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Molecular Principles of Development Summary
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Principles of Development 6th Edition By Lewis Wolpert; Cheryll Tickle; Alfonso Martinez Arias 9780198800569 Chapter 1-14 Complete Guide .
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Principles of Development 6th Edition By Lewis Wolpert; Cheryll Tickle; Alfonso Martinez Arias 9780198800569 Chapter 1-14 Complete Guide .
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Principles of Development 6th Edition By Lewis Wolpert; Cheryll Tickle; Alfonso Martinez Arias 9780198800569 Chapter 1-14 Complete Guide .
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Lecture 1. Basic concepts of developmentForward genetics - phenotype to gene:
= Make random mutations (limited number of mutations) and look at the embryo’s at interesting parts (particular phenotype) -> trying to
identify which gene was hit (mutated)
Reverse genetics - gene to phenotype:
= Interested in one gene -> targeting that gene -> knock it out (mutations) -> see what the effect is
Gap, pair-rule and segment polarity genes in the Drosophila:
• There is a cascade of genes involved in the embryo, which is discovered by genetic screens
• Forward genetics (random mutations) results in messed up segmentation
Gap genes
Pair-rule genes Genes that are involved in the development of the
Segmented polarity genes Drosophila at different times of development
Homeotic genes (Hox) and the identity of body segments:
• Gap genes, pair-rule genes and segmented polarity genes regulate together a fourth group of genes: Hox genes
• A hox gene is highly conserved and is the bar code for where the cells are in the embryo (involved in symmetry)
Mutation? -> homeotic transformation: switched identity
Cells don’t know where they are OR have the wrong information about where they are
• Mutation in the Drosophila?: an extra pair of wings instead of a haltere (function: balance)
Life cycle of Drosophila:
• The fertilized egg is already an elongated shape, which means that there is already some polarity inside
• The polarity corresponds to the future anterior (head) vs posterior (tail) position of the embryo
Forming of a syncytium in early development:
• Externally developing embryos need to be protected against dehydration and consist of a
protective shell, which is a problem for the entry of sperm cells
• However, the sperm cell can enter the oocyte through a micropyle at the anterior side of the oocyte
• After fusion of the sperm and egg nuclei, the cell starts to divide, which creates a syncytium:
Many nuclei with shared cytoplasm (no cell membranes), which allows free diffusion of proteins
Nuclear division without cell division
• The nuclei migrates to the periphery, which creates a syncytial blastoderm
• Cellularization results in the cellular membranes around the nuclei: separated cells
Gastrulation and segmentation:
• From the cellular blastoderm, the zygote is continuing with gastrulation (generating the body plan)
• The gastrulation in Drosophila results in an inverted dorsal/ventral axes compared to vertebrate axes:
Drosophila have the heart on the dorsal side (while vertebrates has this on the ventral side)
Drosophila have the nerve cord (similar to our nervous system) on the ventral side (while vertebrates
have this on the dorsal side)
• There is molecular information (genes) of segmentation that determines where the cells are in the body
Drosophila vs vertebrate development:
• Maternal axes and symmetry
Drosophila: anterior-posterior (A-P) and dorsal-ventral (D-V)
Elongated structure leads to the A-P and D-V axes
Zebrafish/xenopus: animal-vegetal (radial symmetry)
Mammals: no polarity (point-symmetry)
No matter how you rotate the zygote, it is always symmetrical
• Syncytium and cleavages
Drosophila: nuclear divisions, syncytium
Zebrafish: meroblastic divisions, yolk syncytial layer (YSL)
Layer of cells on top of a large mess of yolk: the cleavage won’t go through the whole egg
Xenopus/mouse: holoblastic divisions
Cleavage is going through the whole egg
• Early embryonic cell division time: differences in all species are based on external vs intra uterine development
Embryonic patterning:
= The process of establishing positional information at the molecular level among similar cells
• Body axes:
Dorsal-Ventral (D-V)
Anterior-Posterior (A-P)
Medial-lateral OR left-right
,• Bodies are not point symmetrical, which is caused by breaking of the symmetry; so the patterning starts with polarity and results in
differences in the location and development of the zygote
Asymmetric cell divisions: stuff is not equally divided up after embryonic division
Molecular gradients: determines what the cell is exposed to and responded accordingly
Signaling molecules generate a gradient
• Patterning: the process which establishes differential gene expression (among otherwise similar cells) that is directly related to position
within the embryo, often in a spatially graded way (sides where expression is high vs sides where expression is low)
In summary: the process where cells obtain information about where they are (GPS)
Maternal- vs zygotic genes which set up the body axes:
• Around 50 maternal genes that are expressed in the unfertilized egg, which set up the boxy axes
Bicoid: anterior
Caudal: posterior
• Zygotic genes:
Blastoderm stage: gap genes: a large domain of expression and a region without expression
Gap genes are turning on the pair-rule genes (setting up the stripes)
Segmentation genes
• Cascade of gene activation, that specify the A-P position in a more detailed way
Germ layers - basic cell types:
