Developmental Bio
Friday, January 28, 2022 1:31 PM
C.elegans: Sea urchins: Drosophila melanogaster: Mouse - Mus musculus
Advantages to studying them: Advantages to studying them: Advantages to studying them: Fun Facts:
- Invariant lineage - More closely related to vertebrates than the nematodes and flies - Easy to breed - Mice mature quickly --> 9 weeks from fertilized to adult
○ Pattern development and cell division is the same for every embryo - They are deuterostomes - Quick generation time - Small egg - no yolk
- The embryo is transparent ○ Primary invagination of the gut forms anus - Easy to maintain in a lab - Fertilization occurs in the oviduct where cleavage starts
- Develops rapidly - Transparent embryo - Easy to mutate - Egg surrounded by zona pellucida
- Can watch cell division - Easy handling - Cheap ○ Made of glycoproteins and mucopolysaccharides
- Larva hatch after 15 hours - Dipteran insect ('true flies') - Cleavage produces solid ball of bastomeres = morula
- Mature adults after 50 hours Cleavage - Most genes have similar roles in other organisms - Compaction = where cells increase SA in which they are in contact with ea
- Easy to grow on agar plates - Radial holoblastic cleavage ○ Unique event in mammals
- First 2 cleavages are meridional and perpendicular to each other Life Cycle ○ Happens at 8-cell stage
They are hermaphrodites --> the females can make their own sperm for a short time and then - 3rd cleavage = equatorial - Internal fertilization - Cells polarize after compaction
switch to making oocytes ○ Separates animal and vegetal hemispheres - Syncytial blastoderm ○ Exterior has microvilli
- 4th cleavage = asymmetrical - Cellular blastoderm ○ Interior has smooth surfaces
Invariant lineage: ○ The 4 animal cells divide into 8 equal-volumed blastomeres = mesomeres - Gastrulation --> embryo - Tangential and radial cleavage produces 10 internal cells and 20 outer cells
- Pattern of cell division is the same in every embryo ○ The vegetal cells divide unequally ○ Embryogenesis ~12-15hrs - Cells acquire epidermal characteristics and increase cell adhesion via cadhe
- The larva hatch with exact same complement of 558 cells ▪ Produce 4 large macromeres and 4 small micromeres - Hatches into 1st instar --> 3 instar stages
- Lineage is worked out and the cell fate is drawn --> cell-cell interactions can still change cell - The 8 mesomeres divide to produce 2 animal tiers = an1 and an2 - Third instar larva becomes pupa Morula to Blastocyst
lineage - The micromeres divide again to produce 4 larger micromeres and 4 smaller micromeres - Pupa metamorphosis --> adult fly - After morula is formed, the blastocyst is formed from 2 distinct groups of c
- 131 cells undergo apoptosis Egg is laid on decaying plants --> rotting fruit ○ Trophoectoderm = spherical layer of cells outside embryo
- After 4 molts, 959 cells remain ▪ Forms extraembryonic tissue (placenta, etc.)
- No RNA splicing = each gene produces a single protein General body plan is same in embryo, larva, and adult ○ Inner Cell Mass (ICM) = forms epiblast
- Distinct head and tail with repeating units ▪ Can be cultured as embryonic stem cells
Polar bodies form after fertilization ○ 3 segments form thorax □ Can differentiate into all cell types in vitro
- Abortive cleavage before male and female nuclei fuse ○ 8 segments form abdomen - Cavitation = fluid pumped by trophoectoderm to form blastocoel with ICM
○ Not a true cleavage Genetics - ICM divides into 2 by E4.5-E6.5
○ Unknown reason - 14000 protein coding genes ○ The layer that is in contact with the blastocoel is the primitive endod
- True cleavage happens after nuclei fuse - High incidence of RNA splicing ▪ Contributes to extraembryonic structures
Cleavage: - >1100 genes encoding functional RNAs other than mRNAs ○ The rest of the ICM develops into epiblast
- 1st cleavage = asymmetric ▪ Epiblast is the outermost layer of embryo before it gives rise to
○ Generates AB and P1 cell Drosophila Egg mesoderm
○ P1 = stem cell - Oblong ▪ At this stage, embryo is released from zona pellucida and impla
- 2nd cleavage - Sperm is already activated when it enters uterine layer
○ AB gives rise to AB-a and AB-p ○ Females have storage organ for sperm and releases only a few at a time
▪ These cells give rise to hypodermis, neurons, and some muscle cells - Body axis has already begun being specified before sperm enters egg Blastocyst to Onset of Gastrulation
○ P1 gives rise to P2 and EMS Ecto – blue ○ Maternal determinants help to specify body axis - The mural trophoectoderm that is not in contact with the ICM replicates it
▪ EMS gives rise to MS and E Endo– Yellow - Sperm enter micropyle at anterior end without dividing
□ MS gives rise to muscle, glands, coelomocytes, and some neurons ○ Only one can pass at a time ○ This forms Trophoblast Giant cells
Meso – Red
□ E-cells form the gut - Egg has dorsal appendages ▪ Surrounds whole conceptus and invades the uterine wall to inte
○ At this point, axes have established ○ Aid with gas-exchange maternal tissue --> anchoring the blastocyst to uterine wall
▪ P2 = posterior Embryo has 3 bands of cells along animal-vegetal axis - The uterine wall covers the blastocyst
▪ AB-p = dorsal 1. Small and large micromeres at vegetal pole Embryogenesis - The Polar Trophoectoderm that is in contact with the ICM continues to div
- Actin filaments + myosin motor protein = actomyosin - Gives rise to mesoderm = Forms primary mesenchyme --> skeleton - After nuclei fuse, zygotic nucleus undergoes mitotic divisions every 9 minutes the ectoplacental cone
○ Assembles at egg cortex ○ (9 divisions until syncytial blastoderm) ○ That will then form the Chorion and extraembryonic ectoderm
2. Vegetal plate --> Veg1 and Veg2
○ Interacts with centrosome - Gives rise to endoderm and mesoderm (and some ectoderm) - No initial cleavage of cytoplasm ▪ Both contribute to 'placenta'
○ Contract asymmetrically away from sperm entry point - Secondary mesenchyme --> muscles and connective tissue - Early embryo = syncytial blastoderm - Inside the trophoectoderm, the parietal endoderm (came from cells of pri
- Cortical components flow to anterior ○ Nuclei move to the periphery endoderm) migrates to cover inner surface of mural trophoectoderm
3. Mesomeres (remainder)
- Cytoplasmic components flow to posterior - Give rise to ectoderm --> outer epithelium and neural cells ○ Large proteins can diffuse between nuclei during first 3 hours - The visceral endoderm covers the elongating egg cylinder that has epiblas
- PAR proteins = maternal cortical proteins at posterior and anterior end --> partition cell - Eventually divided into oral and aboral ○ At the syncytial stage, some nuclei localize posteriorly and become surrounded by PM - Epiblast = cup-shaped layer of 1000 epithelial cells --> develops into embr
○ Par genes = protein kinases + other signaling proteins Micromeres = organizer region to form pole cells - The primitive streak appears at E6.5 --> marks A-P axis of embryo at begin
○ Controls placement of mitotic spindle in early embryo ▪ Pole cells will end up on the outside of the blastoderm and eventually give rise gastrulation
○ PAR-3 & PAR-6 = anterior - Cell cleavage = invariant for the first 9-10 divisions to the germ cells ○ At the posterior
○ PAR-1 and PAR-2 = posterior - Development requires both animal and vegetal halves ▪ Blastoderm gives rise to somatic cells ○ Elongates until it reaches bottom of cup
○ PAR proteins regulate the forces that pull on each side of the spindle = helps cleavage - If isolated at the 4-cell stage, any of those cells can give rise to Pluteus ▪ Pole cells are set aside early and are protected from somatic cell inductive ○ By then the Node is visible
- Centrosome: - At the 8 cell stage, animal-vegetal axis is established signals ▪ Condensation of cells
○ Composed of microtubules ▪ If you isolate animal half --> only ectoderm is formed - Cellular blastoderm arises after ~3 hrs (14 mitotic divisions) ▪ Corresponds to Hensen's Node
○ Organizes the microtubule cytoskeleton ▪ If you isolate only vegetal half --> forms large gut and reduced ectoderm = abnormal ○ Membranes form around nuclei = compartmentalize □ Analogous to Spemann's organizer
○ Helps separate chromosomes into the daughter cells - Gastrulation = dramatic cellular migrations to form germ layers
○ Duplicates to form centrosome and nucleates each microtubule spindle pole to The vegetal region organizes the body axis ○ Endoderm = gut Gastrulation and Neurulation
organize chromosome distribution in mitosis and meiosis - Micromeres act as Spermann organizer --> induces formation of body axis ▪ 2 regions: posterior and anterior that will fuse in the middle - Similar to chicken gastrulation but harder to visualize due to having differe
- Maternal factors located in micromeres from 4th cleavage ○ Mesoderm = muscle, connective tissue, heart, and blood ○ Ingression through primitive streak
Antero-posterior Axis - B-catenin --> specifies micromeres ▪ At most ventral region - Epiblasts converge on primitive streak
- Sperm entry determines polarity of axis of fertilized egg - The organizer specifies and sets boundaries for endoderm and ectoderm ▪ Invaginates to form furrow along ventral midline --> internalizes and initially ○ Proliferating cells then migrate and spread laterally & anteriorly be
○ Determines 1st position of cleavage - Signals to macromeres to become endomesodermal cells --> give rise to skeletal rods forms tube ectoderm and visceral endoderm --> forming mesoderm
- Actin filaments help to specify the anterior end --> cap of actin filaments at anterior ▪ Then separates from tube and moves under ectoderm to form muscles and ○ Cells migrating to anterior form Node form prechordal plate mesode
- P-granules specify the posterior Cell fate connective tissue head process
○ P-granules are cytoplasmic constituents containing maternal mRNAs, proteins, etc. - Concentrations of B-catenin are high in the micromere nuclei ▪ Subdivided into Visceral and Somatic ○ Just like chicken, as the primitive streak regresses, the trunk notocho
needed for germline development - B-catenin = coactivator of TF TCF --> activates transcription of vegetal-specific genes □ Visceral = connective tissues associated with gut + organs down --> spinal cord develops above it
○ P-granules remain in P-daughter cells --> P4 gives rise to germline - B-catenin is absent in animal cells --> future ectoderm □ Somatic = muscles ▪ Somites form on either side
