Ryan Lecture 4 Flashcards
What is important in forming a-p axis in chick embryos
Gravity is important
Egg spins in hen ~10-15 times/hr for 20 hrs
What does egg spinning do - a-p chick embryos
Organizes heavier to lighter particles within developing embryo
Lighter yolk components lie on one side of blastoderm = become POSTerior
Primitive streak begins at posterior and extends anteriorly
How many eggs do chicks lay
One egg per day
Only one oviduct develops, not bilateral
What happens during chick gastrulation
Primitive streak regresses
Ant side= hensens node
Describe primitive streak in chick embryos
Extends from post towards ant
Hensens node = organizer region at ant end
What happens when cut chick embryo like a pie
Each wedge will form primitive streak
Has dorsal ventral axis
Early chick embryo is radially symmetrical
Tissues around = competent to respond to signals coming from primitive streak
Describe the new ectopic streak - chick embryos
Forms in ant half, expresses similar molecular markers as endogenous primitive streak (brachyury)
If split into ant and post half =
Ant has new ectopic primitive streak, functions as normal, same gene expression
Post= endogenous primitive streak
What is important for primitive streak - a-p axis formation - exp gen
Vg1
Do exp = vg1 expressing cells placed in ant region, 180 to koller’s sickle (endogenous primitive streak)
Can use cell pellet= groups of cells
Could also use beads soaked in protein vg1
What is important for primitive streak - a-p axis formation - formally set up
Experiment:
1. Transplant cells expressing signalling molecule at the opposite side of the embryo from
Koller’s sickle.
2. Allow embryos to develop until primitive streak is formed.
3. Perform whole mount in situ hybridization experiment to monitor expression of cNodal that is
normally expressed in the primitive streak
What is important for primitive streak - a-p axis formation - results
Monitor nodal expression in primitive streak
If do vg1 + wnt = nodal expression induced by vg1
Multiple genes looked at - many diff results
What happens if place vg1 pellet 90 degrees to koller’s sickle
Can also induce primitive streak BUT no expression endogenous streak
Position important
Summarize effects of pellet vg1 for experiments
Vg1 important for primitive streak formation
Position of pellet matters
If placed at ant end = both ectopic and endogenous primitive streaks form
If placed on lateral margin = only ectopic primitive streak forms, formation of endogenous streak inhibited (by cells, bc only want one streak, if too close=would have to share tissues, think Siamese twins)
What happens if 2 vg1 pellets implanted - results
Implant one then wait and implant second 180 degrees from first
Implanted before endogenous streak formed
If both bead added at t=0 = 2 streaks
If 2nd bead added at t=4hr =1st streak more developed than second
If 2nd bead added at t=6h = only get one streak, no induction of other
What happens if 2 vg1 pellets implanted - conclusions
Depends on timing of second 2nd implant
Suggests that vg1 induces both primitive streak and expression of a molecule that inhibits induction other primitive streak
Describe a-p axis in mouse
1st definitive axis in mouse embryo
Primitive streak and node form on opposite side of embryo from AVE
What does a-p axis in mouse depend on
2 signaling centres are required to pattern anterior end of embryo =
Node (organizer region located at ant end primitive streak)
Anterior visceral endoderm = AVE
Describe AVE - mouse a-p
Derived by distal visceral endoderm (DVE)
Position of AVE determined in part by physical constraints of uterus - if remove physical constraints… embryos develop without a-p axis
Describe expression patterns in a-p polarity of mouse
Dve = shifts to become. AVE
High nodal expression in proximal epiblast
Future post side forms primitive streak
DVE expresses Cerberus and lefty, low nodal in distal epiblast
What does Cerberus do
Inhibits wnt
What does lefty do
Inhibits nodal
What exactly happens - a-p in mouse
DVE tilts and migrates over epiblast in one direction
Which determines future anterior end of the embryo = AVE
WHAT IS AVE NEEDED FOR IN MOUSE
PATTERNINg the anterior end of embryo
Inhibits primitive streak formation (via inhibition of nodal, expression in primitive streak)
At E6.5 = plays a role in patterning anterior primitive streak
Describe mouse gastrulation - a-p
Starts to form at future post end = primitive streak
Endoderm leaves epiblast layer
Then next = mesoderm
On inside = ectoderm
Definitive endoderm on outside
Describe role of node - a-p mouse
Node = responsible for creation of body plan - patterning
Will form at most distal end embryo
Both express many genes also found in chick and frog organizer tissues = Hesens node and spemann mangold organizer
What is patterning along a-p axis dependent on in mouse
hox genes
Describe rna expression patterns in mice of certain proteins
Cdx2
Ant vs post = diff expressions
Mix 1 = long axis
Foxa2 = long axis
Mesp1 = wing like, mesodermal derived, will form underlying layer mesoderm
Describe hox gene clusters - a-p
3’—>5’
3’ genes = expressed more ant and early
5’ genes expressed more post and later
Order evolutionary conserved
How many hox genes clusters do species have
Flies = 1 cluster
Humans = 4 clusters
What do mutations in hox genes do
Cause homeotic transformations = transform one region to another
