Lecture 10+11 Vertebrate Development.pdf

12 Pages
Unlock Document

University of Toronto St. George
Cell and Systems Biology
William Navarre

CSB328H1 © Lisa | Page 13 L E C T U R E 1 0 & 1 1 : E A R L Y A M P H I B I A N ( V E R T E B R A T E ) D E V E L O P M E N T Reading: Gilbert Chapter 8 (frog section only)  fish & amphibians are anamniotic vertebrates (Fig 8.1) FERTILIZATION, CORTICAL ROTATION, & CLEAVAGE  males externally fertilize the eggs as the female lays them  the unfertilized egg has polarity (certain proteins & mRNA are already localized) – dense yolk at the vegetal pole  fertilization can occur anywhere in the animal hemisphere  sperm entry determines the dorsal-ventral polarity – sperm entry marks the ventral pole  the sperm centriole enters the egg w the nucleus & reorganizes the microtubules into parallel cortical rotation involves a parallel array of microtubules tracks in the vegetal cytoplasm (Fig 8.2A,B) st st  this separates the outer cortical cytoplasm from the yolky 50% of 1 cell cycle comp70% of 1 cell cycle complete internal cytoplasm  these microtubular tracks allow the cortical cytoplasm to rotate 30° wrt the internal cytoplasm (Fig 8.2C) – they depolymerise after rotation  this exposes the gray-coloured cytoplasm – gray crescent antibody staining using fluorescent antibodies to tubulin region opposite sperm entry point where gastrulation will begin  amphibian egg undergoes unequal radial holoblastic cleavage (Fig 8.3)  vegetal hemisphere is concentrated w yolk which impedes cleavage st  1 cleavage begins in the animal pole & slowly extends down into the vegetal region  while the 1 cleavage furrow is still cleaving the vegetal pole, nd the 2 cleavage starts at the animal pole (also meridional)  3 cleavage is equatorial but is displaced towards the animal pole bc of the vegetal yolk  animal region becomes packed w many small cells, vegetal region w few large yolky macromeres  amphibian embryo w 16-64 cells is called a morula  128-cell stage = blastula – the blastocoel becomes apparent  EP-cadherin keeps the cleaving blastomeres together  its mRNA is from the oocyte cytoplasm  if this mRNA is destroyed by antisense oligonucleotides, adhesion is dramatically reduced & no blastocoel will form (Fig 8.5B) Page 2|CSB328H1 © Lisa Zhao 2013  Xenopus fate map: (Fig 8.3C)  animal cells  ectoderm (skin & nerves)  vegetal cells  endoderm (gut & associated organs)  cells beneath blastocoel  mesoderm  cells opposite point of sperm entry  neural ectoderm, notochord mesoderm, pharyngeal (head) endoderm  2 functions of the amphibian blastocoel: 1. permits cell migration during gastrulation 2. prevents premature cell interaction bw the cells above it & below it  Nieuwkoop (1973) – placed cells from the animal cap on the vegetal cells below the blastocoel  animal cap cells differentiated into mesodermal tissue instead of ectoderm  the blastocoel prevents the premature contact to keep the animal cap cells undifferentiated th  in the 12 cell cycle, the embryo experiences a mid-blastula transition (MBT) 1. triggered by chromatin remodelling (demethylation of promoters of genes activated at MBT) 2. the zygotic genes begin to be transcribed – diff genes in diff cells 3. blastomeres acquire motility 4. cell cycle acquires gap phases AMPHIBIAN GASTRULATION VEGETAL ROTATION & THE INVAGINATION OF THE BOTTLE CELLS  gastrulation begins in the marginal zone (where the animal & vegetal hemispheres meet, Fig 8.7)  the bottle cells here need to change shape (constrict) to form the blastopore  it brings subsurface marginal cells into contact w the basal region of the surface blastomeres  the marginal cells then begin to migrate along the ECM on the basal region of these surface cells  the major factor in the mvmt of cells into the embryo appears to be the involution of the subsurface cells rather than the invagination of superficial marginal bottle cells  after the bottle cells bring the involuting marginal zone (IMZ) cells into contact w the blastocoel wall, they involute into the embryo  meanwhile, the animal cells undergo epiboly, producing a stream of cells that converge & becomes the dorsal blastopore lip  blastocoel dorsal floor cells are propelled towards the animal cap – vegetal rotation (Fig 8.8)  (B) prospective pharyngeal endoderm (specified by hhex & cerebrus expression) is pushed to the side of the blastocoel  (C,D) vegetal endoderm mvmts push the pharyngeal endoderm fwd, driving the mesoderm passively into the embryo & toward the animal pole, ectoderm begins epiboly CSB328H1 © Lis| Page 3013  sequence of animal involution: (cells constituting the DBL change as the original cells involute) 1. prospective pharyngeal endoderm – transcribe hhex which encodes a transcription factor critical for forming the head & heart 2. prospective prechordal plate cells (precursor of head mesoderm) – transcribe goosecoid which encodes a TF that activates genes controlling head formation 3. chordamesoderm cells (notochord precursor) – express Xbra (Brachyury) which encodes a TF critical for spinal cord formation  the blastocoel becomes displaced to the side opposite the DBL by the archenteron  the DBL expands laterally & ventrally – develops lateral lips & finally a ventral lip (Fig 8.9)  blastopore forms a ring around the exposed endodermal cells on the vegetal surface – yolk plug  the yolk plug is