○ # of germline cells = variable - Dishevelled protein = stabilizes B-catenin in vegetal region ○ Ectoderm = skin and NS - Primitive streak extends to the bottom and the node is formed
○ # somatic cells = 959 - B-catenin and a TF called Otx help to activate the pmar-1 gene in micromeres ▪ Ectodermal cells from ventral region leave individually to form neuroblasts - Anterior ectoderm becomes anterior Neural plate = Brain
- Asymmetry is determined at fertilization --> determined by sperm entry - Pmar-1 = TF exclusive to micromeres between mesoderm and remaining ectoderm ○ Anterior of embryo grows and head fold appears
▪ Helps express organizer function ▪ Forms foregut and hindgut - Notochord begins to be laid down in trunk
Gastrulation: ▪ Helps specify micromeres so that micromeres can specify neighboring cells ▪ Forms epidermis --> produces a thin cuticle made of chitin once the epidermal - Further development = :
- Occurs at 28 cell stage ▪ Helps turn on micromere-specification genes = skeletogenic genes cells stop dividing ○ Distinct head
- 100 mins after fertilization ▪ TF that represses HesC --> HesC represses the genes that specify skeletogenic genes ○ Starts 3hrs after fertilization ○ Neural fold formation
- Occurs when E-cell descendants moves inside embryo and form gut □ Only micromeres express pmar-1 = only micromeres can become skeletal ○ Main nerve cord lies ventrally --> different from vertebrates ○ Fore + hind gut closures
- Apoptosis removes some early cells - Micromeres produce an Early Signal (ES) - Parasegmentation ○ Somite formation
- Directed dilation = increase in hydrostatic pressure - Induces Veg2 cells to adopt endomesoderm state ○ Central blastoderm aka Germ Band After gastrulation + neurulation are complete, embryo undergoes process of Tu
○ Causes shape change - After the 7th cleavage, micromeres send 2nd signal Delta ligand to Notch receptor on ▪ Main trunk region of embryo that extends anterior-posteriorly - Before this happens the embryo is doing an extreme backbend
○ Embryo elongates rapidly along the antero-posterior axis adjacent Veg2 cells = turns on mesoderm specification genes ▪ Segmentation starts here ○ Ventral side (belly) of embryo is on the outside to start
- At 80 cell stage ▪ Specifies them as secondary mesenchyme and pigment cells ▪ Series of evenly-spaced grooves = parasegments - After Turning = embryo can undergo organogenesis
○ Cells from different lineages that will form organs will cluster together - Delta-Notch signal also helps specify endoderm ○ Parasegments = developmental basis of segments
○ Genes responsible for organ identity will be expressed - Wnt-8 signal is secreted by Veg2 = autocrine ▪ Segments = fusion of anterior and posterior halves of parasegments
▪ Pha-4 = pharynx development - Maintains B-catenin stabilization = maintains micromere function ▪ 14 parasegments = 7 segments
- Wnt-8 also acts paracrine = sends signal to Veg1 cells to turn on endoderm specification genes □ 3 form mouth
Dorso-Ventral Axis - If you inhibit degradation of B-catenin = the embryo is vegetalized □ 3 form thorax
- Cell-cell crosstalk determines dorso-ventral axis - All presumptive ectoderm transforms into endoderm □ 8 form abdomen
- Cell-cell interactions can still affect cell fate = dorso-ventral polarity is not fixed - If you inhibit B-catenin accumulation = embryo is animalized
- Left-right axis is not fixed in relation to antero-posterior or dorso-ventral axes - Prevents formation of endoderm and mesoderm Larval Stages
○ Unknown molecular mechanism that determines handedness - Micromeres have a fixed fate = only become primary mesenchyme - Hatch out of egg in 24hrs
- Experiments done to show that these cells are not specified early on - Will still become primary mesenchyme even if micromeres are grafted on another part of the - Head is largely hidden at this stage
○ Push/rotate AB-a = reverse the dorso-ventral axis and P1 daughter EMS is inverted embryo - Acron = specialized head structure at anterior
relative to AB cells - Macromeres do not have a fixed fate when they are formed - Telson = posterior end of larva
▪ Develops normally but inverted axes - At 16-cell stage, they can become endo, meso, or ecto - Denticles = tooth-like outgrowths on ventral side of larva
○ Push left anterior daughter cell of AB-a to be posterior of right AB-a = reverses - At 60-cell stage, Veg2 can only become meso or endo --> never ecto ○ Aids in locomotion
handedness ▪ Meso = secondary mesenchyme - Larva molts twice --> 3 instar stages until pupa
○ Mutations in gpa-16 causes randomization of cleavage spindle orientation = 50% of - Veg1 can only give rise to endo or ecto but never meso - Imaginal discs = group of prospective epidermal cells that will differentiate into organs
worms have right-left interchange ▪ Means that the mesoendoderm specification has already happened and now the endo- during metamorphosis
▪ GPA-16 = G-protein alpha subunit --> works with proteins to position centrosome ecto boundary has yet to be made ○ Imaginal discs for each of the 6 legs, 2 wings, 2 halteres, genital apparatus, eyes,
antennae, etc.
Asymmetric divisions + cell-cell interactions specify cell fate Oral-Aboral Axis - Histoblasts = small group of cells that are dormant in larval stage but proliferate in pupa
- Lineage positions the cells to interact with each other - Perpendicular to animal-vegetal axis stage
- P2 helps specify AB-p - Defines bilateral symmetry of Pluteus larva --> demarcated by larva's mouth ○ Develops to form epidermis of adult abdomen
○ Through interactions with ligand on P2 called APX-1 and receptor on AB-p called GLP-1 - Corresponds to dorso-ventral axis in frogs and fish
▪ GLP-1 = Notch receptor - Animal-vegetal axis = antero-posterior axis Maternal factors guide early stages embryonic development
▪ APX-1 = Delta ligand - Cell cleavage happens in relation to animal-vegetal axis - Maternal mRNAs translate into TFs and transcribe zygotic genes
○ Remove P2 = all AB cells become AB-a - B-catenin accumulation marks the posterior end = vegetal
○ Remove P1 = no AB-a are not made??? - Nodal signaling = oral Antero-Posterior Axis:
- P2 also polarizes EMS cells --> affects daughter cells - BMP signaling = aboral
○ E-cells = posterior - Oral ectoderm cells give rise to Stomodeum 1. Maternal genes --> establish pattern before cellularization
○ MS = anterior - Epithelium that forms the outer layer in the oral region and the neurogenic structures - Bicoid
○ SKN-1 - Aboral ectoderm cells give rise to epithelium that covers larva body - Hunchback
▪ Maternal determinant --> mRNA for skn-1 found uniformly in 2-cell stage but - Oral-aboral axis is related to animal-vegetal axis = important concept - Nanos
higher levels of the SKN-1 protein in the P1 nucleus than AB - Through indirect effect of B-catenin localization in vegetal nuclei - Caudal
▪ Turns on TF med-1 and med-2 - B-catenin accumulation marks posterior end 2. Gap genes = first zygotic patterning genes to be expressed--> establish 'regions'
□ Necessary for MS and E fate - Oral-aboral axis establishes after animal-vegetal axis - Hunchback
▪ Abolish SKN-1 = mesoendodermal cells instead become mesoectodermal - The 2 axes are linked spatially and temporally - Knirps
▪ It's not that SKN-1 is released by P2 to the EMS cells, it's that the skn-1 gene is - Oral ectoderm = organizing region for oral-aboral axis - Kruppel
expressed in EMS cells and is important to give rise to the pharyngeal cells - High Nodal signaling in oral ectoderm - Giant
□ After the 1st cell cleavage --> only P1 cells can give rise to pharyngeal cells - Causes expression of TF Goosecoid = TF that suppresses oral fate in aboral ectoderm - Tailless
□ After 2nd cleavage --> only EMS cells can give rise to pharyngeal - Growth factor BMP-2/4 diffuses from oral ectoderm and induces aboral fate 3. Pair-rule genes --> establish parasegments
□ After 3rd --> only MS has intrinsic ability to generate pharyngeal tissue - Lefty = member of TGF-B family - Eve (even-skipped)
□ Homozygous skn-1 mutants lack pharyngeal and intestinal structures - Antagonizes Nodal signaling - Fushi tarazu (Ftz)
□ The EMS cells are respecified to be similar to C cells --> ectodermal - Restricts Nodal signaling activity to oral ectoderm 4. Parasegment genes --> establish segmentation genes
○ Remove P2 at 4-cell stage = all EMS cells become MS and there will be no E cells (no - If Nodal is blocked, differentiation of ectoderm into oral and aboral is also blocked - Wingless
endoderm) - If Lefty is blocked, all ectoderm is converted to oral - Hedgehog
○ P2 sends Wnt signal (MOM-2) to EMS cell --> received by frizzle receptor (MOM-5) on 5. Hox genes
EMS
▪ Instructs EMS to become E cell Dorso-Ventral Axis:
▪ The goal of this Wnt signaling cascade is to down-regulate expression of the
pop-1 gene in the cell that will become E cell 1. Maternal genes --> establish pattern before cellularization
▪ Embryos that lack pop-1 gene end up producing 2 E daughter cells from the - Pipe
EMS cell - Toll
○ POP-1 protein = TF --> it is the nematodes version of the TCF - Dorsal-cactus
- Spatzle
▪ WRM-1 and SYS-1 are nematode-specific B-catenins
▪ LIT-1 = protein kinase that will phosphorylate POP-1 --> leads to export of POP-1
2. Zygotic genes that further divide D-V axis into dermal layers
from the nucleus - Zerknullt - amnioserosa
○ When MOM-2 binds to MOM-5, the LIT-1 and WRM-1 will enter the nucleus and cause - Decantaplegic + tolloid - dorsal ectoderm
exportation of POP-1 from nucleus - Rhomboid - neuroectoderm
- Twist + snail - mesoderm
▪ The reduced amount of POP-1 in the nucleus will allow for SYS-1 to enter and act
as a coactivator to switch on genes required to specify an E cell - Single-minded - mesoectoderm
○ The MS cell does not have accumulation of LIT-1, WRM-1, or SYS-1 in the nucleus,
therefore, the levels of POP-1 remain high = target genes are suppressed = cell
specified as MS
▪ Higher POP-1 level in anterior vs posterior
Hox Genes
- Specify positional identity of antero-posterior axis
- Evolutionarily conserved --> found in most organisms
- Encode a set of TFs that are involved in specifying positional identity of cells along axis
- Topologically arranged
- Ceh-13 = required for anterior organization of embryo
○ Only Hox gene essential for embryonic development
▪ The other hox genes carry out their functions at larval stage
- Lin-39 = controls mid-body cell fate
○ Also regulates vulva development
○ Mutation in this gene = larval mid-body cells instead express characteristics of anterior
