Embryology 1 : The Genetics of Anterior-Posterior Axis Determination

1. Embryology 1 : The Genetics of Anterior-Posterior Axis Determination

2. Major Axes in Body Plan Development • Embryos develop three axes: the anterior-posterior axis (from head to tail), the dorsal-ventral axis (from back to belly) and the right-left axis • Structures show a specific localization (neural tube is a dorsal structure in the embryo and the heart is on the left side of an adult body) • The specification of axes occurs early during embryonic development

3. Major Axes in Body Plan Development • The specification of early embryonic cells is due to cytoplasmic determinants stored in the oocyte • The cell membranes that form during cleavage establish the region of cytoplasmic determinants that direct different gene expression in every blastomere

4. Drosophila as a Model for Molecular Embryology • Drosophila is easy to breed, cheap to rear in the lab • It is hardy and prolific (genetic studies are possible) • A genome-wide approach has identified the molecules for body plan development

5. A/P Polarity in the Adult ■ The body comprises three thoracic and eight abdominal segments ■ Every segment is specific and has different appendages: T1 has only legs, T2 legs and wings, T3 legs and halteres ■The body is organized accordingly to a “plan”

6. Drosophila Early Embryonic Development ■The zygotic nucleus divides several times without formation of cell membranes ■ The specification of the A/P and D/V axes is accomplished by interaction of molecules in the same single multinucleated cell

7. The Question How do we get from an homogenous egg to a body that is a collection of different segments?

8. General Principles • The determination of the A/P axis is due to a cascade of interacting genes • These genes act sequentially to first divide the embryo in several segments • Segments acquire then their specific identity

9. Cascade of Genes Determining A/P Axis Maternal genes Hunchback Gap genes Pair-rule Segment polarity Homeoticgenes

10. Bicoid and Hunchback at the Anterior Tip Ovary Embryo Bicoid mRNA Nurse Cells Oocyte Bicoid Protein A P In the mother ovary the bicoid mRNA produced by accessory cells, the nurse cells, is deposited in the oocyte and it is tethered to the anterior tip by microtubules The bicoid mRNA is translated into the corresponding protein which forms a gradient with the highest concentration at the anterior tip The same gradient is also created for hunchback, another anterior determinant

11. Nanos and Caudal at the Posterior End Nanos protein P A Similarly, nanos mRNA is given to the egg by the mother and it is bound to the posterior region of the unfertilized egg by interaction with the cytoskeleton A gradient for the corresponding protein is created as reported in the picture, with the highest concentration at the posterior end A similar gradient is also created for Caudal

12. Early A/P Determinants Four gradients are present: an A/P gradient for Bicoid and Hunchback and a P/A gradient for Nanos a Caudal All these proteins function as transcription factors that can activate the expression of the following genes in the cascade, the gap genes

13. Summary • Positional information has been generated: the presence of Bicoid and Hunchback “label” the anterior end, Nanos and Caudal the posterior one • Embryos defective (mutant) for Bicoid lack the anterior structures while embryos mutant for Nanos lack the posterior part

14. THE GAP GENES giant Kruppel Knirps ■The Gap Genes are expressed into broad regions of the embryo ■ Mutations in the gap genes produce embryos lacking a series of contiguous segments corresponding to the segments where the gap gene is expressed

15. The Question How do we get from an embryo with smooth gradient of proteins to an embryo characterized by proteins expressed in broad stripes?

16. Regulation of Gap Gene Expression I II Stripe I requires high level of Hb protein Stripe II requires high level of Caudal Kr requires low levels of Hb II Stripe II requires low levels of Caudal

17. The Pair-Rule Genes Even-skipped Stripe 2 The pair rule genes are expressed in a zebra-like pattern dividing the embryo in 15 sub-regions where a vertical band of nuclei express the gene and the next one does not The stripe 2 of eve is repressed by both high levels of giant and Kruppel

18. Regulation of eve stripe 2 The expression of eve stripe II is limited to the “valley” region between high levels of giant and Kruppel proteins since both are repressors Bicoid and Hunchback activates the expression of eve in the same stripe

19. We are half-the way done!

20. Drosophila Early Embryonic Development ■The zygotic nucleus divides several times without formation of cell membranes ■ The specification of the A/P and D/V axes is accomplished by interaction of molecules in the same single multinucleated cell

21. The Segment Polarity Genes ■ The segment polarity genes reinforce and maintain the periodicity generated by the pair-rule genes ■ They are expressed in 14 stripes along the A/P axis ■ Their expression is regulated by the pair-rule genes

22. Regulation of Segm-Polarity by Eve, Ftz Fushi-tarazu (Ftz) and even-skipped (eve) are expressed in close, non overlapping regions High levels of Ftz and Eve induce the expression of engrailed (en), a Segment-polarity gene expressed in 14 stripes Conversely Eve and Ftz inhibit wingless (wg). The expression of wg will be confined in stripes running between the expression of Ftz and Eve where there is no Ftz or Eve

23. Maintenance of en and wg Expression by Reciprocal Interaction Wg is a secreted protein. It binds to wg receptor on the adjacent en cells and it activates en. En is a transcription factor and it activates the expression of hedgehog (Hh). Hh is a secreted protein that binds to its own receptor on wg expressing cells and it activates the expression of wg. En and wg establish a polarity in every segment

25. Homeotic genes Colinearity between the position of the gene on the chromosome and the sequence of the segment they specify: Scr specifies T1 and precedes on the chromosome Antp that specifies T2 Every homeotic gene represses the expression of the previous gene in the segment they specify. Ubx identifies T3 and represses Antp in T3

26. When Something Goes Wrong… Normal fly head In Ubx mutants T3 becomes T2 Ectopic expression of Antp in the head induces the formation of legs attached to the head Extra legs

27. Mammals and Drosophila Every homeotic gene in Drosophila has its homolog in mammals (Scr corresponds to a5, b5 and c5 but the three genes in mammals have the same function as Scr in Drosophila) Colinearity is also seen in mammals (panel B)

28. When Something Goes Wrong… Thoracic vertebra Extra rib Lumbar vertebra The function of Homeotic genes in mammals is the same as in flies: theKO of Hoxc8 in mouse causes an homeotic transformation: the first lumbar vertebra becomes a rib. A rib is associated with the thoracic vertebra anterior to it

29. Retinoic acid as a teratogen • RA can become a teratogen if present in large amounts or at particular times during development • It is a secreted molecule involved in A/P axis formation in mammals and in forming the jaws • It affects Hox gene expression in A/P axis determination and it inhibits neural crest cell migration from the cranial region of the neural tube

30. The role of cell-adhesion molecules in creating boundaries between tissues

31. Cells do not sort randomly Cells from the neural plate and from the epidermis were dissociated in alkaline solution. When cells were mixed together, they re-aggregated and they became spatially segregated: the neural cells are inside while the epidermal cells stay at the periphery

32. How boundaries between tissues are established • Boundaries between tissues can be created by different cell types having both different types and different amounts of cell-adhesion molecules • The most common cell-adhesion molecules are cadherins • Cadherins establish and maintain intercellular connections and they are crucial in the spatial segregation of cell types

33. Cadherins Cadherins are Ca-dependent adhesion molecules They are anchored to the cell by a complex of proteins called catenins Catenins interact with actin cytoskeleton They have an adhesive recognition site to bind to similar cadherins Cadherins join cells together by binding to the same type of cadherins on another cell (homophilic interaction)

34. Cadherins Cells with E-cadherins (Epidermal cells) stick together and they will sort out from cells containing N-cadherins (Neural Cells) in their membranes

35. Cadherins during gastrulation During gastrulation the presumptive neural tube expresses N-cadherins while the presumptive epidermis expresses E-cadherins These tissues separate: the cells expressing N-cadherins invaginate to form the neural tube while the cells expressing E-cadherins will form the epidermis If the epidermis is experimentally manipulated to inactivate the E-cadherins the cells will not hold together. If the N-cadherins are inactivated in the cells of the presumptive neural tube, the neural tube will not form

36. The Extracellular Matrix as a Source of Developmental Signals • EM consists of macromolecules secreted by cells into their immediate environment • Cell adhesion and cell migration is mediated by the EM • EM is made up of proteoglycans, collagen and specialized glycoproteins such as fibronectin and laminin

37. Fibronectin • FN functions as an adhesion molecule linking cells to one another or to other molecules (collagen and proteoglycans) • FN has an important role in cell migration • The roads over which migrating cells travel are paved with FN • FN leads germs cells to the gonads and heart cells to the midline of the embryo • Cells expressing integrins can bind adhesion molecules

38. INTEGRINS On the extracellular side integrins bind to the sequence Arg-Gly-Asp found in adhesion molecules including fibronectins On the intracellular side they bind Vinculin and a-Actinin, these proteins bind to Actin filaments This dual binding allow cells to move by contracting Actin filaments against the EM