CAPS 306 Module 2

Carnegie Stage 1

Fertilization gives rise to zygote. Pronuclei fuse and zygote divides to produce 2 blastomeres. Division continues.

Day 1 (No need to memorize)

Carnegie stage 2

Zygote is now called the morula (cleavage to 16~32 cells). At 8 cells, morula increases cell-cell adhesion and cell polarity. 2 layers form.

Inner embryoblast becomes embryo proper + yolk sac.

Outer trophoblast becomes extraembryonic tissues

Days 2-3

Carnegie Stage 3

Morula becomes blastocyst when the blastocoel (fluid filled sac) forms due to cavitation.

Blastocoel formation pushes inner cell mass to one side (toward polar trophoblasts).

Days 4-6

Carnegie Stage 4

Blastocyst hatching. Trophoblast pushes out through zona pellucida first, then the zona pellucida bursts, and the blastocyst fully hatches.

Adplantation (adhering to endometrial lining of uterus) of blastocyst occurs.

Day 6

Carnegie Stage 5 - Initial Implantation

Adhered blastocyst slowly starts to invade into maternal uterine tissue.

Embryoblasts differentiate into epiblast and hypoblast. Amniotic cavity forms in epiblast.

Trophoblasts differentiate into cytotrophoblasts and syncytiotrophoblast.

Days 7-12

Embryoblast

Inner layer of cells of morula/blastocyst. Differentiates into epiblast and hypoblast during implantation.

Trophoblast

Outer layer of morula/blastocyst.

Differentiates into inner cytotrophoblast and outer syncytiotrophoblast (digest maternal tissue, rupture maternal capillaries, secrete hCG, promote progesterone production, inhibit maternal immune system attack on blastocyst). 

Epiblast

Dorsal layer of embryoblast.

Form embryo (embryo proper).

Hypoblast

Ventral layer of embryoblast.

Forms yolk sac.

Cytotrophoblasts

Immediately surrounds blastocyst.

Syncytiotrophoblast

Acts as a barrier between fetal and maternal tissue.

Forms when many syncitiotrphoblasts that are interfacing the maternal uterine tissue fuse together to make a large multi-nucleated cell.

Digests maternal tissue, ruptures blood vessels, produces hCG, promotes progesterone production, influences maternal immune system to tolerate embryo.

Carnegie Stage 5 - 1st Hypoblast Migration

Hypoblast proliferates spreading out around the inside of the cytotrophoblast. Create primary yolk sac. Extraembryonic mesoderm forms between primary yolk sac and cytotrophoblast.

Amniotic cavity increases in size.

Syncytiotrophoblast fully surrounds blastocyst, and blastocyst is fully engulfed into uterine tissue. Lacunae form.

Lacunae

Gaps in the syncytiotrophoblast. Blood from ruptured maternal blood vessels can collect here.

Carnegie Stage 5 - 2nd Hypoblast Proliferation

Hypoblast proliferates along extraembryonic mesoderm.

Pushes primary yolk sac out of the way, and eventually creates secondary yolk sac. Primary yolk sac degenerates.

The extraembryonic mesoderm splits: forms the chorionic cavity which surrounds the embryoblast layers.

Lacunae fuse with maternal blood vessels.

Secondary yolk sac

Endodermal lining of the definitive yolk sac.

Amniotic cavity formation

Wraps around the yolk sac and becomes a lining surrounding the embryo.

Gastrulation

Process of forming layers of tissue (endo, meso, ectoderm)

Gastrulation: Primitive streak

Cells at primitive streak express a novel set of genes (signal ligands, transcription regulators).

Activate Slug at primitive streak

Gastrulation: MET + EMT

1) Primitive streak cells producing Slug/Snail inhibit E-cadherins in nearby epiblast cells. Epiblast cells near primitive streak delaminate and migrate towards hypoblast.

2) Migrating cells displace hypoblast cells. Eventually forms definitive endoderm. (MET)

3) Slug/Snail and Twist lead to expression of mesoderm genes. Migrating cells differentiate into mesoderm and insert themselves into mesoderm layer.

Cells that migrate through primitive node…

Migrate cranially to form prechordal plate.

Migrate along midline to form notochord.

Notochord formation

Epiblast cells migrating at the primitive node migrate cranially (towards prechordal plate). Forms hollow rod (notochordal process).