Fate maps (progeny of cells) & fate, specified, and determined:
• Fate: predicted outcome based on position in the embryo
We know what the cells (most likely) will give rise to, but the cells do not
Cells don’t received the molecular information or instructions
• Specified: signals received that steer lineage decisions, but still reversible
Cells know what they will be, but chan change their mind
Cells received instructions
• Determined: cells have become irreversibly committed
Cells received the information and determine in that type of cell
Irreversible committed
Spatial gene expression patterns with whole mount (whole embryo) in situ hybridization:
• Detection of RNA with RNA probes
1a. Sequence gene of interest by cloning in a plasmid
2a. Linearize the plasmid
om
3a. Incorporation of a nucleotide that is chemically modified: DIG-AP (=digoxigenin)
(=molecular label)
1b. Fertilization of eggs and collect the zygotes
2b. Remove the chorion (protective shell) with enzymes or pincets and fixate the
embryo with formaldehyde cross linking
3b. Permeabilize the emrbyos (depending on the stage) with soap: permeabilize the
embryo and linked molecules inside
5. Add the modified nucleotide probe together with the emrbyo
6. Hybridize to RNAs that are present within the embryo (if the cells at that location expressed for that gene)
7/8: Specific antibody recognizes the DIG-AP on the probe (linked to an alkaline phosphate), resulting in chromogenic reaction that forms
the precipitate
, Lecture 2. Origin and specification of the germ layers in vertebrates
Cell division (cleavage):
• Process immediately after fertilization, which increases the number of cells without the increasing of the total cytoplasmic volume
Cells become smaller and smaller with each cycle of division
Maternal to zygotic transition (MZT):
• In xenopus and zebrafish, the MZT starts at the mid-blastula stage, so the embryo is taking
over control
• In other species, the timing of the MZT is different, but also results in transcribing and expression mwan
of the zygote own genes aoomun
mmumm
• MZT is a period of time with:
Maternal RNA degradation: inheritance of molecules from the mother
This proces starts AFTER ZGA, which means that the zygote has at a certain point
both (maternal and own) RNA.
Zygotic genome activation (ZGA): earliest RNA copies being transcribed from its own genome
• Mid blastula transition (MBT): period between maternal and zygotic transcription (at the blastula
stage)
Determined by ZGA, loss of cell cycle synchrony, and cells that become more motile (because of gastrulation)
How is the MBT regulated?:
• Because of the cell division, the amount of repressor activity per nucleus would reduced
Repressor activation in the embryo remains constant, but the number of nuclei increases, which results in a smaller amount of
repressor per nucleus over time
• In conclusion: the nucleus-to-cytoplasm ratio changes during early development, causing MBT and at the end MZT.
Germ layers and boxy-axes - maternally determined? (xenopus):
• Fertilized egg:
Animal-vegetal axes
Ectoderm and endoderm
= Maternally determined (cannot be altered): mosaic
• Blastula (just before gastrulation):
The marginal zone (which is faithed to become mesoderm)
D-V and A-P axes
= Zygotic determined (post-ZGA) (specified during development): regulative
Animal-vegetal axes in zebrafish and xenopus:
• Specific mRNAs that are located to the animal OR vegetal halve
Most mRNAs in the animal halve, because there is more cytoplasm and space for the mRNAs
Also some mRNAs in the vegetal halve
gdf1 mRNA (Vg-1): part of the TGF-beta family of signaling factors
wnt11b mRNA (Xwnt-11): part of the Wnt family mRNAs important for breaking radial symmetry (A-P and
vegt mrRNA: t-box transcription factor D-V axes) and mesoderm/endoderm specification
Breaking radial symmetry by fertilization in xenopus:
• Sperm enters in the animal halve, which results in breaking the symmetry
and triggers the cortex to activate microtubules
• Microtubules start to rotate the cortex 30 degrees towards the future dorsal
region
Dorsalizing factors of the Wnt pathway (wnt11 and dishevelled) are
relocated from their initial position at the vegetal pole to a position
approximately opposite to the site of sperm entry
• Dorsalizing factors are transported along microtubules and the Wnt pathway is activated during cleavage
Activation of the Wnt pathway result in an accumulation of beta-catenin in nuclei on the future dorsal side of the blastula
• In the late blastula, the Spemann organizer is formed in the dorsal region and this is the place where gastrulation will start
Wnt-pathway has a role in establishing D-V axes:
• Wnt absent
Kinase (GSK3) is phosphorylating beta-catenin, which results in degradation of beta-catenin
Transcriptional co-repressors (CTBP, HDAC, and Groucho) bind to the TCF transcription factors and
prevent gene expression
• Wnt present
Wnt binds to the frizzle receptor and forms a complex of proteins (incl. GSK3)
Beta-catenin is not phosphorylated and degraded, and is transported to the nucleus, where it binds
and activates T-cell specific factors (TCF), resulting in expression of target genes
• TCF-3 (TCF7L1): transcription factor which translocate beta-catenin to the nucleus
= Transcription factor that uses beta-catenin as an activator to activate its targets
Wnt signaling for dorsal structures:
• Beta-catenin makes the specific body structure (head and the dorsal side of the embryo)
Over expression of beta-catenin on the ventral side leads to twinned embryo development
• Wnt inhibitors expressed ventrally (ventral side) by over expression, to avoid a twinned embryo development
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