and posterior
- Mab-5 and egl-5 = controls posterior regions and provides positional info for posterior cells
- Php-3 and nob-1 = posterior group genes
Temporal aspect of gene expression and development
- Heterochronic mutations
○ Mutations that alter the timing of developmental events
○ Lin-4 and Lin-14 = 1st 2 heterochronic genes
○ Lin-14 is a TF that controls lineage of T-blast cells in larval stages 1-4
▪ High levels of LIN-14 = larval 1 stage
▪ Medium levels = larval 2 stage
▪ Low levels = L3 stage
○ Mutations in lin-14 affect timing of development The determination of body plan begins in the ovary
▪ Gain of function mutation = over-development, non-sequential, and repeating
- Germarium contains stem cells (2n) that divide asymmetrically to produce 1 stem cell and 1
□ Kind of 'stuck' or repeating L1 stage where there was high levels of LIN-14
cystoblast
▪ Loss of function mutation = precocious development
- Cystoblast is also a stem cell, but it will divide mitotically and give rise to the egg
□ Kind of like skipping steps in development
▪ Has 4 mitotic divisions --> produces 16 cells with cytoplasmic bridges = germline
□ Similar to L3 levels of LIN-14
○ Lin-4 is a microRNA that controls expression of lin-14 by repressing it cyst
▪ The posterior cell becomes the egg and the other 15 cells become nurse cells
▪ Loss of function mutation in lin-4 leads to over-expression of lin-14
□ Nurse cells produce maternal messages --> mRNAs and proteins to
□ Similar effect as GOF mutation in lin-14
support embryo development
Maternal messages sent to egg through cytoplasmic bridges
Vulval Development
- Develops in last larval stage ◊ Example: gurken
- Follicle cells (somatic ovarian cells) form a sheath around nurse cells and egg = Egg
- Contains 22 cells derived from AB blastomeres --> ectodermal AB
chamber
- P5p, P6p, P7p = give rise to vulva
○ Different P cells than in early embryo ▪ Multiple egg chambers are strung together by stalks of follicle cells
○ P6p = primary ▪ Egg chambers have distinct polarity
- The antero-posterior polarity of the egg is the result of signalingfrom the anterior of the
○ P5p and P7p = secondary
older egg chamber to the posterior of the younger egg chamber
○ Tertiary cells = P cells that fuse to become hypodermis --> non-vulval
- Through Notch-Delta
- Lin-39 = hox gene that prevents the P5p, P6p, and P7p from fusing into the hypodermis
- Germline cells produce Delta which binds to Notch on follicle cells
Developmental Bio Page 1
, support embryo development
Maternal messages sent to egg through cytoplasmic bridges
Vulval Development
◊ Example: gurken
- Develops in last larval stage
- Follicle cells (somatic ovarian cells) form a sheath around nurse cells and egg = Egg
- Contains 22 cells derived from AB blastomeres --> ectodermal AB
chamber
- P5p, P6p, P7p = give rise to vulva
○ Different P cells than in early embryo ▪ Multiple egg chambers are strung together by stalks of follicle cells
○ P6p = primary ▪ Egg chambers have distinct polarity
- The antero-posterior polarity of the egg is the result of signalingfrom the anterior of the
○ P5p and P7p = secondary
older egg chamber to the posterior of the younger egg chamber
○ Tertiary cells = P cells that fuse to become hypodermis --> non-vulval
- Through Notch-Delta
- Lin-39 = hox gene that prevents the P5p, P6p, and P7p from fusing into the hypodermis
- Germline cells produce Delta which binds to Notch on follicle cells
- The 3 cell lineages that give rise to the vulva divide, move, and then fuse to become 7
▪ Specifies anterior follicle cells as Polar follicle cells
concentric rings stacked on each other = forms conical structure
- Polar cells release signal called Unpaired --> stimulates receptors on adjacent follicle
- Gonadal anchor cell specifies the fate of the 3 cells (P5p, P6p, P7p) with inductive signal
cells to activate the JAK-STAT pathway
○ Secretes a growth factor (similar to EGF in animals)
▪ Causes follicle cells to become stalk
▪ Product of lin-3 gene = LIN-3 inductive signal
▪ Specifies anterior and posterior follicle cells
▪ Binds to nematode version of tyrosine kinase receptor EGF (LET-23)
- Stalk cells upregulate cadherin expression to keep the younger egg at the posterior
▪ Mutations in lin-3 = no vulva formation
end
○ Most of the signal is received by P6p = most strongly activated
▪ P6p sends lateral signal to P5p and P7p = received by LIN-12 (Notch receptor)
□ Lateral signal = 3 proteins from Delta family
- Wnt signaling is also involved in this --> involves another B-catenin called BAR-1
As a germline cyst buds from the germarium, it signals through the Delta–Notch pathway
(small red arrows) to induce the formation of anterior polar cells (red). These, in turn, signal
Questions for final exam prep:
to the adjacent cells anterior to them and induce them to become stalk (green). Signals from
the stalk induce adjacent follicle cells in the younger cyst to become posterior polar cells
- What are some differences between the animal and vegetal poles of
(red), and induce the younger cyst to round up and the oocyte to become positioned at the
the Xenopus egg?
posterior of the egg chamber. Signals from anterior and posterior polar cells then specify
- What kinds of maternal determinants are laid down in the frog egg and
adjacent follicle cells as posterior and anterior follicle cells, respectively (shown in more
how does that differ from drosophila?
detail in Fig. 2.18). The yellow arrows indicate the overall direction of signaling from older to
○ Maternal mRNAs and proteins
younger egg chambers.
○ Drosophila don’t have proteins laid down by the mother
- Where does ectoderm, mesoderm, and endoderm arise from? Maternal mRNAs are involved in antero-posterior specification
○ Ectoderm from animal cells
- The egg moves to the posterior end of the egg chamber with the help of maternal mRNAs
○ Endoderm from vegetal cells
from the future anterior end
○ Mesoderm comes from signals from vegetal cells acting on - mRNAs are deposited by nurse cells to the future posterior end
animal cells - This is done by reorientation of microtubule cytoskeleton (on which maternal mRNAs
▪ Band of cells around the equator are transported)
- When is most of the embryo's mRNA transcribed? ▪ Dependent on PAR proteins
○ Mid-blastula transition stage - Egg nucleus is also positioned posteriorly
- How do maternal mRNAs localize to their respective places on the - Maternal gurken mRNA is located posterior --> translated to give local concentration of
animal-vegetal axis? (2 routes) Gurken near posterior end of egg nucleus
○ METRO - message transport organizer region - Gurken = member of TGF-a family
▪ Involves association of mRNAs with the cytoskeleton and - Gurken protein is secreted across the egg membrane --> induces adjacent terminal
small vesicles of ER follicle cells to adopt posterior fate
○ Microtubules through kinesin motor proteins ▪ This is done through locally stimulating the Torpedo receptor that is present on
- What maternal mRNA is localized in the vegetal cortex of mature follicle cells
oocytes? Gurken activates follicle cells = initiates formation of both axes
○ Vg1 - Gurken = key initiator for both A-P and D-V formation
- When is Vg1 translated and what is it a signal for? - Diffusible protein ligand
○ Translated after fertilization - In all cases, gurken mRNA is first transcribed by follicle cells and transferred to the egg
○ Signals mesoderm induction (maternal message!)
- What is one of the earliest signals required for D-V specification? - The mRNA is moved to posterior then it gets translated, secreted, and the signal
○ Wnt11 diffuses to bind to torpedo receptors on nearby follicle cells
○ Dorsalizing factor deposited in dorsal end after fertilization
- What transcription factor is localized in the vegetal pole and specifies Maternal mRNA movement in the egg is dependent on microtubule re-orientation
endoderm and mesoderm? - Bicoid mRNA is produced by nurse cells then moved to anterior
○ VegT - Not translated until after fertilization
○ Translated after fertilization - Transported along microtubules by action of motor protein dynein
- What determines D-V axis? - Oskar mRNA moves to posterior end
○ Sperm entry - At posterior, oskar gets translated and organizes the posterior pole cytoplasm (pole
- What is cortical rotation and why is it important? plasm)
○ 30 degree rotation of cortex after sperm penetrates oocyte - Promotes germ cell formation
○ Causes an array of microtubules under the cortex to reorient - Recruits patterning proteins - including nanos mRNA
themselves so that their plus ends are pointing away from the - Transported along microtubules by action of motor protein kinesin
site of sperm entry - Maternal gurken accumulates near egg nucleus --> involved in posterior follicle patterning
▪ This acts as tracks for maternal mRNAs to localize to their - New posterior follicle cells signal to egg --> moves nucleus toward anterior via
respective poles microtubules
○ Without this process, there would be no dorsal structures, as this
is necessary for dorsalizing factors to localize to dorsal end Dorso-ventral axis is initiated in egg
- Dsh and Wnt11 are maternal mRNAs that are dorsalizing factors - Achieved after posterior end of the egg is specified
- Why is it important to set up the D-V axis? - Gurken is involved in D-V axis initiation too because the posterior localization of Gurken
○ Need the D-V axis so the organizer region (dorsal lip) can be specifies the posterior follicle cells which express torpedo (receptor for gurken)
established - The newly specified posterior follicle cells signal to the egg to move the nucleus towards
○ Need to do this before germ specification anterior
- What are the components of the Wnt/B-catenin pathway? - Gurken induces dorsal follicle cells because torpedo causes dorsal morphology
○ Wnt11 = secreted ligand - There is a torpedo signaling cascade in the follicle cells that restricts the production ofPipe
○ TCF3 = transcription factor that B-catenin activates protein to the ventral side
○ GSK-B = kinase that tags B-catenin for degradation - Gurken binds to torpedo = inhibition of pipe synthesis --> dorsal cell fate
○ Dsh = prevents formation of destruction complex when activated - No gurken = no gurken binding to torpedo = no inhibition of pipe synthesis = pipe
▪ Prevents degradation of B-catenin synthesis --> ventral cell fate
- What would be the implication if the Frizzled receptor were blocked?