As bilaminar disc elongates, so does notochordal process. Notochordal process will fuse with endoderm, creating notochordal plate.

Notochord then detaches itself from endoderm. It is no longer hollow or connected to the amniotic cavity.

Mesoderm around notochord differentiates into paraxial, intermediate, and lateral. Paraxial becomes somites.

Differential gene expression achieved by…

Unique sets of resident (nuclear) transcription factors being expressed for each cell type.

Also depends on unique sets of signals.

Axes (Embryonic Development)

Dorsal/Ventral = Back/Belly

Anterior/Posterior = Head/Butt

Lateral = Left/Right

IN HUMANS: Dorsal = Posterior = Back, Ventral = Anterior = Belly. Head/Butt = Cranial/Caudal.

Potency of stem cells

Capacity of stem cell to differentiate into more specialized cells.

Potency increases if the cell can differentiate into more cells: the more specialized the cell, the less potent it is.

Gene Regulation

Different parts of DNA remain available for transcription.

1) RNA Pol II and general TFs bind promoter.

2) cis-regulatory modules (enhancers/silencers) are bound by activators(decondense DNA)/repressors(condense DNA).

Mediator complex links regulatory TFs to general TFs.

DNA looping during transcription

Caused by regulatory TFs binding to promoter via mediator complex. There are multiple enhancers per gene (usually 4

cis-regulatory modules

Enhancers + Silencers.

Bound by multiple regulatory TFs in a sequence specific manner (i.e. TFs must be complimentary to DNA sequence).

TF binding to enhancers/silencers cause chromatin decondensation/condensation via interaction with chromatin remodeling complexes.

Synergy

Non-linear increase in transcription activation and specificity (occurs when multiple regulatory TFs bind enhancer).

Combinatorial Coding

Cis-regulatory modules in different cell types have unique DNA sequences (sequence specificity) and thus require unique sets of TFs to activate the same gene.

Ways chromatin can be loosened/tightened

Chromatin remodeling complex:

1) Nucleosome remodeling (slide nucleosomes down DNA) via SWI/SNF

2) Histone removal

3) Replacement of histones with histone variants that bind DNA loosely.

Histone-modifying enzyme:

4) Modify histone tails to destabilize chromatin and attract transcription machinery.

Histone tail modification

Acetylation or methylation of positively charged Lysine (K) and Arginine (R) residues. e.g. K27 of Histone 3

Will directly alter histone/DNA binding or recruit other complexes that open/compact chromatin.

Acetylation of histone

Activates (loosens histone/DNA binding)

Methylation of histone

Deactivates (tightens histone/DNA binding)

H3K27 activation

1) Acetylation by histone acetyltransferase (e.g. P300 + CBP)

2) Demethylation by histone demethylase (e.g. KDM6A,B)

H3K27 repression

1) Methylation via histone methyltransferase (e.g. EZH2 = subunit of PRC2)

2) Deacetylation via histone deacetylase (e.g. HDAC1,2,3)

Pioneer TFs

Can open compacted chromatin to initiate transcription in a previously inaccessible gene.

Also known as master regulators. Drive cell differentiation.

e.g. Sox2

Repressor TFs interfere with activator TFs by…

1) Compete with activator TF to bind to same/overlapping cis-regulatory region

2) Mask activation surface of activator (bind to activator active site)

3) Interact with general TFs and prevent activator interaction with general TF.

Repressor TFs close chromatin by…

1) Recruit chromatin remodeling complexes

2) Recruit histone deacetylases

3) Recruit histone methyltransferases

Chromatin modifier complex types

Writers: add modification

Erasers: remove modification

Readers: turn genes on and off through other proteins after reading modifications on tails.

Things to consider when choosing a model organism

1) Size and expense of housing

2) Generation time

3) Experimental tractability (embryo accessibility and manipulability)

4) Organism type and phylogenetic position (relative to humans)

5) Legacy (how much data is there already on the organism?)

Neurulation

Process through which CNS is formed in an embryo

Gastrulation to Neurulation

Gastrulation prepares the embryo for neurulation.

Neural plate, formed through gastrulation (the ectoderm just above the notochord for humans).

Spemann-Mangold Organizer (found in the DBL)

Equivalent of the primitive node. Found in Xenopus (forgs) and Zebrafish.

Is part of the head organiser of the presumptive neural ectoderm.

