Lecture 3 De Robertis August 17, 2011

1. Lecture 3 De Robertis August 17, 2011 MOLECULAR REGULATION OF DEVELOPMENT GROWTH FACTOR SIGNALING, HOX GENES AND THE BODY PLAN

2. Two parts: • Pattern formation by morphogen gradients of growth factors and the dorsal-ventral (D-V) axis. BMP receptors. B) Hox genes and the antero-posterior (A-P) axis. - Colinearity - Activation by retinoic acid. Retinoid receptors. - Hox genes in Evolution and Development

3. During development groups of inducing cells called organizing centers secrete graded growth factor signals. The concentration gradient of a “morphogen” can induce multiple cell differentiation choices. Fig. 1

4. D-V patterning can be studied in the frog embryo. Fig. 2

5. The best example of a morphogen is the gradient of BMP signaling that controls D-V tissue differentiation. Fig. 3a

6. Mesoderm cell differentiation is determined by a gradient of BMPs BMP signaling ventral dorsal Lateral plate Somite Notochord Fig. 3b Bone Morphogenetic Proteins are growth factors discovered here at UCLA by Dr. Marshall Urist

7. Genes specifically expressed in the dorsal blastopore lip (Spemann organizer) of the gastrula were cloned. Organizer-specific Genes Fig. 4a

8. Chordin mRNA is expressed in Spemann’s organizer. Chordin protein is secreted and diffuses in the embryo. Fig. 4b

9. Chordin is an antagonist that binds BMP growth factors in the extracellular space, inhibiting binding to cell surface receptors. Chordin establishes a BMP4 activity gradient at gastrula. Another protein, Noggin, has similar activity. Secreted antagonists are used in development to generate morphogen gradients. Chordin inhibits Fig. 5

10. Signal transduction: membrane receptors transduce the signal so that transcription factors are activated through phosphorylation. TGFβ family members (30 different ligands in humans) activate cell surface receptors (serine-threonine kinases). Xnr BMP4 I will try to show a movie of this. No need to remember any details; it is just to illustrate that activated cell membrane receptors can cause changes in gene expression. Fig. 6a

11. Fig. 6b

12. Visualizing a morphogen gradient: phosphorylated Smad1 forms a gradient, maximal in the ventral, in the Xenopus gastrula Dorsal Ventral Blastopore Transverse section at the level of white arrows Side view Fig. 7a

13. Epidermis CNS Dorsal Ventral Spemann’s Organizer Mesoderm Endoderm At gastrula a gradient of BMP4 is established by a ventral source of BMP4 and a dorsal source of Chordin and Noggin, two BMP antagonists secreted by the dorsal organizing center. Fig. 7b

14. The BMP gradient induces different tissues in mesoderm and ectoderm (because the DNA-binding partners are different) Ectoderm differentiation Mesoderm differentiation BMP signaling BMP signaling Neural crest Lateral plate Somite Notochord Epidermis CNS Fig. 7c

15. Conclusion; a morphogen gradient can be generated by a source of growth factor (such as BMP) or by a localized source of inhibitor (such as Chordin). Both mechanisms are used. This is how organizing centers work in embryonic induction. Fig. 8

16. Cell-cell communication is controlled by surprisingly few signal transduction pathways: • TGFβ/BMP Serine/Threonine kinase receptors • Receptor Tyrosine kinases such as FGF, EGF, IGF, Insulin • Wnts • Sonic Hedgehog • Notch • G protein-coupled receptors (7-transmembrane receptors) • Nuclear hormone receptors Only a few signaling pathways pattern the embryo, but there are hundreds of differentiated cell types in the human body. The same signals can trigger different types of cell differentiation responses in cells of different developmental history. Fig. 9

17. A-P patterning outline: 2a) Hox genes: colinearity between the body plan and gene order in genomic DNA 2b) Hox genes and Retinoic acid 2c) Hox genes in Evolution and Development (Evo-Devo) Fig. 10

18. 2) Hox genes and the development of body plans Homeotic transformations in humans. A cervical vertebra transformed into a thoracic one with ribs. Fig. 11

19. Homeotic Mutations – the Homeobox story Fig. 12

20. Edward B. Lewis Homeotic genes specify body segment identity in Drosophila. Edward Lewis predicted Hox genes would be duplicated. Fig. 13

21. Homeobox refers to nucleic acid. Homeodomain refers to protein. The homeodomain is a 60 aa helix-turn-helix DNA-binding domain that is very conserved during evolution. It fits into the major groove of the DNA. Define Hox, homeobox The term homeobox is reserved for the nucleic acid sequences that encode homeodomains. Since they are highly conserved, and can be detected by low-stringency hybridization across species. Fig. 14

22. Hox complexes are conserved between Drosophila and mammals (from De Robertis et al., Scientific American, 1990) Fig. 15

23. Vertebrates have four Hox complexes, with about 10 genes each. • They display colinearity: • Temporal colinearity: genes on one end of the complex are expressed first, those on the other (posterior) end are turned on last. • Spatial colinearity: the more anteriorly expressed genes are in one end, the more posterior ones at the other end of the gene complex. • Anterior Hox genes are activated sequentially by retinoic acid. • Hox genes can be aligned in 13 groups of paralogues. Fig. 16

24. Extensive conservations between Drosophila and the four human Hox complexes High RA response Low RA response From De Robertis, E.M. Evo-Devo: Variations on Ancestral themes. Cell 132, 185-195 (2008) Fig. 17

25. Hox-C6 protein is seen in eight thoracic segments of the mouse embryo. Translation is blocked in the tail region, probably through the action of microRNAs. The inset shows that Hox-C6 mRNA is expressed all the way to the tip of the tail. From De Robertis, Cell 132, 185-195 (2008) Fig. 18

26. Temporal and spatial colinearity: order of Hox genes in DNA follows the antero-posterior body axis. Why have Hox genes stayed together in a complex? Fig. 19

27. Hox knockouts in mice cause homeotic transformations, in this case an extra rib in the lumbar region (HoxC-8 mutant). Treatment with retinoic acid can also cause lumbar ribs. Your patient this week has 13 ribs. Fig. 20

28. 2b) Retinoic acid activates HOX genes sequentially in cultured human teratocarcinoma cells Fig. 21

29. nuclear receptor structure/function LBD DBD Dimerization corepressor ligand coactivator Fig. 22

30. Retinoic acid receptor is a DNA-binding protein that works as a ligand-activated transcription factor. Many hydrophobic hormone receptors work in this way. Nuclear receptors work very differently from cell surface receptors. (RA) RA Fig. 23

31. Hox complexes have a retinoic acid receptor response element (RARE) in the DNA before paralogue 1. This DNA enhancer element controls expression of many genes in the complex. In retinoic acid teratogenesis, Hox gene expression borders move into more anterior regions. RARE Fig. 24

32. Pharyngeal arch 1 does not express any Hox gene. It gives rise to maxillary and mandibular structures. Retinoic acid can cause cleft palate and micrognathia Fig. 25

33. Urbilateria used Hox genes and Chordin/BMP to pattern the embryo. Developmental control genes placed evolutionary constraints on the types of animal shapes that evolved by Natural Selection. Fig. 26 From: De Robertis, E.M., Evo-Devo: Variations on Ancestral themes.Cell 132, 185-195 (2008)

34. Evo-Devo: Urbilateria had a Hox gene complex. Developmental constraints channeled evolution. High RA response Low RA response Fig. 27

35. Overview Langman’s Medical Embryology Fig. 28