○ Wnt pathway would be inhibited Specifying A-P extremities of embryo begins in egg
○ Dorsal structures would not develop - Trunk gets fragmented and binds to Torso at extremities
- What maternal protein/gene prevents Wnt signaling in ventral region? - Torso is technically found everywhere on egg surface but trunk is only fragmented at
Wnt antagonist the poles (unknown why)
○ Dickkopf1 (Dkk1) - Trunk is secreted into perivitelline space by nurse cells
- Activity of Wnt11, Wnt5a, and B-catenin sets up what important region - Trunk binds to torso receptor = signaling cascade at pole regions
in the vegetal region? - Produces specialized cytoplasmic region at poles of the egg
○ The Blastula organizer aka Nieuwkoop center
- What 2 signals specify the Nieuwkoop center? Body axes are set up while embryo is still a syncytium
○ VegT and B-catenin - A-P and D-V axes become fully established and start patterning while embryo issyncytial
- What transcription factor is activated by VegT and B-catenin and what blastoderm
does it do? - A-P is divided into head, thorax, abdomen
○ Siamois - Thorax and abdomen get divided further into segments
○ Tells the Nieuwkoop center to induce the Spemann organizer - 2 endoderm regions invaginate to form gut during gastrulation
right above it - D-V axis is divided into 4 regions in early embryogenesis
- Which proteins does VegT signal to induce mesoderm at equator? - Mesoderm = muscle and connective tissue
○ Xenopus Nodal-related proteins (Xnr) - Ventral ectoderm/neuroectoderm = NS and larval epidermis
- What is similar about D-V patterning in fish and frog? - Dorsal ectoderm = larval epidermis
○ Both have cortical rotation that localizes maternal mRNA on - Amnioserosa = extra embryonic membrane on dorsal side
dorsal side
○ B-catenin activates dorsal program in blastoderm Establishment of D-V axis via Dorsal transcription factor
- What is different about D-V patterning in fish and frog? - Dorsal = TF for ventral patterning
○ In the fish, it is unknown how sperm entry affects cortical - Member of NFkB family
rotation - Maternal dorsal mRNA is transcribed and sent to egg
○ Only Wnt5a is a known dorsalizing factor in fish - Localized throughout cytosol
- What do you know about the TGF-B signaling cascade? - Translated 90 after fertilization (late syncytium - syncytial blastoderm stage)
○ Ligands, receptors, and intracellular proteins (SMADs) dimerize - Dorsal protein is bound to Cactus in cytoplasm = dorsal cannot enter nucleus and turn on
○ Short signaling cascade transcription of ventral genes
- What is the ectoderm maternal determinant that is localized in all - The maternal signal Spatzle is secreted into perivitelline space but can only be cleaved on
animal cells? ventral side due to presence of pipe (established in egg by lack of gurken-torpedo signaling)
○ Ectodermin - The Spatzle fragment is a ligand for Toll receptor
○ Adds ubiquitin to Smad4 = degradation = shutting down - The intracellular signaling from Toll receptor causes the release of Dorsal from Cactus
- Through Cactus kinase
pathways that lead to mesoderm formation (BMP and TFG-B)
- What transcription factor maintains regional identity of animal cells? ▪ Leads to degradation of Cactus
○ Fox11e - Therefore, dorsal can enter nucleus and transcribe ventral patterning genes
- Dorsal gradient from ventral to dorsal side of syncytium--> intranuclear concentration that
- What is the endoderm maternal determinant localized in all vegetal
lasts about 2 hours while blastoderm becomes cellular
cells?
○ VegT - Multiple TFs are activated by the presence or absence of Dorsal
- Now the embryo is in cellular blastoderm stage
- True or False: VegT promotes the expression of zygotic genes (like Hox)
- Ventral genes transcribed by Dorsal:
that enable endoderm development and separation from mesoderm?
○ True ▪ Twist = transcription regulator that reinforces and maintains ventral genes
▪ Snail = mesoderm patterning
- Describe the experiment that showed that mesoderm is induced by
▪ Single-minded = mesoectoderm --> small layer between mesoderm and
interactions between vegetal and animal cells in early blastula
neuroectoderm
○ Cultured animal cap in contact with vegetal cells
▪ Rhomboid = neuroectoderm patterning
○ Mesodermal tissues formed
- Dorsal genes (no Dorsal protein)
○ Animal region was pre-labelled with lineage marker
▪ Decapentaplegic = dorsal ectoderm
○ Showed that labeled cells form the mesoderm
□ Forms heterodimer with Screw to signal more strongly in most dorsal
○ Conclusion: vegetal cells induce animal cells to form mesoderm
regions
○ Even when animal and vegetal cells are separated by filter,
□ Many forms of receptors for Dpp and they respond differently as Dpp
mesoderm induction occurs diffuses ventrally
▪ Means that the mesoderm induction does not require □ Inhibited by other secreted proteins in mid-region --> limits range of Dpp
direct cell-cell contact via cell junctions □ Activated in the absence of Dorsal TF
▪ Signals are diffusing from cell-cell through ECM ▪ Tolloid = dorsal ectoderm
- What is the community effect? □ 'protects' Dpp by degrading Sog and Tsg and releasing Dpp
○ A few animal cells placed on vegetal tissue will not be induced to
□ Metalloproteinase
become mesoderm ▪ Zerknult = amnioserosa
○ Needs a larger aggregate of animal cells to respond to induction
▪ Short gastrulation + Twisted gastrulation
- What does 'competence' mean in this context? □ Bind to Dpp and prevent it from binding to its receptors
○ Ability of the animal cells to respond to the mesoderm-inducing
signal Antero-Posterior Patterning
○ Cells lose their competence 11hours after fertilization - bicoid mRNA localizes at anterior end of egg
- At what stage is the mesodermal induction starting? - oskar and nanos mRNA localize to posterior end of egg
○ Mid-gastrula stage - Hunchback and caudal are evenly distributed
○ 5hrs after end of competence period - Protein synthesis starts at fertilization
- At what stage is zygotic expression turned on? - Gradients of bicoid and nanos established first
○ Mid-blastula stage - Bicoid triggers activity of hunchback and inhibits caudal
○ Actually occurs late blastula stage just before gastrulation - Nanos inhibits hunchback synthesis
○ 12-cleavage stage --> embryo has 4096 cells - = gradients of caudal and hunchback are established
- How is zygotic gene expression triggered? - Next step is activation of zygotic genes at cellularization --> Gap genes
○ Increased concentration of DNA
○ As blastula divides, cytoplasm stays the same but the DNA Gap genes further subdivide A-P axis
concentration increases - Gap genes = 1st zygotic genes to be expressed
○ A concentration threshold of DNA has to be reached for - Expression of gap genes is induced by the gradients of maternal bicoid and hunchback genes
transcription of zygotic genes to be initiated - Gap genes are restricted to stripes of expression
- Which genes are the exception to this rule? - This is due to combination of short half -life + inhibition by other gap genes
○ Nodal-related genes - Anterior:
- What kind of tissue does the dorsal vegetal tissue induce? - High levels of Hb + Bcd induce expression of anterior Giant
○ Notochord, muscle - Kruppel is expressed where Hb is declining --> middle of A-P axis (posterior end of
- What kind of tissue does the ventral vegetal tissue induce? anterior region)
○ Blood-forming tissue, little muscle - High levels of Hb without Bcd prevents posterior Knirps and Giant expression in
- Endoderm arises from cells expressing which gene? anterior
○ Ndr ▪ Still have anterior Knirps and Giant expression
- Which genes are activated by VegT? - Caudal protein is highest at posterior --> activates posterior Knirps and Giant
○ Ndr and Derriere (aka GDF3)
- Where is the highest concentration of Ndr and why?
○ In the dorsal region because that’s where you have B-catenin
and VegT
○ B-catenin stimulates ndr transcription
○ This corresponds to the induction of the Spemann organizer
- What gene does ndr stimulate?
○ Smad2
○ Produces a gradients of Smad2 from dorsal to ventral during
early-mid gastrulation
- Which transcription factors are induced by mesoderm-inducing and
patterning signals in all mesoderm cells?
○ Brachyury
○ Eomesodermin Pair-rule genes further divide embryo into 'parasegments'
- Pair-rule genes are laid down in response to gap gene patterns
- What maintains brachyury expression?
- Expressed in a series of 7 stripes
○ FGF family expressed in mesoderm
- Each stripe corresponds to 2nd parasegment
- What is the first zygotic gene to be expressed? Where is it expressed?
- Even skipped (eve) = defines odd-number parasegments
What do those cells become?