Induces neural tissue.

BMP antagonists

Noggin, Chordin (CNS)

Secreted by the Spemann-Mangold Organizer.

Needed to induce CNS tissue

BMP antagonist pathway

Antagonist binds ligand, prevent BMP from binding to TGF-B receptor.

No TGF-B signaling occurs, SMADS not activated.

Wnt antagonists

Cerberus, dickkopf1…

Needed for development of the head (forebrain).

Formation of Neural Plate

Notochord stimulates production of FGF and inhibition of BMP4 through noggin, chordin, Wnt3a (in the caudal part = spine).

Causes ectoderm to thicken.

Head organiser

Gives rise to the prechordal mesoderm = where the head will be.

Expresses both BMP and Wnt antagonists.

Trunk/Tail organizer

Induces spinal cord (notochord).

Only BMP antagonists.

Head formation

Wnt blockage →

Induce TFs Otx2 and Lim1 →

Forms anterior nervous system structure.

Wnt silences Otx2 and Lim1: no Wnt blockage = no head.

Neural Tube Closure

Neural folds fold over the neural plate, creating the neural tube.

In humans: pretty much the same. Cells of neural plate at the primitive streak divide at different rates, creating a groove in the neural plate. The ridges eventually meet and fuse, creating the neural tube which houses the central canal.

Anterior-Posterior Patterning of Brain

How the boundaries between different regions of the brain are made.

MHB

Midbrain hindbrain boundary.

Regional organiser (the part that makes the boundary) of MHB is the isthmic organiser.

How is the regional organiser positioned?

Opposing gradients of Wnt and Wnt antagonists (dickkopf1 and cerberus):

Regions with net Wnt antagonism activate Otx2

Regions with net Wnt activation activate Gbx2

Resolution of MHB

1) Broadly define regions using Wnt vs Wnt antagonist gradients

2) Otx2 or Gbx2 are preferentially expressed as a result of Wnt:Wnt antagonist gradient in each cell

3) Otx2 and Gbx2 silence each other, generating a sharp boundary: even slightly more Otx2 means Gbx2 not expressed, and vice versa.

4) Otx2 and Gbx2 produce Wnt1 and Fgf8 respectively. The TF Engrailed 1 is also expressed.

Fgf8+Otx2 = midbrain dopaminergic neurons

Fgf8 + Fgf4 + Shh + Gbx2 = serotonergic neurons

Anterior-Posterior Patterning of CNS

Established by Hox genes in response to gradients of Wnt, Retinoic Acid, and Fgf.

Hox genes code for homeodomain transcription factors.

Anterior-Posterior Patterning of CNS: Gradients

Opposing Retinoic acid vs Fgf gradients determine anterior (head) vs posterior (tail) (respectively)

Drosophila Anterior-Posterior CNS Patterning: Drosophila Gene Complexes

Antennapedia (AntP - equivalent to Hox6) and Ultrabithorax (Ubx - equivalent to Hox7)

Co-linearity

Expression pattern parallels position of gene on the chromosome

Dorsal Ventral Patterning of Spinal Cord

Notochord secretes Shh, while ectoderm and roofplate of neural tube produce BMPs (2,4,7).

BMP to Shh gradient determines dorsal-ventral.

Shh activity

Secreted ventrally (by notochord and floorplate).

Inhibit Class I TFs (Pax6, Dbx2), Activate Class II TFs (Nkx2.2, Nkx6.1, Nkx6.2, Olig2).

Pax6 vs Nkx2.2 and Dbx2 vs Nkx6.1

Pairwise groups of TFs inhibit each other.

Resolution of boundaries in neural tube

Cross repression at borders.

If wrong type of cell is in the wrong place, they won’t stick well because the cell-cell adhesion molecules produced by each type of cell is different. Cell signaling also encourages movement to right domain.

Neurogenesis

Making neurons.

Neurons arise from:

1) Neuroectoderm for Drosophila

2) Neural Tube for humans

Drosophila neurogenesis: Unique overlaps of spatial patterning TFs in neurogenesis

Unique proneural clusters form, producing unique neuroblasts from each cluster.

Drosophila neurogenesis: Establishing proneural clusters

Form as a consequence of different combinations of TFs. These combinations change as you move from the posterior end to the anterior end (Hox genes) and from the midline out to the left and right.

