1. Early Development of Vertebrates
Fish, Birds, and Mammals

2. Zebrafish Development

3. Cleavage in Zebrafish What kind of cleavage occurs in fish?
discoidal meroblastic cleavage; first 12 divisions are synchronous; cells are initially connected to each other and to yolk

4. Cleavage in Zebrafish
At the time of the mid-blastula transition three cell populations form:
yolk syncytial layer (YSL)
enveloping layer (EVL)
deep cells
What is the importance of each?
YSL – directs some cell movements;formed at 9-10 cell cycle
EVL – becomes extraembryonic periderm for protection
deep cells – embryo proper
fate of cells determined shortly before gastrulation

5. Gatrulation in Fish How does gastrulation begin?
epiboly of blastoderm over yolk – caused by expansion of YSL and dragging of attached EVL – dependent upon microtubules in YSL; deep cells move outward and intercalate with superficial cells
one side thickens – dorsal

6. Gastrulation in Fish What is the germ ring in the developing embryo?
How does hypoblast form?
thickening composed of two layers – epiblast and hypoblast
hypoblast may form from involution of superficial cells or by ingression of epiblast cells
embryonic shield – intercalation of epi and hypoblast cells on dorsal surface – equivalent to dorsal lip

7. Gastrulation in Fish What is the role of convergence and extension?
brings hypoblast and epiblast cells to dorsal side to form embryonic shield (likened to dorsal lip)
intercalation extends chordamesoderm toward animal pole
epiblast gives rise to ectoderm while hypoblast gives rise to mesoderm and endoderm

8. Axis Formation in Fish
How has it been demonstrated that the embryonic shield is critical to dorsal-ventral axis formation?
What structures form from it?
notocord and prechordal plate

9. Axis Formation in Fish
How does notocord induce neural structures from overlying ectoderm?
secretes Chordino which binds BMP2B blocking ventral and lateral fates

10. Axis Formation in Fish
Where is the Nieuwkoop center and how does it function?
dorsal part of yolk cell – accumulates B-catenin – activates embryonic shield (organizer)
B-catenin of embryonic shield combines with zebrafish version of Tcf3 to become transcription factor that activates genes making squint and bozozok. Squint is nodal-like paracrine factor while bozozok is similar to siamois
nodal-like signaling also important for endoderm formation
B-catenin activation of stat3 coordinates movements of mesoderm & endoderm during gastrulation
wnt proteins
What determines the anterior-posterior axis?

11. Cleavage in Birds What kind of cleavage occurs in birds?
discoidal meroblastic

12. Cleavage in Birds
What is the area pellucida, area opaca and subgerminal cavity?
pellucida – one-cell thick layer in center of blastoderm
opaca – peripheral ring of blastoderm cells that contain deep cells
subgerminal cavity – fluid-filled space between blastodisc and yolk

13. Gastrulation in Birds How is the two-layered blastoderm formed?
ingression of epiblast cells in to subgerminal cavity and migration of marginal zone cells form hypoblast
embryo comes from epiblast; hypoblast forms some parts of extraembryonic membranes (yolk sac)

14. Gastrulation in Birds What is the primitive streak?
seen in birds reptiles and mammals
establishes axes of embryo
cells ingress through primitive streak into blastocoel

15. Gastrulation in Birds What is important about the primitive groove?
the primitive knot or Henson’ node?
How does the primitive streak define the embryonic axes?
depression in streak; groove – migrating cells enter here; node – similar to dorsal lip and embryonic shield
extends posterior to anterior; cells move from dorsal to ventral side and is dividing line for left-right

16. Gastrulation in Birds
What is the fate of cells migrating through the primitive streak?
first cells through Henson’s node migrate anteriorly – pharyngeal endoderm – displace hypoblast cells
hypoblast becomes confined to anteriormost part of area pellucida (germinal crescent)
next cells move anteriorly but not as ventrally – head mesenchyme, prechordal plate mesoderm – will form head process
then cells form chordamesoderm
cells passing through more posterior parts of streak –endoderm & extra-embryonic membranes and mesoderm

17. Gastrulation in Birds What happens to the primitive streak?
regresses; Henson’s node move posteriorly- formation of notocord
anterior end of embryo develops faster than posterior

18. Gastrulation in Birds
What is needed to complete the process of gastrulation?
epiboly of ectoderm – yolk becomes enclosed by ectoderm

19. Axis Formation in Birds
How does the pH influence the formation of the dorsal-ventral axis?
How do we know this is true?
above blastoderm – pH is basic from albumin; acidic in subgerminal cavity below
get transport of water and sodium ions from albumin through cells to subgerminal cavity creating a potential difference of 25 mv across epiblast layer – positive at ventral surface and negative at dorsal surface
reverse pH – reverse axis

20. Axis Formation in Birds
How goes gravity influence formation of anterior –posterior axis?
egg spinning in reproductive tract shifts yolk components and tips up blastoderm – posterior marginal zone (where gastrulation is initiated) equivalent of Nieuwkoop center

21. Axis Formation in Birds
How does formation of Nieuwkoop center in birds compare with amphibians?
forms when B-catenin and Vg1 converge – gravity restricts Vg1 cells to posterior region of embryo

22. Axis Formation in Birds
From where does the “organizer” in the chick form?
anterior part of Koller’s sickle
posterior part is Nieuwkoop center
genes expressed in posterior part of Koller’s – Vg1, nodal
genes expressed in anterior part of streak and Henson’s node – chordin, sonic hedgehog (antagonize BMP’s)
to get neural induction must not only block BMP but also have FGF’s

23. Axis Formation in Birds
How is left-right axis formed?
How has this been demonstrated experimentally?
block activin with follistatin – heart can be on either side, etc.

24. Early Mammalian Development
What makes mammalian cleavage unique?
slowness, rotational, asynchronous in early stages, early activation of mammalian genome, compaction

25. Early Mammalian Development
What is compaction and when does it occur?
formation of tight junctions between outside cells of ball – in mice after third cleavage

26. Early Mammalian Development
How is trophoblast different from inner cell mass?
How does blastocyst form from morula?
trophoblast – from extenal cells – chorion and placenta – produces eomesodermin
inner cell mass – from internal cells – embryo, yolk sac, allantois, amnion – produces Oct4,Foxd3 – pluripotentiallity
cavitation – troph cells secret fluid to form blastocoel

27. Early Mammalian Development
What enables the blastocyst to attach to the uterus?
strypsin lysis zona; integrins in trophoblast cells attach to collagen, laminin, fibronectin, etc of endometrium
protein digesting enzymes enable blastocyst to bury into wall

28. Gastrulation in Mammals
Development is internal. What modifications to the anatomy does this involve?
expansion of oviduct to form uterus; development of chorion to obtain nutrients from mother
Chorion derived from trophoblast and supplemented with mesoderm from inner cell mass; chorion induces uterine cells to form decidua

29. Derivation of Mammalian Tissues
epiplast + hypoblast = bilaminar germ disc; hypoblast forms by delamination

30. Gastrulation in Mammals
gastrulation begins at the posterior end of the embryo
node is same as Hensen’s node in birds and is at posterior end
mesoderm and endoderm migrate through primitive streak – ingression
FGF’s – downregulate E-cadherin; regulate snail, Brachury and Tbx6 genes that influence specification
What appears to control cell migration and specification?

31. Gastrulation in Mammals
precursors of ectoderm are anterior to primitive streak
human hypoblast cells are replaced by endoderm precursors

32. Extraembryonic Membranes
trophoblast gives rise to cyto and synctiotrophoblast
synctiotrophoblast digests uterine tissue; uterus sends blood vessels to this area
mesodermal tissue extends outward from embryo – from yolk sac and primitive streak derived cells
connecting stalk of extraembryonic mesoderm connecting embryo to trophoblast forms vessels of umbilical cord

33. Extraembryonic Membranes
How does placenta provide nutrients and oxygen to fetus?

34. Anterior-Posterior Axis Formation
Where are the two signaling centers located in the mammalian embryo?
node and anterior visceral endoderm (AVE)
node – formation of all of the body; two signaling centers work together to form anterior region

35. Anterior-Posterior Axis Formation
How is mouse node and AVE similar to chick and frog organizer tissues?
expresses similar genes; node – chordin and noggin; AVE – Hesx1, Lim-1, Otx-2 (head genes) and cerberus
picture – above expression of BMP antagonists in node; below – wild type embryo, chordin gene knocked out, missing chordin and noggin

36. Anterior-Posterior Axis Formation
What kind of interactions occur between visceral endoderm and epiblast in mice?
nodal protein from epiblast patterns visceral endoderm
cells of epiblast synthesize Oct4, Otx2 and Crypto transcription factors
head forming genes (cerberus, Otx2) expressed at distal tip of epiblast – begin to pattern it
distal visceral endoderm prevents crypto expression in distal epiblast
rotation of visceral endoderm causes distal region to become the AVE, crypto factor becomes reduced here which forms anterior region

37. Anterior-Posterior Axis Formation
What is believed to be the role of the Hox genes?
different combinations of Hox genes specify a particular part of the a-p axis; genes start to be expressed in mesodermal cells at early stage of gastrulation when they begin to leave primitive streak
anterior genes expressed first
four copies of Hox genes on four different chromosomes

38. Anterior-Posterior Axis Formation
What kind of evidence has enabled us to break the Hox gene code?
knockout experiments
experiments with retinoic acid
comparative anatomy studies

39. Anterior-Posterior Axis Formation
What was shown to occur when the Hoxa-3 gene was “knocked out”?
the Hoxa-2 gene?
abnormal neck cartilage, thymus, thyroid and parathyroid glands
affects structures of second pharyngeal arch – stapes, stylioid bones missing (get duplicate structures of first arch (incus, malleus)
different sets of genes are needed to specify any region of a-p axis; members of a paralogous group may be responsible for different subsets of organs within a region; defects caused by knocking out a gene occur in most anterior region of that gene’s expression.

40. Anterior-Posterior Axis Formation
How has the application of retinoic acid provided us information about hox genes?
B) embryo treated with RA – see effect on first pharyngeal arch – short flat & fused with second arch
gradient of RA exists in embryo – highest in posterior
enhancers of several hox genes have RA receptors in enhancers – when exposed to exogenous RA can activate genes that would normally not be turned on (hoxa-7)

41. Anterior-Posterior Axis Formation
What is the connection between hox gene expression and the type of vertebrae formed?
Which hox genes are expressed will determine the type of vertebrae formed

42. Dorsal-Ventral Axis Formation
What is the connection between a-v pole and d-v axis?
first cleavage plane correlates with sperm entry and defines border between embryonic and abembryonic pole

43. Left-Right Axis
What appears to be responsible for specifying the left-right axis?
similar to chick but genes play different roles – fgf8 gene specifically