Describe the types of homeotic transformations possible
Loss of function = cause post —> anterior transformations, to make more ant phenotype
Gain of function mutations = cause anterior to posterior transformations, bring more posterior gene forward
Describe antennapedia mutation in drosophila
Gain of function = express antennapedia in more anterior segment
Bring segment forwards = expression in ant end
Describe ultrabithorax mutation drosophila
Lose of function = lose ultrabithorax expression in T3 segment
Hox gene expression pattern in T3 looks like the segment that is anterior to it
Post transformed to ant
So get 2 pairs of wings=extra segment with wings
Describe hox genes in vertebrates - gen `
3’ cluster = more ant expression than genes at 5’ end cluster
Not all clusters have same fam members
Some have additional
Have to knockout multiple members
Describe anterior boundary of expression of hox genes
Gets more anterior the lower the number of cluster it is
See boundary of hind lim bud = in situ hybridization
What are paralogs - describe
Members of diff hox clusters, show similar anterior boundary in somites
So like C4 more ant than C6
What happens when you extend boundary of Hoxa10 anteriorly
Shape of ribs patterned by hox genes
So makes ant look more like post
So lose ribs
What happens if you delete hox A10, C10, D10
Normally extend to pattern lumbar region
So now look more anterior
Have to knockout all to get pattern = A,C, & D
Lumbar vertebrae now have ribs = look like thoracic vertebrae
Posterior segment looks more like anterior segment = posterior to anterior transformation
Are vertebrates externally and internally asymmetric
NOOO
Externally bilaterally symmetrical
But internally there are many asymmetries
Describe normal vertebrate internally presentation
Situs solitus
Heart on left
Left lung bilolobed, right lung trilobed
Liver mostly on right
Stomach on left
Kidneys sit on diff a-p axis, not even across
Happens early in embryo
Evolutionarily conserved pattern
Allows for efficient packing of organs within body cavity
Describe situs Inversus
Switched - mirror image, higher incidence heart defect but properly packed and connected
Could also be just one cavity - abd or thoracic
Describe other internal abnormal presentations of vertebrates
Left isomerism = polysplenia
Right isomerism = asplenia
= lungs have same number of lobes
Situs inversus thoracalis
Situs inversus abdmoninalis
= Spleens could be missing
Describe why we get many diff presentations of internal organs of vertebrates
Each organ as forming on a-p axis of body receives L-R cues independently as they develop= patterned differently
Describe antisymmetry
Snails
Dextral (clockwise) and sinistral (counterclockwise) are equally common
Almost never inherited = random, dextral and sinistral phenotypes in offspring are equally common regardless of parental phenotype
29 cases in plants and animals = mostly
Describe directional asymmetry
Humans
Most individuals are asymmetrical in the same direction
Typically inherited
Genetic mutations
Describe initial morphological asymmetries
Early chick embryos
Bilaterally symmetric = ~24hrs —> heart looping = ~ 39hrs, rightwardly looping, first l-p we see patterning —> axial rotation = ~50hrs
Mouse has to contort extra = since endoderm outside, = flips inside out, axial rotation
Name the 4 steps of L-R patterning
Initiation
Stabilization of signal
Propagation of signal
Translation of signal
Describe step 1 = initiation - L-P chick
Disruption of bilateral symmetry
Occurs prior to node formation and at level of the node
Very species specific
What is needed for initiation - describe
H+/K+ - atpase = Xenopus and chick = pump protons, move ions, gives l-r patterning, active during cleavage and dwindles at gastrulation
Gap junctions = xenopus, chick, some cleavage but mostly gastrulation, cells at midline do not connect by gap junctions, know they are on diff sides, just not which side
What is a critical early step in establishing l vs r
Asymmetric cell movement at hensen’s node
Cell move diff = from right to left side
If put glue in embryo and block celll movements = will change l and r patterning
What is needed at level of node
Asymmetric gene expression - early chick = starts during gastrulation, more during gastrulation/neurulation, dwindles before organogenesis
Node monocilia = mouse, chick, xenopus, zebrafish = during gastrulation
Describe asymmetric gene expression at node - chick
Shh on left side of hensens node
Cldn10 on right side hensens node = similar at first then gradually increased, so only on right
Very important for l and r patterning and early
Tjs = block paracellualr movements of ions, so will block ability of ions to move from surface between cells to signal to underlying tissue
Describe cilia at the node
Mouse rabbit zebrafish, xenopus
Bit diff in mouse
Node = teardrop shaped
Planar cell polarity
Ant and post
Each of cells have a cilia that sits at post end cell and at particular angle
Describe kif3b knockout embryos
Kinesin motors
LR patterning defects
Mutants have stunted cilia = D,H,G which are important in early embryos
Mutant cilia immotile
Stumper cilia
Describe cilia at mouse node
Actually move and turn
12 hr cycles in mouse = have standard times
At 2-6 am on days of dev cilia rotate on node
How is cilia moving important = evidence for nodal flow
Artificial flow can affect situs