- Induced by bicoid and hunchback
○ Goosecoid
- Fushi tarazu (ftz) = defines even numbered parasegments
○ Expressed at dorsal-most mesoderm
- Parasegments are important for laying down segment pattern during gastrulation
○ Becomes Spemann organizer
- They don't align perfectly though
- Which axis replaces the animal-vegetal? - Each segment will eventually include an anterior region from one parasegment and a
○ A-P
posterior region from an adjacent one
- Explain the transplant experiment involving the cells from the
Developmental Bio Page 2
Friday, January 28, 2022 1:31 PM
C.elegans: Sea urchins: Drosophila melanogaster: Mouse - Mus musculus
Advantages to studying them: Advantages to studying them: Advantages to studying them: Fun Facts:
- Invariant lineage - More closely related to vertebrates than the nematodes and flies - Easy to breed - Mice mature quickly --> 9 weeks from fertilized to adult
○ Pattern development and cell division is the same for every embryo - They are deuterostomes - Quick generation time - Small egg - no yolk
- The embryo is transparent ○ Primary invagination of the gut forms anus - Easy to maintain in a lab - Fertilization occurs in the oviduct where cleavage starts
- Develops rapidly - Transparent embryo - Easy to mutate - Egg surrounded by zona pellucida
- Can watch cell division - Easy handling - Cheap ○ Made of glycoproteins and mucopolysaccharides
- Larva hatch after 15 hours - Dipteran insect ('true flies') - Cleavage produces solid ball of bastomeres = morula
- Mature adults after 50 hours Cleavage - Most genes have similar roles in other organisms - Compaction = where cells increase SA in which they are in contact with ea
- Easy to grow on agar plates - Radial holoblastic cleavage ○ Unique event in mammals
- First 2 cleavages are meridional and perpendicular to each other Life Cycle ○ Happens at 8-cell stage
They are hermaphrodites --> the females can make their own sperm for a short time and then - 3rd cleavage = equatorial - Internal fertilization - Cells polarize after compaction
switch to making oocytes ○ Separates animal and vegetal hemispheres - Syncytial blastoderm ○ Exterior has microvilli
- 4th cleavage = asymmetrical - Cellular blastoderm ○ Interior has smooth surfaces
Invariant lineage: ○ The 4 animal cells divide into 8 equal-volumed blastomeres = mesomeres - Gastrulation --> embryo - Tangential and radial cleavage produces 10 internal cells and 20 outer cells
- Pattern of cell division is the same in every embryo ○ The vegetal cells divide unequally ○ Embryogenesis ~12-15hrs - Cells acquire epidermal characteristics and increase cell adhesion via cadhe
- The larva hatch with exact same complement of 558 cells ▪ Produce 4 large macromeres and 4 small micromeres - Hatches into 1st instar --> 3 instar stages
- Lineage is worked out and the cell fate is drawn --> cell-cell interactions can still change cell - The 8 mesomeres divide to produce 2 animal tiers = an1 and an2 - Third instar larva becomes pupa Morula to Blastocyst
lineage - The micromeres divide again to produce 4 larger micromeres and 4 smaller micromeres - Pupa metamorphosis --> adult fly - After morula is formed, the blastocyst is formed from 2 distinct groups of c
- 131 cells undergo apoptosis Egg is laid on decaying plants --> rotting fruit ○ Trophoectoderm = spherical layer of cells outside embryo
- After 4 molts, 959 cells remain ▪ Forms extraembryonic tissue (placenta, etc.)
- No RNA splicing = each gene produces a single protein General body plan is same in embryo, larva, and adult ○ Inner Cell Mass (ICM) = forms epiblast
- Distinct head and tail with repeating units ▪ Can be cultured as embryonic stem cells
Polar bodies form after fertilization ○ 3 segments form thorax □ Can differentiate into all cell types in vitro
- Abortive cleavage before male and female nuclei fuse ○ 8 segments form abdomen - Cavitation = fluid pumped by trophoectoderm to form blastocoel with ICM
○ Not a true cleavage Genetics - ICM divides into 2 by E4.5-E6.5
○ Unknown reason - 14000 protein coding genes ○ The layer that is in contact with the blastocoel is the primitive endod
- True cleavage happens after nuclei fuse - High incidence of RNA splicing ▪ Contributes to extraembryonic structures
Cleavage: - >1100 genes encoding functional RNAs other than mRNAs ○ The rest of the ICM develops into epiblast
- 1st cleavage = asymmetric ▪ Epiblast is the outermost layer of embryo before it gives rise to
○ Generates AB and P1 cell Drosophila Egg mesoderm
○ P1 = stem cell - Oblong ▪ At this stage, embryo is released from zona pellucida and impla
- 2nd cleavage - Sperm is already activated when it enters uterine layer
○ AB gives rise to AB-a and AB-p ○ Females have storage organ for sperm and releases only a few at a time
▪ These cells give rise to hypodermis, neurons, and some muscle cells - Body axis has already begun being specified before sperm enters egg Blastocyst to Onset of Gastrulation
○ P1 gives rise to P2 and EMS Ecto – blue ○ Maternal determinants help to specify body axis - The mural trophoectoderm that is not in contact with the ICM replicates it
▪ EMS gives rise to MS and E Endo– Yellow - Sperm enter micropyle at anterior end without dividing
□ MS gives rise to muscle, glands, coelomocytes, and some neurons ○ Only one can pass at a time ○ This forms Trophoblast Giant cells
Meso – Red
□ E-cells form the gut - Egg has dorsal appendages ▪ Surrounds whole conceptus and invades the uterine wall to inte
○ At this point, axes have established ○ Aid with gas-exchange maternal tissue --> anchoring the blastocyst to uterine wall
▪ P2 = posterior Embryo has 3 bands of cells along animal-vegetal axis - The uterine wall covers the blastocyst
▪ AB-p = dorsal 1. Small and large micromeres at vegetal pole Embryogenesis - The Polar Trophoectoderm that is in contact with the ICM continues to div
- Actin filaments + myosin motor protein = actomyosin - Gives rise to mesoderm = Forms primary mesenchyme --> skeleton - After nuclei fuse, zygotic nucleus undergoes mitotic divisions every 9 minutes the ectoplacental cone
○ Assembles at egg cortex ○ (9 divisions until syncytial blastoderm) ○ That will then form the Chorion and extraembryonic ectoderm
2. Vegetal plate --> Veg1 and Veg2
○ Interacts with centrosome - Gives rise to endoderm and mesoderm (and some ectoderm) - No initial cleavage of cytoplasm ▪ Both contribute to 'placenta'
○ Contract asymmetrically away from sperm entry point - Secondary mesenchyme --> muscles and connective tissue - Early embryo = syncytial blastoderm - Inside the trophoectoderm, the parietal endoderm (came from cells of pri
- Cortical components flow to anterior ○ Nuclei move to the periphery endoderm) migrates to cover inner surface of mural trophoectoderm
3. Mesomeres (remainder)
- Cytoplasmic components flow to posterior - Give rise to ectoderm --> outer epithelium and neural cells ○ Large proteins can diffuse between nuclei during first 3 hours - The visceral endoderm covers the elongating egg cylinder that has epiblas
- PAR proteins = maternal cortical proteins at posterior and anterior end --> partition cell - Eventually divided into oral and aboral ○ At the syncytial stage, some nuclei localize posteriorly and become surrounded by PM - Epiblast = cup-shaped layer of 1000 epithelial cells --> develops into embr
○ Par genes = protein kinases + other signaling proteins Micromeres = organizer region to form pole cells - The primitive streak appears at E6.5 --> marks A-P axis of embryo at begin
○ Controls placement of mitotic spindle in early embryo ▪ Pole cells will end up on the outside of the blastoderm and eventually give rise gastrulation
○ PAR-3 & PAR-6 = anterior - Cell cleavage = invariant for the first 9-10 divisions to the germ cells ○ At the posterior
○ PAR-1 and PAR-2 = posterior - Development requires both animal and vegetal halves ▪ Blastoderm gives rise to somatic cells ○ Elongates until it reaches bottom of cup
○ PAR proteins regulate the forces that pull on each side of the spindle = helps cleavage - If isolated at the 4-cell stage, any of those cells can give rise to Pluteus ▪ Pole cells are set aside early and are protected from somatic cell inductive ○ By then the Node is visible
- Centrosome: - At the 8 cell stage, animal-vegetal axis is established signals ▪ Condensation of cells
○ Composed of microtubules ▪ If you isolate animal half --> only ectoderm is formed - Cellular blastoderm arises after ~3 hrs (14 mitotic divisions) ▪ Corresponds to Hensen's Node
○ Organizes the microtubule cytoskeleton ▪ If you isolate only vegetal half --> forms large gut and reduced ectoderm = abnormal ○ Membranes form around nuclei = compartmentalize □ Analogous to Spemann's organizer
○ Helps separate chromosomes into the daughter cells - Gastrulation = dramatic cellular migrations to form germ layers
○ Duplicates to form centrosome and nucleates each microtubule spindle pole to The vegetal region organizes the body axis ○ Endoderm = gut Gastrulation and Neurulation
organize chromosome distribution in mitosis and meiosis - Micromeres act as Spermann organizer --> induces formation of body axis ▪ 2 regions: posterior and anterior that will fuse in the middle - Similar to chicken gastrulation but harder to visualize due to having differe
- Maternal factors located in micromeres from 4th cleavage ○ Mesoderm = muscle, connective tissue, heart, and blood ○ Ingression through primitive streak
Antero-posterior Axis - B-catenin --> specifies micromeres ▪ At most ventral region - Epiblasts converge on primitive streak
- Sperm entry determines polarity of axis of fertilized egg - The organizer specifies and sets boundaries for endoderm and ectoderm ▪ Invaginates to form furrow along ventral midline --> internalizes and initially ○ Proliferating cells then migrate and spread laterally & anteriorly be
○ Determines 1st position of cleavage - Signals to macromeres to become endomesodermal cells --> give rise to skeletal rods forms tube ectoderm and visceral endoderm --> forming mesoderm
- Actin filaments help to specify the anterior end --> cap of actin filaments at anterior ▪ Then separates from tube and moves under ectoderm to form muscles and ○ Cells migrating to anterior form Node form prechordal plate mesode
- P-granules specify the posterior Cell fate connective tissue head process
○ P-granules are cytoplasmic constituents containing maternal mRNAs, proteins, etc. - Concentrations of B-catenin are high in the micromere nuclei ▪ Subdivided into Visceral and Somatic ○ Just like chicken, as the primitive streak regresses, the trunk notocho
