MCBl41 Developmental Biology Lecture 1 February 20,2014

Middle third of the course: Early development of vertebrates from the oogonium to thelate neurula stage.Professor John Gerhart, 395 LSA; iqerhart@berkelev.edu. Office hours: TBALecture schedule and optional readings. Use Gilbert as a supplement and reference:Developmental Biology, Tenth edition, 2014; [Ninth, 2010]; or (Eighth 2006).

Lecture 1, Thursday, Feb 20. Chordates and vertebrates: defining traits, outline of amphibiandeveloprnent 6-1 2 ; [6- 1 1 ] ; @2-a6; 21 6-217 ; 7 22: 7 25-7 31 ; 7 42-7 44)

Lecture 2, Tuesday, Feb 25. Fate, competence, specification, determination. Discovery ofSpemann's organizer. 19-23; 107-113,241-245,252-255; t19-21; 112-113; 241-244;255-2561, (10-13;25-31 ; 57 -62. 304-306).

Lecture 3, Thursday , Feb 27 . Cytoplasmic localizations, cortical rotation, and axis formation in theamphibian egg 90-91,2422-244; [92-9.3.242-2441; (81-83; 136; 153-158;615-619; 291-294;302-310)

Lecture4, Tuesday, Mar4. Endo-mesoderm induction 92-93,256-262; [9a-95; 257-2621; (155-158;311-312).

Lecture 5, Thursday, Mar 6. Formation of Spemann's organizer 262-266; [261-263], (312-324).Lecture 6, Tuesday, Mar 11. Gastrulation9,245-251;19;244-2521; (29;295-302).Lecture 7, Thursday, Mar 13. Neural induction and neurulation 79-82, 266-270,336-227; [79-80;

263-272; 336-3371 (312-324; 375-38 1Lecture 8, Tuesday, Mar 18. Chick development and extraembryonic development2S6-298; l2B7-

297 ; 480-4821; (336-3a8; 501 -503; 45).Lecture 9, Thursday, Mar 20. Mammalian early development 298-311; [300-313]; (92-96; 348-368).Spring Break March 24-28Lecture 1; Tuesday, Apr 1. Professor Richard Harland: Summary of amniote and mammalian

gastrulation, overview of organogenesis - materibl will not be on Midterm 2.Midterm 2: Thursday, Apr 3,11:10-12:30. Reviews at time TBA

Final third of the course: late development of vertebrates-organogenesis,cytodifferentiation. Lecture notes and reading assignments will be posted later. ,

(Lecture l,Tuesday, Apr 1. Summary and overview-see above(Midterm 2: Thursday, Apr 3 [see above]) rLecture 2, Tuesday, Apr 8. Neural Tube: induction, morphogenesis, pattern, differentiation.Lecture 3, Thursday, Apr 10. Neural tube continuedLecture 4, Tuesday, Apr 15. Somite differentihtionLecture 5, Thursday, Apr'17. Neural crest and placodes.Lecture 6, Tuesday, Apr 22. Muscle developmentLecture 7, Thursday, Apr 24. Limb development.Lecture 8, Tuesday, Apr 29. Endodermal derivatives and kidney.Lecture 9, Thursday, May 1. Summary of organogenesisReading/RevieWRecitation week May 5-9.

Prof. Harland will hold a 2-hour review session prior to the final exam.Final Exam: Final Exam Group 13 - Thursday, May 15, 2014,8-1 1 am.

DATE TRAITS (14 are the main ones).Chordates = vertebrates + uiochordates + cephalochordates (sie p. 3)

1. Notoohord hollow nerye cord (CNS)

7. pituitary 6. somites

0004. post-analtail

7

E9E'u.lFv)o(Lmouth

EqtrluFz

DORSALEctodermepidermisneuraltube

Mesoderm

kidneygonadmelomheart

Endodermgutpharynxgillpouches

5. endostyle/thyroid. 3. pharyngeal apparatus' (gill slits, pharyngeal arches and pouches)

B. left-right differences in gut andheart coiling, and brain regions

DORSAL

VENTRAL in gonadVENTRAL

CHARDATES AS METAZOADEUTEFOSTOMESChodatesHemichordatesEchinoderms

LOPHOTROCHOZOA14 phylaAnnelids, Mollusks,Flatworms...

ECDYSOZOA ..9 phylaArthrcpods,Nematodes...

CnidariansCtenophorcsSponges '

Elght classes of the Chordate phylum 2

The advar,rtages of each for studying develomenl.1. Cepf,Flochordates-amphioxus (lancelet) 1 genome done. Basal chordate.