Drosophila neurogenesis: Signaling in Neuroblast Formation

Delta/Notch signaling pathway - usually the cell in the middle of the cluster will become neuroblast. It then delaminates.

The pathway inhibits proneural gene expression of receptor cells.

Delta/Notch signaling for Drosophila neurogenesis

Initially balanced:

Proneural genes produce Delta and Notch. Are expressed in equal levels within cluster.

Then becomes unbalanced:

Proneural genes in one cell are expressed more, inhibiting proneural gene expression in nearby cells and removing inhibition of its own proneural genes.

Drosophila neurogenesis: Proneural Genes

Encode for a type of bHLH transcription factor.

Master regulators for neurogenesis.

Drosophila neurogenesis: Neuroblast Division (signalling)

Asymmetric division mediated by segregation of Par6 and Pins to apical cortex, and segregation of Numb and Prospero to basal cortex.

Drosophila neurogenesis: Neuroblast Division (Products)

Initial round:

Produces a neuroblast and a GMC.

Neuroblast has Par6, while all Numb and Prospero are packaged into GMC.

2nd round and onward:

Again produces neuroblast and GMC, but new GMC may have different temporal TFs.

Drosophila neurogenesis: Prospero in the GMC

Translocated into the nucleus. Affect genes.

1) Turns off stem cell genes

2) Turns off Notch gene (which is the receptor for Delta/Notch proneuron suppression pathway)

3) Works with proneural genes to activate neuron genes

Drosophila neurogenesis: Numb in the GMC

Is in the cytosol.

Binds existing Notch proteins and ubiquitinates them, signaling for destruction.

Drosophila neurogenesis: GMC division

Produces neurons.

Neuronal fate is consolidated by:

1) Prospero remains transiently in the nucleus after division.

2) Numb is retained to keep Notch off.

Drosophila neurogenesis: Direct vs Indirect Neuroblast Division

Direct:

1) Type 0: NB divides, creating neuron and another NB

2) Type 1: NB divides, create GMC and NB. GMC divides to make 2 neurons.

Indirect:

3) Type 2: NB divides, create INP and NB. INP (Intermediate neuronal precursor) is a “mini neuroblast”: divides and creates GMC and another INP.

Neuroectoderm

A pseudostratified neuroepithelium. Each neuroepithelial cell contacts the ventricular and pial surface (Inner and outer membranes of neural tube).

Neural tube is made up of neuroectoderm

Interkinetic Nuclear Migration

During the cell cycle, the nuclei of neuroepithelial cells will to the pial surface and back.

Mitosis occurs at ventricular surface, S phase occurs at pial.

Direct neurogenesis (type 0): amplification of neuroepithelial cell populations

Neuroepithelial cells will divide symmetrically. Self renews the cell to amplify population.

Direct neurogenesis (type 0): Neurogenic stage

Asymmetric division of neuroepithelial cells occurs, making 1 neuroepithelial cell and 1 neuron. Numb and Numb-like is packaged into neuron.

Neuron migrates away from ventricular zone (to pial surface, forming mantle/intermediate zone).

Transition to glial cell production

Temporal process. Shh signaling changes, causing:

Notch signaling increase, TF profile change to turn off proneural gene expression.

Activate Sox9 (activate gliogenic program).

pMN (progenitor of motor neuron) TFs

Pax6 and Nkx6.1, along with Olig2 turn on motor neuron determinant TFs and proneural genes. Including:

Ngn2 (proneural genes)

Hb9 (i.e. Mnx1)

Lhx3, Isl1 = MN determinant neurons

Temporal switch in neurogenesis example - pMN

Shh expression increases over time.

Increases Nkx2.2 in pMN domain. Without Pax6, Olig2 and Sox9 work with Nkx2.2 to make oligodendrocytes (Ngn2 decrease)

Neurogenesis in Cortex

Thickening of cortex.

Neurogenesis lasts a long time because more cells must be generated, and then gliogenesis follows.

Neurogenesis in cortex steps

1) Neuroepithelial cells

2) Radial glia

3) Intermediate progenitor cells + radial glia

4) Ependymal cells

Intermediate Precursor Cells

Responsible for massive expansion in primate brains.

Cux2 TF onset

primary determinant of later born, upper layer neurons.

(4 upper layers, I being highest up, IV being right above lower layers)