needed for germline development - B-catenin = coactivator of TF TCF --> activates transcription of vegetal-specific genes □ Visceral = connective tissues associated with gut + organs down --> spinal cord develops above it
○ P-granules remain in P-daughter cells --> P4 gives rise to germline - B-catenin is absent in animal cells --> future ectoderm □ Somatic = muscles ▪ Somites form on either side
○ # of germline cells = variable - Dishevelled protein = stabilizes B-catenin in vegetal region ○ Ectoderm = skin and NS - Primitive streak extends to the bottom and the node is formed
○ # somatic cells = 959 - B-catenin and a TF called Otx help to activate the pmar-1 gene in micromeres ▪ Ectodermal cells from ventral region leave individually to form neuroblasts - Anterior ectoderm becomes anterior Neural plate = Brain
- Asymmetry is determined at fertilization --> determined by sperm entry - Pmar-1 = TF exclusive to micromeres between mesoderm and remaining ectoderm ○ Anterior of embryo grows and head fold appears
▪ Helps express organizer function ▪ Forms foregut and hindgut - Notochord begins to be laid down in trunk
Gastrulation: ▪ Helps specify micromeres so that micromeres can specify neighboring cells ▪ Forms epidermis --> produces a thin cuticle made of chitin once the epidermal - Further development = :
- Occurs at 28 cell stage ▪ Helps turn on micromere-specification genes = skeletogenic genes cells stop dividing ○ Distinct head
- 100 mins after fertilization ▪ TF that represses HesC --> HesC represses the genes that specify skeletogenic genes ○ Starts 3hrs after fertilization ○ Neural fold formation
- Occurs when E-cell descendants moves inside embryo and form gut □ Only micromeres express pmar-1 = only micromeres can become skeletal ○ Main nerve cord lies ventrally --> different from vertebrates ○ Fore + hind gut closures
- Apoptosis removes some early cells - Micromeres produce an Early Signal (ES) - Parasegmentation ○ Somite formation
- Directed dilation = increase in hydrostatic pressure - Induces Veg2 cells to adopt endomesoderm state ○ Central blastoderm aka Germ Band After gastrulation + neurulation are complete, embryo undergoes process of Tu
○ Causes shape change - After the 7th cleavage, micromeres send 2nd signal Delta ligand to Notch receptor on ▪ Main trunk region of embryo that extends anterior-posteriorly - Before this happens the embryo is doing an extreme backbend
○ Embryo elongates rapidly along the antero-posterior axis adjacent Veg2 cells = turns on mesoderm specification genes ▪ Segmentation starts here ○ Ventral side (belly) of embryo is on the outside to start
- At 80 cell stage ▪ Specifies them as secondary mesenchyme and pigment cells ▪ Series of evenly-spaced grooves = parasegments - After Turning = embryo can undergo organogenesis
○ Cells from different lineages that will form organs will cluster together - Delta-Notch signal also helps specify endoderm ○ Parasegments = developmental basis of segments
○ Genes responsible for organ identity will be expressed - Wnt-8 signal is secreted by Veg2 = autocrine ▪ Segments = fusion of anterior and posterior halves of parasegments
▪ Pha-4 = pharynx development - Maintains B-catenin stabilization = maintains micromere function ▪ 14 parasegments = 7 segments
- Wnt-8 also acts paracrine = sends signal to Veg1 cells to turn on endoderm specification genes □ 3 form mouth
Dorso-Ventral Axis - If you inhibit degradation of B-catenin = the embryo is vegetalized □ 3 form thorax
- Cell-cell crosstalk determines dorso-ventral axis - All presumptive ectoderm transforms into endoderm □ 8 form abdomen
- Cell-cell interactions can still affect cell fate = dorso-ventral polarity is not fixed - If you inhibit B-catenin accumulation = embryo is animalized
- Left-right axis is not fixed in relation to antero-posterior or dorso-ventral axes - Prevents formation of endoderm and mesoderm Larval Stages
○ Unknown molecular mechanism that determines handedness - Micromeres have a fixed fate = only become primary mesenchyme - Hatch out of egg in 24hrs
- Experiments done to show that these cells are not specified early on - Will still become primary mesenchyme even if micromeres are grafted on another part of the - Head is largely hidden at this stage
○ Push/rotate AB-a = reverse the dorso-ventral axis and P1 daughter EMS is inverted embryo - Acron = specialized head structure at anterior
relative to AB cells - Macromeres do not have a fixed fate when they are formed - Telson = posterior end of larva
▪ Develops normally but inverted axes - At 16-cell stage, they can become endo, meso, or ecto - Denticles = tooth-like outgrowths on ventral side of larva
○ Push left anterior daughter cell of AB-a to be posterior of right AB-a = reverses - At 60-cell stage, Veg2 can only become meso or endo --> never ecto ○ Aids in locomotion
handedness ▪ Meso = secondary mesenchyme - Larva molts twice --> 3 instar stages until pupa
○ Mutations in gpa-16 causes randomization of cleavage spindle orientation = 50% of - Veg1 can only give rise to endo or ecto but never meso - Imaginal discs = group of prospective epidermal cells that will differentiate into organs
worms have right-left interchange ▪ Means that the mesoendoderm specification has already happened and now the endo- during metamorphosis
▪ GPA-16 = G-protein alpha subunit --> works with proteins to position centrosome ecto boundary has yet to be made ○ Imaginal discs for each of the 6 legs, 2 wings, 2 halteres, genital apparatus, eyes,
antennae, etc.
Asymmetric divisions + cell-cell interactions specify cell fate Oral-Aboral Axis - Histoblasts = small group of cells that are dormant in larval stage but proliferate in pupa
- Lineage positions the cells to interact with each other - Perpendicular to animal-vegetal axis stage
- P2 helps specify AB-p - Defines bilateral symmetry of Pluteus larva --> demarcated by larva's mouth ○ Develops to form epidermis of adult abdomen
○ Through interactions with ligand on P2 called APX-1 and receptor on AB-p called GLP-1 - Corresponds to dorso-ventral axis in frogs and fish
▪ GLP-1 = Notch receptor - Animal-vegetal axis = antero-posterior axis Maternal factors guide early stages embryonic development
▪ APX-1 = Delta ligand - Cell cleavage happens in relation to animal-vegetal axis - Maternal mRNAs translate into TFs and transcribe zygotic genes
○ Remove P2 = all AB cells become AB-a - B-catenin accumulation marks the posterior end = vegetal
○ Remove P1 = no AB-a are not made??? - Nodal signaling = oral Antero-Posterior Axis:
- P2 also polarizes EMS cells --> affects daughter cells - BMP signaling = aboral
○ E-cells = posterior - Oral ectoderm cells give rise to Stomodeum 1. Maternal genes --> establish pattern before cellularization
○ MS = anterior - Epithelium that forms the outer layer in the oral region and the neurogenic structures - Bicoid
○ SKN-1 - Aboral ectoderm cells give rise to epithelium that covers larva body - Hunchback
▪ Maternal determinant --> mRNA for skn-1 found uniformly in 2-cell stage but - Oral-aboral axis is related to animal-vegetal axis = important concept - Nanos
higher levels of the SKN-1 protein in the P1 nucleus than AB - Through indirect effect of B-catenin localization in vegetal nuclei - Caudal
▪ Turns on TF med-1 and med-2 - B-catenin accumulation marks posterior end 2. Gap genes = first zygotic patterning genes to be expressed--> establish 'regions'
□ Necessary for MS and E fate - Oral-aboral axis establishes after animal-vegetal axis - Hunchback
▪ Abolish SKN-1 = mesoendodermal cells instead become mesoectodermal - The 2 axes are linked spatially and temporally - Knirps
▪ It's not that SKN-1 is released by P2 to the EMS cells, it's that the skn-1 gene is - Oral ectoderm = organizing region for oral-aboral axis - Kruppel
expressed in EMS cells and is important to give rise to the pharyngeal cells - High Nodal signaling in oral ectoderm - Giant
□ After the 1st cell cleavage --> only P1 cells can give rise to pharyngeal cells - Causes expression of TF Goosecoid = TF that suppresses oral fate in aboral ectoderm - Tailless
□ After 2nd cleavage --> only EMS cells can give rise to pharyngeal - Growth factor BMP-2/4 diffuses from oral ectoderm and induces aboral fate 3. Pair-rule genes --> establish parasegments
□ After 3rd --> only MS has intrinsic ability to generate pharyngeal tissue - Lefty = member of TGF-B family - Eve (even-skipped)
□ Homozygous skn-1 mutants lack pharyngeal and intestinal structures - Antagonizes Nodal signaling - Fushi tarazu (Ftz)
□ The EMS cells are respecified to be similar to C cells --> ectodermal - Restricts Nodal signaling activity to oral ectoderm 4. Parasegment genes --> establish segmentation genes
○ Remove P2 at 4-cell stage = all EMS cells become MS and there will be no E cells (no - If Nodal is blocked, differentiation of ectoderm into oral and aboral is also blocked - Wingless
endoderm) - If Lefty is blocked, all ectoderm is converted to oral - Hedgehog
○ P2 sends Wnt signal (MOM-2) to EMS cell --> received by frizzle receptor (MOM-5) on 5. Hox genes
EMS
▪ Instructs EMS to become E cell Dorso-Ventral Axis:
▪ The goal of this Wnt signaling cascade is to down-regulate expression of the
pop-1 gene in the cell that will become E cell 1. Maternal genes --> establish pattern before cellularization
▪ Embryos that lack pop-1 gene end up producing 2 E daughter cells from the - Pipe
EMS cell - Toll
○ POP-1 protein = TF --> it is the nematodes version of the TCF - Dorsal-cactus
- Spatzle
▪ WRM-1 and SYS-1 are nematode-specific B-catenins
▪ LIT-1 = protein kinase that will phosphorylate POP-1 --> leads to export of POP-1
2. Zygotic genes that further divide D-V axis into dermal layers
from the nucleus - Zerknullt - amnioserosa
○ When MOM-2 binds to MOM-5, the LIT-1 and WRM-1 will enter the nucleus and cause - Decantaplegic + tolloid - dorsal ectoderm
exportation of POP-1 from nucleus - Rhomboid - neuroectoderm
- Twist + snail - mesoderm
▪ The reduced amount of POP-1 in the nucleus will allow for SYS-1 to enter and act
as a coactivator to switch on genes required to specify an E cell - Single-minded - mesoectoderm
○ The MS cell does not have accumulation of LIT-1, WRM-1, or SYS-1 in the nucleus,
therefore, the levels of POP-1 remain high = target genes are suppressed = cell
specified as MS
▪ Higher POP-1 level in anterior vs posterior
Hox Genes
- Specify positional identity of antero-posterior axis
- Evolutionarily conserved --> found in most organisms
- Encode a set of TFs that are involved in specifying positional identity of cells along axis
- Topologically arranged
- Ceh-13 = required for anterior organization of embryo
○ Only Hox gene essential for embryonic development
▪ The other hox genes carry out their functions at larval stage