Small marine chordate with notochord, dorsal hollow nerve cord, gill slits,somites, and post-anal tail.

2. Urochordates-ascidians, larvaceans. 4 genomes done. Small, fast developinglarva with chordaletraits. Good methods to introduce and express genes; goo(lineage tracing.

3. Agnathans $awless fish)-lamprey, hagfish. 2 genomes done. Basalvertebratewith neural crest, peripheral nervous system, adaptive immune system.

4. Flsh (awed fish)- cartilaginous (sharks, rays, skates) and bony fish (ray linnedand sarcopterygian). 5 genomes done. Zebrafish are small, fast developing,good genetics and transgenesis. r

5. Amphlblans-anurans (frogs) and urodeles (salamanders, newts). 2 genomesdone. Eggs are big, robust; and easy to get and to inject; a century ofexperimental work. Transgenesis. mRNA knockdown.

6. Reptlles-allig-ators, lizards, turtles. Cleiodoic egg, elitraembryonic tissues. 1genome done. Largely unstudied.

7. Blrds-2 genorhes done (chlcken, zebrafinch). Mammal-like development(epiblast, hypoblast, primitive streak), yet egg bigger and easier to get.

8. Mammals-Monotrgrnes (echidna, platypus) 1 genome done. Marsupials.(kangaroo, opossum) 1 genorne done. Eutherians (mouse, rat, dog). 52genornes done. For mouse, good genetics, gene knockout, and transgenesis.

AlIveftehra,'teshalve about25,000 genes, asdo many otherbilatera,lanlmals. Andmost of thegenes aresiftr'lar ta thosein other anirnals.

tetrapod ancestorinvasion of wetlands

limbs

I

tI

Phylogenetic tree of the Ghordate classesI

I

amniote ancestorinvasion of dryland

cleidoic eggextraembryonic fibsues

B. mammals (t4 genomes done). -l

monotremes (echidna, platypus) |marsupials (kangaroo, opossum) Ieutheria (human, mouse)

|7. birds (2 genomes done) |chicken,.quail, zebra tinch

I6. reptiles ( genome done) |alligators Isnakes, tunles IIizards I

5. amphibia ( genome done) | frfrogs (Xenopus laevis) | >satamanders

l*4c. fish: sarcopterygian lHcoelacanth, lungfish

| tr4b. fish: ray finned (s senomes done) | >zebrafish, stickleback, medaka

I4a. fish: cartilaginous Iskafes, rays, sharks I

I3. jawless fish 1t genome done) |Iamprey, hagfish

-|

Ibony ftshancestor

iawed ancestorpreditio n, mo re m oti I e,

elaborate head'paired sense organs'

Paired fins

E5*t0.

Ha,qoto

vertebrate ancestorbone, verlebrae, neural cresl

pertpheral neruous sYstemadaptive immune sYstem

' exPanded genomegreater motilitY

4x genome inerease,then gene ioss

dorsal nerve cordgrllslits

post-anal tailsomifes

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u,

oo(JG'o,()att.cur

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2. urochordat€s 73 genom" oon"I , F- ascidians,iarvaceans

lE F1. cephatochordates ( genomeaonq | tr

- amphioxus >

300 200 100

approx. 0.1 aa substitutions(depends on the specific protelns and lineage)

millions of years from present

600? 500

XenoPus devel'oPment:

1.3 mmdiameter

1cell , 1 mg

Freesttdmrnlng;'hdpole .r '

'*"' :"' U Ferritizarion0 min

OoqenesismRItAs localized, animal-vegetal axis

B months-1year:sexual maturity

10!o cel ls j100 grams (100,06Ox egg weight)

Metamorphosis (vegetarian to carnivore)3 monthst , : ' ,^a - .

New gene exPresslonMesoderm inducticn

10 hrs.20,000 cells

GastrulationMorphogenesis;Neural inductionand dorsalizationby the organizer

n€urallolds

ts.*t [lFl| - tGrey

Cleavage

,e eo min

Cortical rotation: second axis 360 min (6 hrS)

@ts#/ i 1,.\##

$o.7in Gilbert gth Ed

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. Esg.-Aninrt ml€

Gastrula (seclionl

Adult

|;;'."l /@6lodem lulufe gtn

f;,";l(sectionf

rrv' ' Lr 'e ' r n5 to 1oa cellsfeeding, growth ]"--

r r l tgtolg

hatching

f\ 72 hrs (3daYs)

Cvtodifferentiation-'oig"nog"nesis i#-- loo,ooo cells

o*-_Histogenesisrr'---\= t7-" : { -o ' - ' '

rish.l

FfiI(dorsal view!

