptor binding Gilbert: Developmental Biology, 7 th ed (2003) Fig

19 Organizer gene activity chordin mrna is localized in the organizer : chordin mrna is found in the dorsal lip late in gastrulation, chordin is localized in the dorsal mesoderm of the notochord chordin protein binds to BMP4 and BMP2 inhibits receptor BMP receptor binding inhibition of BMP4 & BMP2 induces formation of the neural tube in adjacent ectoderm Gilbert: Developmental Biology, 7 th ed (2003) Fig

20 Figure 9.8 Mesoderm induction Xenopus

21 Figure 9.8 Mesoderm induction Xenopus

22 Figure 9.8 Mesoderm induction Xenopus

23 Mesoderm induction Xenopus mesoderm inducers: Vg1 bfgf Figure 9.9a, b activin

24 Mesoderm induction, Organizer formation β catenin Nodal related high BMP4 low Organizer VegT, Vg1 Nodal related low BMP4 high Ventral mesoderm 1. β catenin acts with VegT and Vg1 to activate Xnr genes (Xenopus Nodal related) 2. Organizer originates in the region where VegT & Vg1 and β catenin overlap 3. Gradient of Xnr protein specifies mesoderm: low Xnr ventral mesoderm 4. High Xnr levels activate goosecoid and other organizer genes

25 Left right asymmetry Most animals are bilaterally symmetrical ( Bilateria) however, individuals deviate to some degree from true bilateral symmetry: fluctuating asymmetry: non heritable minor left right differences antisymmetry: heritable morphological left right differences sidedness is randomly distributed (ca. 50% each) regular asymmetry or directed asymmetry: sidedness is fixed for a species or for a higher taxon e.g. in humans: heart on left side stomach curves to the left liver & spleen on right side

26 Left right asymmetry Deviation from directed asymmetry is often lethal! situs inversus: complete reversal of left right symmetry in all organs heterotaxis: some organs reversed isomerism: normally asymmetrical organs duplicated or missing

27 Left Right Asymmetry Mechanistic basis for establishing asymmetry: chiral molecules may cause symmetry breaking event (specific orientation of stereoisomeric molecules relative to the body axes) translated into left right differences at the level of cells, tissues and the whole organism Candidate chiral molecule: Dynein motor protein complex associated with axonemes, cilia

28 Left Right Asymmetry Dyneins microtubule associated motor protein complexes Axonemal dyneins: Fig 2.7. chiral: curve clockwise (from base) = handedness mediate sliding between adjacent microtubules in cilia or flagella cause cilia to rotate in a specific direction (clockwise) monocilia (at Hensen s node mouse) generate oriented flow of signal molecules to the left side of the embryo signal molecules activate or inhibit patterning genes on left side

29 Iv + and Inv + iv+: situs inversus viscerum iv protein is a left right dynein iv /iv = no motility, no fluid flow randomized L R asymmetry (lethal) inv+: inversion of embryonic turning wild type & heterozygous embryos turn clockwise inv /inv turn counterclockwise in amniotic cavity 100 % of homozygotes for inv show situs inversus mechanism of inv action is unknown

30 Nodal Nodal activated by iv,inv intracellular protein TGF β family nodal gene activated by iv and inv genes nodal protein synthesized in left lateral plate mesoderm mesoderm adjacent to nodal expression develops into asymmetrical organs ectopic expression of nodal on right side randomizes location of the heart nodal is involved in determining left right asymmetry in mice, frogs, chicken & zebrafish nodal expression in mouse: wild type ectopic

31 Pitx2 & lefty activated by iv, ivn, nodal pitx2+ and lefty+ genes : pitx2 expression depends on iv, ivn and nodal genes pitx2 and lefty encode homeobox transcription factors that regulate genes both are expressed primarily on left side of vertebrate embryos have been found in all vertebrates studied injection of ptx2 on right side of embryo can cause a complete reversal of gut coiling and heart looping nodal, pitx2 and lefty form an evolutionary conserved signaling system that is involved in regulating left right asymmetry in all vertebrates pitx2 injection in Xenopus: