2. Chapter 8 - Early Development in invertebrates The next chapters examine early development in several models, including invertebrates (Ch. 8-9) amphibians (ch 10) and then vertebrates (ch. 11) 1 frog egg becomes 37,000 cells in 43 hours! Fig. 8.1

3. 1.Cleavage- One cell is subdivided into many cells to form a blastula 2. Gastrulation- Extensive cell rearrangement to form endo-, ecto- and meso-derm 3. Organogenesis- Cells rearranged to produce organs and tissue 4. Gameteogenesis- produce germ cells (sperm/egg) Note: Somatic cells denote all non-germ cells General Animal Development Recall from lecture 1

4. How does egg undergo cleavage without increasing it’s size? Answer- it abolishes G1 and G2 phases of cell cycle Do you need a cell cycle primer?? Four cell cycle phases M- mitosis G1- Gap 1 S- DNA Synthesis G2- Gap 2

5. Reminder- mitosis occurs in M phase, DNA replication in interphase From Mol. Biol of the Cell by Alberts et al, p864

6. Cyclin dependent kinases (cdks, cdcs) drive the cell cycle Cyclins (e.g cycli A, B…) regulate cdk (cdc) activity Mitosis Promoting Factor (MPF= cyclin B+cdc2) Example- MPF

7. How does egg undergo cleavage without increasing it’s size? Answer- it abolishes G1 and G2 phases of cell cycle Cyclins are synthesized in eggs Cyclin is degraded S phase M phase

8. What actually drives the cleavage process? Answer- Two processes- 1. Karyokinesis (mitotic division of the nucleus) The mitotic spindle (microtubules composed of tubulin) does this 2. Cytokinesis (mitotic division of the cell) Contractile rings “pinch off” (microfilaments composed of actin) Fig. 8.3 Cytochalasin B prevents cytokineses

9. 1.Cleavage- One cell is subdivided into many cells to form a blastula 2. Gastrulation- Extensive cell rearrangement to form endo-, ecto- and meso-derm 3. Organogenesis- Cells rearranged to produce organs and tissue 4. Gameteogenesis- produce germ cells (sperm/egg) Note: Somatic cells denote all non-germ cells General Animal Development Recall from lecture 1

10. Gastrulation- cells of blastula are dramatically rearranged Three germ layers are produced Five types of movements 12 3

11. Gastrulation Five types of movements 45

12. Axis formation Three axes must be determined- Anterior-posterior (front-back) Dorsal-ventral (back-belly) Right-left (right side-left side) Fig. 8.7

13. Now let’s take a look at one beast- the sea urchin Cleavages 1 and 2 are through animal/vegetal poles Cleavage 3 results in four vegetal and four animal cells 12 3 Fig. 8.8 Cleavage 4 results in four macromeres and and four micromeres only in vegetal cells 4 1. Cleavage

14. Post cleavage 5Fig. 8.9 Cell fate map

15. Micromeres signal other cells via  -catenin to influence fate Micromere cell fate is autonomous- these become skeletal tissue if placed in a dish All other cells have conditional specification Example- Transplant micromeres to animal pole at 16 cell stage Micromeres cause a second invagination Animal pole cells become vegetal cells Fig. 8.13

16. Sea urchin (continued) Egg Late blastula Gastrula Later stages Fig. 8.16 Sea urchin development 2. Gastrulation Note-micromemeres produce primary mesenchyme which will become larval skeleton

17. How do mesenchyme cells know to migrate inside blastocoel? Answer- changing cell attachment proteins Hyaline layer Extracellular matrix Basal lamina Primary mesenchyme cell- Blastocoel 98% decrease in hyaline affinity 100-fold increase in EM/basal lamina affinity Fig. 8.19

18. How does invagination occur? Terms- Invagination region is called archenteron Opening created is called the blastopore Answer- swelling of inner lamina layer Inner layer Outer layer Vegetal cells secrete chondroitin sulfate proteoglycan, causing inner layer to swell and cause buckling Hyaline

19. Now let’s take a look at another creature- the nematode C. elegans 959 cells at maturity 1mm long Produces eggs and sperm (hermaphrodyte) Transparent 16 hour from egg to hatch Entire genome sequences- 19,000 genes What a great model! 1mm

20. Mature eggs passes through the sperm on the way to the vulva Germ cells undergo mitosis, then begin meiosis as travel down oviduct Oviduct Cleavage C. elegans 1. Cleavage

21. Cleavages 1 produces founder cell (AB) and stem cell (P1) Cleavage 2 results in three founder cells and one stem cell (P2) 12 3 Remaining cleavages result in a single stem cell and more founder cells Fig. 8.42 C. elegans

22. How is the anterior- posterior axis determined? Answer- P-granules- ribonucleoprotein complexes P-granules always stay associated with the “P” cell Fig. 8.43 C. elegans 1 3 5 PAR proteins- these specify polarity, cell division and cytoplasmic localization What directs the P granules?

23. C. elegans 1. Cleavage P1 develops autonomously AB does not (thus is conditional) What drives P1 lineage? P granules? No, these do not enter nucleus! Other possibilities SKN-1- a bZIP family transcription factor that control EMS cell fate PAL-1 - a transcription factor required for P2 lineage PIE-1- inhibits SKN-1 and PAL-1 in P2

24. Yes- P2 produces a signal that tells ABp to only neurons and hypodermal cells not neurons or pharynx like ABa GLP-1 is the receptor on ABp, and APX-1 is the ligand on P2 GLP-1 is a Notch family protein APX-1 is a Delta family protein Does P2 dictate fate of adjacent cells?

25. Chapter 9 - Axis specification in Drosophila Drosophila genetics is the groundwork for developmental genetics Cheap, easy to breed and maintain Drosophila geneticists take pride in being different and in sharing information Problems- fairly complex, non-transparent Fig. 8.1

26. Insects tend to undergo superficial cleavage- cleavage occurs at rim of the egg In contrast to other creatures, insects form nuclei, then create cells Fig. 9.1 Drosophila 1. Cleavage 17 10 Mitotic divisions #1-#9 - duplicate nuclei (8 min/division Mitotic division #10 – nuclei migrate to rim Termed a syncytial blastoderm Mitotic division #11-14 – progressively slower divisions

27. Fig. 9.3 Drosophila 1. Cleavage 14 Mitotic divisions #14 – cells created with each nuclei to create the cellular blastoderm Note – each nuclei has a territory of cytoskeletal proteins Nuclei staun Tubulin stain Egg plasma membrane folds between nuclei to create individual cells Cycle 11-14- midblastula transition- nuclear division slows and transcription increases

28. 2. Gastrulation VentralDorsal Ventral furrow (from mesoderm) It becomes the ventral tube Segments 3 thorax 8 abdominal Head Fig. 9.6 Fig. 9.7

30. 2. Gastrulation Establishment of anterior-posterior polarity-protein gradients rule the day Maternal effect- in specific region of egg Gap- among 1 st gene transcribed in embryo Pair rule – result in 7 bands Segment polarity – result in 14 segments Gene family Fig. 9.8 Examples bicoid Nanos caudel fushi tarazu hairy Kruppel hunchback engrailed wingless

31. 2. Gastrulation Active during creation of syncytial blastoderm Fig. 9.10 Bicoid mRNA injected in anterior nanos mRNA injected, localize to posterior Hunchback (diffuse) Caudel (diffuse) Bicoid prevents caudel mRNA translation Nanos prevents hunchback mRNA translation Maternal effect genes

32. Oocyte mRNAs Syncytial Blastoderm proteins Mechanism Anterior Posterior Maternal effect genes Fig. 9.11

33. What if we mess up the bicoid gradient? Bicoid - mutant Wild- type Two tails Inject bicoid into: Anterior Wild-type Bicoid -/- MiddlePosterior Normal Head in middle Two heads Thus, bicoid specifies head development Maternal effect genes Fig. 9.14

34. How does nanos specify posterior? Answer- By preventing hunchback translation Anterior (no nanos) Mechanism In anterior, Pumilio binds 3’UTR (untranslated region) of hunchback mRNA, and mRNA is polyadenylated and translated In posterior, nanos prevents polyadenyltation, and thus prevents translation Posterior (with nanos) Fig. 9.16

35. 2. Gastrulation Segmentation genes Two steps in Drosophila development Bicoid, nanos, hunchback, caudel, etc. Determination genes Segmentation genes Gap genes Pair-rule genes Segment polarity genes SpecificationDeterminationEgg (Cell fate is flexible)(Cell fate is determined) Maternal effect genes activate gaps genes, which activate pair-rule genes, which activate segment polarity genes Segmentation genes establish boundaries GapPair-ruleSegment polarity Fig. 9.19

36. a. Gap Genes Gap genes respond to maternal effect proteins Gap proteins interact to define specific regions of embryo Four major gap proteins- hunchback, giant, Kruppel and knirp These are all DNA binding proteins- activate or repress transcription Fig. 9.21 b. Pair-rule genes Gap genes activate and repress pair-rule genes in every other stripe, resulting in seven stripes Three major pair rule proteins- hairy, evenskipped, runt These are all DNA binding proteins- activate or repress transcription Cells in each parasegment contains a unique set of pair rule genes expression unlike any other parasegment Gap Pair-rule

37. Why do we observe expression of pair-rule proteins in every other segment? Answer- pair-rule genes use different enhancer elements Example- even-skipped (a pair-rule gene) has several enhancers, but only one is active in a given stripe Fig. 9.22 This enhancer is only active in stripes #1 b. Pair-rule genes Different concentrations of gap proteins determine pair- rule gene expression

38. c. Segment polarity genes Pair-rule Segment polarity Segment polarity genes act once cells are formed syncytial blastoderm Maternal, gap and pair-rule genes operate before cells are formed 14 Fig. 9.1

39. c. Segment polarity genes Segment polarity genes encode proteins that make up Hedgehog and Wingless signal transduction pathways One cell produces wingless The adjacent cell produces hedgehog Wingless and hedgehog activate expression of each other Fig. 9.25

40. 2. Gastrulation Homeotic selector genes Responsible for directing structure formation of each segment These genes are clustered on chromosome 3 in the homeotic complex (also called Hom-C) in two regions- The Antennapedia complex- The bithorax complex- Chromosome 3 1. The order of these genes on the chromosome matches order of segmental expression 2. Homeotic genes are regulated by all gene products expressed posterior to it Two amazing features

41. What about dorsal ventral polarity?? This occurs after cells are created (post syncytial blastoderm) Dorsal (a transcription factor) gradient is established Dorsal is found throughout syncytial blastoderm, but only in nuclei of ventral cells How does this occur? By a very complex pathway involving gurkin and torpedo proteins ( and a host of other proteins) Organs form at the intersection of dorsal-ventral and anterior-posterior regions of gene expression