/ - - -Wswl

ffiN1W .r.*;\$ ItB [-ffi*[Spemann's

. \v \lj'l ../ orqanlzer

, f6 \ /

I -- retgpg(e

Iailbud stage:fembryo I

(lateral viewlJ

hoxl0 hoxl2

"H1'ubmp2l4l7

The tailh.rd stage is also called the pharyngula slage. lt is the earliest stage possessing all the main traits of the phylum, namely, notochord,apost.anattai t , that is,aphytotypicbodyplansypica|of thephy|um).At this stage the hox genes and >20O other "seleclor genes" are first expressed, making a dense map of gene expr€ssion domains covering theootoderm, rnesderm, and endodermal layers of the body. Seleotor genes encode transcription factors or intercellular signaling oomponentsthat s€lect what developrnent will subsoquenlly odcur in the domain of each.

bmp2I4l7

4Fate Map of the early gastrula stage (Amphibian, Xenopus).This is worth learning in some detai l . Five weeks from now, you wil l be able to explain

why each of the different parts of the gastrula develops the way it does, in terms ofthe maternal mRNAs it contained earl ier and the inductive signals i t receives in thenext stage.

Why make a map of this stage? lt has al l the organization of the egg, though nowcleaved to 10,000 cells, yet i t is ful ly prepared to undergo gastrulation andneurulation, two great events of morphogenesis that wil l establish embryonicorganization, with all the chordate traits in place, an organization very different fromthat of the egg.

To make a fate mgp. you need to be ab,te to place dye spots on the egg surface (e.9., nile blue) orinject dye into the interior (e.g., injected fluorescent dextran) at identifiable locations. Cleavage stageembryos have the animalpote, vegetalpole and grey crescent as landmarks. The early gastrula has theanimal pqle, vegetal pole, and upper blastopore lip as landmarks. Every dye spot can be locatedaccurately relative to the three landmarks, which are reliably the same in all embryos of the population.

After the marked cleavage sJage embryo or marked gastrula develops to a tadpole, you can locate the dyespot in a specific part of the tadpole and conclude that the cells you marked earlier have developed to thatpart. Then, you make a fate map of the cleavage stage embryo or gastrula by combining data on the fatesin the tadpole of all marks at all points made earlier.

blus la hlnclbrala

red in axial mugclo

(somit6s)

blastopore l lp,lusl startlng to form

ECTOOEBM6pldermls

tube (CNS)

MESODERMs {lo muscls)

plate (coelom)

notochord

L.

!oo' .

'C 6)96ct o.lt tl<tL

ENDODERMgur

t€oc,s

\*ootu

tr

*ot

fi

LoC'

J

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epldermis

gutroof \=

'*:: -/

glt l sl i tendoderm

mesoderfll

. -hear l

bloodG-erm cells (precursors /Fatemaps, early gastrula, left side viewof eggs orsperm cel ls)

---r- ' - -" ' re

(\,

er 4'-1)nt'"VLy'-'/''2*n'

{H.cr}il

CloHl6NrOCl: mol. wt 353 855; also

tCt. f r ' .H.OtrSO.: mol wt ' 732 865U oii"Jv": absotption maxima 635-645)

nl le blue'

Inject fluorescein-dextran solutioninto on.e cellof a 32.cell embryo.

Early gastrula stage embryo10 hrs post fertillzatlon

20,000 cells, lust asgastrulatlon movements start

Conclude: Fate mappinq shows us that prospective ectoderm is at the top (animal hemisphere),prospective endoderm is at the bottom (vegetal hemisphere), and prospective mesoderm is in themiddle (equatorial level) in an internal subsurface layer in Xenopus. Mesoderm is not on thesurface. (This is unusual forvertebrate embryos). Every tadpole parthas a corresponding priorlocus in the early gastrula, and before that, in the ferti l ized egg. The fate map of the early gastrulagives a very convoluted view of the tadpole. Gastrulation and neurulation wil l de-convolute themap.What is FATE: In English, the word connotes pre-destination, as in destiny. But here it onlymeans "outcome". Fate maps tell what the gastrula cells wil l become in the tadpole, but we don'tieam whether those gastrula cells are yet autonomously (on their own) capable of reaching thatoutcome, that is, whether they are yet determined for that fate. How can we find out if the gastrulacells are "determined"? See next page.