- Lin-39 = controls mid-body cell fate
○ Also regulates vulva development
○ Mutation in this gene = larval mid-body cells instead express characteristics of anterior
and posterior
- Mab-5 and egl-5 = controls posterior regions and provides positional info for posterior cells
- Php-3 and nob-1 = posterior group genes
Temporal aspect of gene expression and development
- Heterochronic mutations
○ Mutations that alter the timing of developmental events
○ Lin-4 and Lin-14 = 1st 2 heterochronic genes
○ Lin-14 is a TF that controls lineage of T-blast cells in larval stages 1-4
▪ High levels of LIN-14 = larval 1 stage
▪ Medium levels = larval 2 stage
▪ Low levels = L3 stage
○ Mutations in lin-14 affect timing of development The determination of body plan begins in the ovary
▪ Gain of function mutation = over-development, non-sequential, and repeating
- Germarium contains stem cells (2n) that divide asymmetrically to produce 1 stem cell and 1
□ Kind of 'stuck' or repeating L1 stage where there was high levels of LIN-14
cystoblast
▪ Loss of function mutation = precocious development
- Cystoblast is also a stem cell, but it will divide mitotically and give rise to the egg
□ Kind of like skipping steps in development
▪ Has 4 mitotic divisions --> produces 16 cells with cytoplasmic bridges = germline
□ Similar to L3 levels of LIN-14
○ Lin-4 is a microRNA that controls expression of lin-14 by repressing it cyst
▪ The posterior cell becomes the egg and the other 15 cells become nurse cells
▪ Loss of function mutation in lin-4 leads to over-expression of lin-14
□ Nurse cells produce maternal messages --> mRNAs and proteins to
□ Similar effect as GOF mutation in lin-14
support embryo development
Maternal messages sent to egg through cytoplasmic bridges
Vulval Development
- Develops in last larval stage ◊ Example: gurken
- Follicle cells (somatic ovarian cells) form a sheath around nurse cells and egg = Egg
- Contains 22 cells derived from AB blastomeres --> ectodermal AB
chamber
- P5p, P6p, P7p = give rise to vulva
○ Different P cells than in early embryo ▪ Multiple egg chambers are strung together by stalks of follicle cells
○ P6p = primary ▪ Egg chambers have distinct polarity
- The antero-posterior polarity of the egg is the result of signalingfrom the anterior of the
○ P5p and P7p = secondary
older egg chamber to the posterior of the younger egg chamber
○ Tertiary cells = P cells that fuse to become hypodermis --> non-vulval
- Through Notch-Delta
- Lin-39 = hox gene that prevents the P5p, P6p, and P7p from fusing into the hypodermis
- Germline cells produce Delta which binds to Notch on follicle cells
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, support embryo development
Maternal messages sent to egg through cytoplasmic bridges
Vulval Development
◊ Example: gurken
- Develops in last larval stage
- Follicle cells (somatic ovarian cells) form a sheath around nurse cells and egg = Egg
- Contains 22 cells derived from AB blastomeres --> ectodermal AB
chamber
- P5p, P6p, P7p = give rise to vulva
○ Different P cells than in early embryo ▪ Multiple egg chambers are strung together by stalks of follicle cells
○ P6p = primary ▪ Egg chambers have distinct polarity
- The antero-posterior polarity of the egg is the result of signalingfrom the anterior of the
○ P5p and P7p = secondary
older egg chamber to the posterior of the younger egg chamber
○ Tertiary cells = P cells that fuse to become hypodermis --> non-vulval
- Through Notch-Delta
- Lin-39 = hox gene that prevents the P5p, P6p, and P7p from fusing into the hypodermis
- Germline cells produce Delta which binds to Notch on follicle cells
- The 3 cell lineages that give rise to the vulva divide, move, and then fuse to become 7
▪ Specifies anterior follicle cells as Polar follicle cells
concentric rings stacked on each other = forms conical structure
- Polar cells release signal called Unpaired --> stimulates receptors on adjacent follicle
- Gonadal anchor cell specifies the fate of the 3 cells (P5p, P6p, P7p) with inductive signal
cells to activate the JAK-STAT pathway
○ Secretes a growth factor (similar to EGF in animals)
▪ Causes follicle cells to become stalk
▪ Product of lin-3 gene = LIN-3 inductive signal
▪ Specifies anterior and posterior follicle cells
▪ Binds to nematode version of tyrosine kinase receptor EGF (LET-23)
- Stalk cells upregulate cadherin expression to keep the younger egg at the posterior
▪ Mutations in lin-3 = no vulva formation
end
○ Most of the signal is received by P6p = most strongly activated
▪ P6p sends lateral signal to P5p and P7p = received by LIN-12 (Notch receptor)
□ Lateral signal = 3 proteins from Delta family
- Wnt signaling is also involved in this --> involves another B-catenin called BAR-1
As a germline cyst buds from the germarium, it signals through the Delta–Notch pathway
(small red arrows) to induce the formation of anterior polar cells (red). These, in turn, signal
Questions for final exam prep:
to the adjacent cells anterior to them and induce them to become stalk (green). Signals from
the stalk induce adjacent follicle cells in the younger cyst to become posterior polar cells
- What are some differences between the animal and vegetal poles of
(red), and induce the younger cyst to round up and the oocyte to become positioned at the
the Xenopus egg?
posterior of the egg chamber. Signals from anterior and posterior polar cells then specify
- What kinds of maternal determinants are laid down in the frog egg and
adjacent follicle cells as posterior and anterior follicle cells, respectively (shown in more
how does that differ from drosophila?
detail in Fig. 2.18). The yellow arrows indicate the overall direction of signaling from older to
○ Maternal mRNAs and proteins
younger egg chambers.
○ Drosophila don’t have proteins laid down by the mother
- Where does ectoderm, mesoderm, and endoderm arise from? Maternal mRNAs are involved in antero-posterior specification
○ Ectoderm from animal cells
- The egg moves to the posterior end of the egg chamber with the help of maternal mRNAs
○ Endoderm from vegetal cells
from the future anterior end
○ Mesoderm comes from signals from vegetal cells acting on - mRNAs are deposited by nurse cells to the future posterior end
animal cells - This is done by reorientation of microtubule cytoskeleton (on which maternal mRNAs
▪ Band of cells around the equator are transported)
- When is most of the embryo's mRNA transcribed? ▪ Dependent on PAR proteins
○ Mid-blastula transition stage - Egg nucleus is also positioned posteriorly
- How do maternal mRNAs localize to their respective places on the - Maternal gurken mRNA is located posterior --> translated to give local concentration of
animal-vegetal axis? (2 routes) Gurken near posterior end of egg nucleus
○ METRO - message transport organizer region - Gurken = member of TGF-a family
▪ Involves association of mRNAs with the cytoskeleton and - Gurken protein is secreted across the egg membrane --> induces adjacent terminal
small vesicles of ER follicle cells to adopt posterior fate
○ Microtubules through kinesin motor proteins ▪ This is done through locally stimulating the Torpedo receptor that is present on
- What maternal mRNA is localized in the vegetal cortex of mature follicle cells
oocytes? Gurken activates follicle cells = initiates formation of both axes
○ Vg1 - Gurken = key initiator for both A-P and D-V formation
- When is Vg1 translated and what is it a signal for? - Diffusible protein ligand
○ Translated after fertilization - In all cases, gurken mRNA is first transcribed by follicle cells and transferred to the egg
○ Signals mesoderm induction (maternal message!)
- What is one of the earliest signals required for D-V specification? - The mRNA is moved to posterior then it gets translated, secreted, and the signal
○ Wnt11 diffuses to bind to torpedo receptors on nearby follicle cells
○ Dorsalizing factor deposited in dorsal end after fertilization
- What transcription factor is localized in the vegetal pole and specifies Maternal mRNA movement in the egg is dependent on microtubule re-orientation
endoderm and mesoderm? - Bicoid mRNA is produced by nurse cells then moved to anterior
○ VegT - Not translated until after fertilization
○ Translated after fertilization - Transported along microtubules by action of motor protein dynein
- What determines D-V axis? - Oskar mRNA moves to posterior end
○ Sperm entry - At posterior, oskar gets translated and organizes the posterior pole cytoplasm (pole
- What is cortical rotation and why is it important? plasm)
○ 30 degree rotation of cortex after sperm penetrates oocyte - Promotes germ cell formation
○ Causes an array of microtubules under the cortex to reorient - Recruits patterning proteins - including nanos mRNA
themselves so that their plus ends are pointing away from the - Transported along microtubules by action of motor protein kinesin
site of sperm entry - Maternal gurken accumulates near egg nucleus --> involved in posterior follicle patterning
▪ This acts as tracks for maternal mRNAs to localize to their - New posterior follicle cells signal to egg --> moves nucleus toward anterior via
respective poles microtubules
○ Without this process, there would be no dorsal structures, as this
is necessary for dorsalizing factors to localize to dorsal end Dorso-ventral axis is initiated in egg
- Dsh and Wnt11 are maternal mRNAs that are dorsalizing factors - Achieved after posterior end of the egg is specified
- Why is it important to set up the D-V axis? - Gurken is involved in D-V axis initiation too because the posterior localization of Gurken
○ Need the D-V axis so the organizer region (dorsal lip) can be specifies the posterior follicle cells which express torpedo (receptor for gurken)
established - The newly specified posterior follicle cells signal to the egg to move the nucleus towards
○ Need to do this before germ specification anterior
- What are the components of the Wnt/B-catenin pathway? - Gurken induces dorsal follicle cells because torpedo causes dorsal morphology
○ Wnt11 = secreted ligand - There is a torpedo signaling cascade in the follicle cells that restricts the production ofPipe
○ TCF3 = transcription factor that B-catenin activates protein to the ventral side
○ GSK-B = kinase that tags B-catenin for degradation - Gurken binds to torpedo = inhibition of pipe synthesis --> dorsal cell fate
○ Dsh = prevents formation of destruction complex when activated - No gurken = no gurken binding to torpedo = no inhibition of pipe synthesis = pipe
▪ Prevents degradation of B-catenin synthesis --> ventral cell fate
- What would be the implication if the Frizzled receptor were blocked?