eventually internalized  in the end, all the endodermal precursors are inside, the ectoderm has encircled the surface, & the mesoderm has been brought bw them; the first cells into the blastopore are the most anterior EPIBOLY OF THE PROSPECTIVE ECTODERM  epiboly is accomplished by: 1. increase in cell number (cell division, Fig 8.12A&B stained nuclei of cells undergoing mitosis) 2. intercalation – integration of several deep layers into one (Fig 8.12C)  assembly of fibronectin into fibrils by the blastocoel roof (presumptive ectoderm) cells (Fig 8.13A&B fibronectin lattice, embryonic cells)  fibronectin-containing ECM provides a substrate for adhesion & cues for the direction of cell migration  involuting mesodermal precursors travel on fibronectin lattice  Boucaut et al. (1984) – if embryo is injected w a synthesized peptide that competes w fibronectin, cells binding to that peptide will stop migrating & cease involution (Fig 8.13D) Page 4|CSB328H1 © Lisa Zhao 2013 PROGRESSIVE DETERMINATION OF THE AMPHIBIAN AXES SPECIFICATION OF THE GERM LAYERS  vegetal cells differentiate into endoderm AND induces the cells immediately above them to become mesoderm – “bottom-up” specification  mechanism for this “bottom-up” specification lies in the mRNAs in the vegetal cortex:  VegT mRNA is critical in generating the endodermal AND mesodermal lineages  VegT mutants  whole embryo becomes epidermis (no mesoderm/endoderm)  VegT activates a gene that codes for Sox17 transcription which activates the genes that specify cells to be endoderm  VegT also activates genes that encode Nodal paracrine factors that instruct the cell layers above them to become mesoderm o Nodal signals the cells to express phosphorylated Smad2 which activates eomesodermin & Brachyury (Xbra) & specifies those cells as mesoderm o Eomesodermin & Smad2 work together to activate zygotic genes for the VegT proteins – this positive feedforward loop sustains the mesoderm (Fig 8.14)  Vg1 mRNA produces Vg1 Nodal protein to activate other genes in the dorsal mesoderm  specification of the germ layers by mRNAs:  vegetal cells are specified as endoderm by Sox17  equatorial cells are specified as mesoderm by Eomesodermin  animal cap becomes specified as ectoderm THE DORSAL-VENTRAL & ANTERIOR-POSTERIOR AXES  β-catenin is localized in the region opposite the point of sperm entry  β-catenin induces expression of certain genes that initiate the mvmt of the involuting mesoderm, establishing the AP axis  the formation of the AP axis is linked to the formation of the DV axis CSB328H1 © Lis| Page 5013 THE WORK OF HANS SPEMANN & HILDE MANGOLD AUTONOMOUS SPECIFICATION VS INDUCTIVE INTERACTIONS  early amphibian blastomeres have identical nuclei, each capable of producing an entire larva  lassoed the zygote in the plane of the first cleavage to constrict all the nuclear divisions to one half  a nucleus would escape across the constriction after a few divisions  Spemann tightened the constriction until the two halves were separated  twin larvae developed, one slightly more advanced than the other  BUT when lassoed perpendicular to the plane of the first cleavage (still longitudinally), one side produced a normal larva while the other, an unorganized tissue mass of ventral cells (belly piece, Fig 8.16B)  when 2 blastomeres are separated such that only one of the two cells contains the gray crescent, only the blastomere containing the crescent develops normally  the gray crescent gives rise to the cells that form the DBL – critical in invagination of cells  the importance of the gray crescent material lies in its ability to initiate gastrulation, & crucial changes in cell potency occur during gastrulation  cells of the early gastrula were uncommitted, but fates of late gastrula cells were determined  Spemann transplanted prospective epidermal cells from an early gastrula to an area in an early gastrula of another species where neural tissue normally formed  transplanted cells gave rise to neural tissue  transplantation of prospective neural tissue to belly region  transplanted neural tissue became epidermal (Fig 8.17A)  thus, cells of the early newt gastrula exhibit conditional specification – their fate depends on location  cells of the late gastrula exhibit autonomous specification – their fate was determined & the cells developed independent of location (Fig 8.17B) PRIMARY EMBRYONIC INDUCTION  only the dorsal lip of the blastopore has its fate autonomously determined in the early gastrula  dorsal lip was transplanted to presumptive ventral epidermis (belly skin) region (Fig 8.18)  DBL tissue invaginated (showing self-determination) & continued to self-differentiate  the host cells began to participate in the production of the new embryo  eventually, a secondary embryo formed conjoined to its host  the DBL cells & their derivatives (notochord & head endomesoderm) make up the organizer 1. they induced the host’s ventral tissues to change their fates to form a neural tube & dorsal mesodermal tissue 2. they organized host & donor tissues into a secondary embryo – instruct the formation of axes  primary embryonic induction: the key induction in which the progeny of DBL cells induce the dorsal axis & the neural tube Page 6|CSB328H1 © Lisa Zhao 2013 MOLECULAR MECHANISMS OF AMPHIBIA
More Less

Related notes for CSB328H1

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.