○ Wnt pathway would be inhibited Specifying A-P extremities of embryo begins in egg
○ Dorsal structures would not develop - Trunk gets fragmented and binds to Torso at extremities
- What maternal protein/gene prevents Wnt signaling in ventral region? - Torso is technically found everywhere on egg surface but trunk is only fragmented at
Wnt antagonist the poles (unknown why)
○ Dickkopf1 (Dkk1) - Trunk is secreted into perivitelline space by nurse cells
- Activity of Wnt11, Wnt5a, and B-catenin sets up what important region - Trunk binds to torso receptor = signaling cascade at pole regions
in the vegetal region? - Produces specialized cytoplasmic region at poles of the egg
○ The Blastula organizer aka Nieuwkoop center
- What 2 signals specify the Nieuwkoop center? Body axes are set up while embryo is still a syncytium
○ VegT and B-catenin - A-P and D-V axes become fully established and start patterning while embryo issyncytial
- What transcription factor is activated by VegT and B-catenin and what blastoderm
does it do? - A-P is divided into head, thorax, abdomen
○ Siamois - Thorax and abdomen get divided further into segments
○ Tells the Nieuwkoop center to induce the Spemann organizer - 2 endoderm regions invaginate to form gut during gastrulation
right above it - D-V axis is divided into 4 regions in early embryogenesis
- Which proteins does VegT signal to induce mesoderm at equator? - Mesoderm = muscle and connective tissue
○ Xenopus Nodal-related proteins (Xnr) - Ventral ectoderm/neuroectoderm = NS and larval epidermis
- What is similar about D-V patterning in fish and frog? - Dorsal ectoderm = larval epidermis
○ Both have cortical rotation that localizes maternal mRNA on - Amnioserosa = extra embryonic membrane on dorsal side
dorsal side
○ B-catenin activates dorsal program in blastoderm Establishment of D-V axis via Dorsal transcription factor
- What is different about D-V patterning in fish and frog? - Dorsal = TF for ventral patterning
○ In the fish, it is unknown how sperm entry affects cortical - Member of NFkB family
rotation - Maternal dorsal mRNA is transcribed and sent to egg
○ Only Wnt5a is a known dorsalizing factor in fish - Localized throughout cytosol
- What do you know about the TGF-B signaling cascade? - Translated 90 after fertilization (late syncytium - syncytial blastoderm stage)
○ Ligands, receptors, and intracellular proteins (SMADs) dimerize - Dorsal protein is bound to Cactus in cytoplasm = dorsal cannot enter nucleus and turn on
○ Short signaling cascade transcription of ventral genes
- What is the ectoderm maternal determinant that is localized in all - The maternal signal Spatzle is secreted into perivitelline space but can only be cleaved on
animal cells? ventral side due to presence of pipe (established in egg by lack of gurken-torpedo signaling)
○ Ectodermin - The Spatzle fragment is a ligand for Toll receptor
○ Adds ubiquitin to Smad4 = degradation = shutting down - The intracellular signaling from Toll receptor causes the release of Dorsal from Cactus
- Through Cactus kinase
pathways that lead to mesoderm formation (BMP and TFG-B)
- What transcription factor maintains regional identity of animal cells? ▪ Leads to degradation of Cactus
○ Fox11e - Therefore, dorsal can enter nucleus and transcribe ventral patterning genes
- Dorsal gradient from ventral to dorsal side of syncytium--> intranuclear concentration that
- What is the endoderm maternal determinant localized in all vegetal
lasts about 2 hours while blastoderm becomes cellular
cells?
○ VegT - Multiple TFs are activated by the presence or absence of Dorsal
- Now the embryo is in cellular blastoderm stage
- True or False: VegT promotes the expression of zygotic genes (like Hox)
- Ventral genes transcribed by Dorsal:
that enable endoderm development and separation from mesoderm?
○ True ▪ Twist = transcription regulator that reinforces and maintains ventral genes
▪ Snail = mesoderm patterning
- Describe the experiment that showed that mesoderm is induced by
▪ Single-minded = mesoectoderm --> small layer between mesoderm and
interactions between vegetal and animal cells in early blastula
neuroectoderm
○ Cultured animal cap in contact with vegetal cells
▪ Rhomboid = neuroectoderm patterning
○ Mesodermal tissues formed
- Dorsal genes (no Dorsal protein)
○ Animal region was pre-labelled with lineage marker
▪ Decapentaplegic = dorsal ectoderm
○ Showed that labeled cells form the mesoderm
□ Forms heterodimer with Screw to signal more strongly in most dorsal
○ Conclusion: vegetal cells induce animal cells to form mesoderm
regions
○ Even when animal and vegetal cells are separated by filter,
□ Many forms of receptors for Dpp and they respond differently as Dpp
mesoderm induction occurs diffuses ventrally
▪ Means that the mesoderm induction does not require □ Inhibited by other secreted proteins in mid-region --> limits range of Dpp
direct cell-cell contact via cell junctions □ Activated in the absence of Dorsal TF
▪ Signals are diffusing from cell-cell through ECM ▪ Tolloid = dorsal ectoderm
- What is the community effect? □ 'protects' Dpp by degrading Sog and Tsg and releasing Dpp
○ A few animal cells placed on vegetal tissue will not be induced to
□ Metalloproteinase
become mesoderm ▪ Zerknult = amnioserosa
○ Needs a larger aggregate of animal cells to respond to induction
▪ Short gastrulation + Twisted gastrulation
- What does 'competence' mean in this context? □ Bind to Dpp and prevent it from binding to its receptors
○ Ability of the animal cells to respond to the mesoderm-inducing
signal Antero-Posterior Patterning
○ Cells lose their competence 11hours after fertilization - bicoid mRNA localizes at anterior end of egg
- At what stage is the mesodermal induction starting? - oskar and nanos mRNA localize to posterior end of egg
○ Mid-gastrula stage - Hunchback and caudal are evenly distributed
○ 5hrs after end of competence period - Protein synthesis starts at fertilization
- At what stage is zygotic expression turned on? - Gradients of bicoid and nanos established first
○ Mid-blastula stage - Bicoid triggers activity of hunchback and inhibits caudal
○ Actually occurs late blastula stage just before gastrulation - Nanos inhibits hunchback synthesis
○ 12-cleavage stage --> embryo has 4096 cells - = gradients of caudal and hunchback are established
- How is zygotic gene expression triggered? - Next step is activation of zygotic genes at cellularization --> Gap genes
○ Increased concentration of DNA
○ As blastula divides, cytoplasm stays the same but the DNA Gap genes further subdivide A-P axis
concentration increases - Gap genes = 1st zygotic genes to be expressed
○ A concentration threshold of DNA has to be reached for - Expression of gap genes is induced by the gradients of maternal bicoid and hunchback genes
transcription of zygotic genes to be initiated - Gap genes are restricted to stripes of expression
- Which genes are the exception to this rule? - This is due to combination of short half -life + inhibition by other gap genes
○ Nodal-related genes - Anterior:
- What kind of tissue does the dorsal vegetal tissue induce? - High levels of Hb + Bcd induce expression of anterior Giant
○ Notochord, muscle - Kruppel is expressed where Hb is declining --> middle of A-P axis (posterior end of
- What kind of tissue does the ventral vegetal tissue induce? anterior region)
○ Blood-forming tissue, little muscle - High levels of Hb without Bcd prevents posterior Knirps and Giant expression in
- Endoderm arises from cells expressing which gene? anterior
○ Ndr ▪ Still have anterior Knirps and Giant expression
- Which genes are activated by VegT? - Caudal protein is highest at posterior --> activates posterior Knirps and Giant
○ Ndr and Derriere (aka GDF3)
- Where is the highest concentration of Ndr and why?
○ In the dorsal region because that’s where you have B-catenin
and VegT
○ B-catenin stimulates ndr transcription
○ This corresponds to the induction of the Spemann organizer
- What gene does ndr stimulate?
○ Smad2
○ Produces a gradients of Smad2 from dorsal to ventral during
early-mid gastrulation
- Which transcription factors are induced by mesoderm-inducing and
patterning signals in all mesoderm cells?
○ Brachyury
○ Eomesodermin Pair-rule genes further divide embryo into 'parasegments'
- Pair-rule genes are laid down in response to gap gene patterns
- What maintains brachyury expression?
- Expressed in a series of 7 stripes
○ FGF family expressed in mesoderm
- Each stripe corresponds to 2nd parasegment
- What is the first zygotic gene to be expressed? Where is it expressed?
- Even skipped (eve) = defines odd-number parasegments
What do those cells become?
- Induced by bicoid and hunchback
○ Goosecoid
- Fushi tarazu (ftz) = defines even numbered parasegments
○ Expressed at dorsal-most mesoderm
- Parasegments are important for laying down segment pattern during gastrulation
○ Becomes Spemann organizer
- They don't align perfectly though
- Which axis replaces the animal-vegetal? - Each segment will eventually include an anterior region from one parasegment and a
○ A-P
posterior region from an adjacent one
- Explain the transplant experiment involving the cells from the
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