Cilia at post end and move in v coordinated fashion
Exp = positioned chambers, mutant embryos w/o and did experiment so flow would move l—>r or r—>l
Peristaltic pump experiment
Usually flows from r—>l but can switch to l —> r
How is cilia moving important = evidence for nodal flow = Results
Reverse of situs of wild type embryos at presomite stage by fast rightward flow = override endogenous flow
Artificial flow can correct situs inversus defects in embryos with immotile cilia
Describe idea of cilia
Do not know what molecule is that is being asymmetrically distributed
Immotile cilia = crown cells
Motile cilia = pit cells
Create flow/movement bc surrounding node = crown cells,
If abandon immotile cilia = get calcium flux released = important
What is the role of cilia
Nodal flow
Particles moves and smash open and release
Describe cilia in lr patterning
Motile cilia at the node have now been found in mice, xenopus (gastrocoel roof plate), zebrafish (kuppfer’s vesicle) and rabbits (node)
What do cilia defects do
Defects that affect cilia motility, structural proteins and other comments affect laterality
Experiments indicate that monociliary movement is important but do not indicate that nodal flow initiates asymmetry
What is important for positioning cilia
Planar cell polarity
Describe xenopus l-r
Movement of particles across gastrocoel roof plate
If put glue = disrupt lr patterning
DESCRribe chick and pig embryos
Chick have cilia on cells of hensens node but no flow, rounded shape, due to #?, shape of node? = asymmetric gene expression at hensens node in chick
Similarly = no nodal flow at node in pig embryos
What about humans - nodal flow and cilia
Humans with ciliary defects have associated laterality defects but nothing is known about if there is flow
Kartagener syndrome = many cilia defects, infertile, cannot smell, have lr patterning defects = situs inversus
Know cilia important but not specifics
Describe step 2 = stabilization of the signal - lp
Maintenance of the signal that broke bilateral symmetry - patterning event conserved
At node and/or peirnodal region
Asymmetric expression of nodal in chick
Describe step 3 = propagation of signal - lp
Transfer and maintenance of info on left and right sides embryo
Midline acts as barrier = molecular and physical, midline = notochord, super strong solid bar, do not want lr signals to mix
Asymmetric gene expression in lateral plate mesoderm
Conserved in species
Describe chick - nodal
Expressed only on left side of perinodal region
Patterning on left mostly
Describe asymmetric nodal and lefty-2 in lat plate mesoderm
Conserved in all species
Controls morphogens by inhibition
Lefty 1 = expressed in midline, inhibits ndoal
Lefty 2 = expressed in lateral plate mesoderm
How does lefty act
Classical feedback inhibitor of nodal signaling
Describe lefty - reaction diffusion model hypothesis
Interactions between an activator and an inhibitor control signalling gradients within a field of cells and subsequently pattern them
Activators turns on its own expression and activates its own inhibitor, also turns on other genes
Describe principles of the reaction diffusion model
Activator activates its own production
Activator activates its inhibitor
Inhibitor blocks auto activation of activator
Inhibitor acts at a long range to restrict the effective range of the activator, can work at longer range
Describe nodal left feedback loop
Relative expression levels of nodal and lefty determine intensity of signal
Nodal turns own expression but also turns on lefty
Diff doses nodal signalling
Describe pitx2 expression
Asymmetric in left lateral plate mesoderm
Pitx2 produces multiple transcripts
Induced by nodal signalling
Conserved in all species
Stays in asymmetrically position tissues = left gonad - recall oviduct, one egg
Pitx2c expressed in lat plate mesoderm
Pitx2 isoforms = same homeodomain and transactivation domain
Pitx2c = produced from internal promoter = starts only at exon 4
What does bilateral pitx2 expression do
Randomized direction of heart looping in chick
Endogenous or ectopic
Which side saw higher level = threshold and that side made loop
Describe pitx2 in xenopus - bilateral pitx2 expression
Frogs
Reverse direction of heart and gut looping
Counter clockwise
Pitx2 = downstream, effectors of model, clockwise
Describe step 4 = translation of signal
Conversion of info into asymmetric morphogenesis
Occurs along entire AP axis and at level of individual organs
Pitx2c asymmetrically expressed in organs that exhibit asymmetry = independent interpretation of Pitx2c info, organs respond different to decreasing doses of Pitx2c, need diff doses
Describe generation of anatomical asymmetries- morphogenesis
Directional looping- diff loop = heart, stomach, intestine
Asymmetric development = lung, liver
Asymmetric regression = blood vessels,right oviduct = if overexpress pitx2 = can retain gonads on both sides
Describe cellular basis of asymmetric organogenesis
Differential Cell death - regression
Differential Proliferation = smp, spleen
Differential Cell elongation = chick gut (one side = cells stay columnar or other = get flat and elongated, important for tissue to shape and band)
Differential Migration = zebrafish gut
Rearrangement of actin-cytoskeleton
Intrinsic/extrinsic forces = heart tube
Describe LR patterning cascade in chick embryos
Asymmetric expression of Pitx2c in developing organs asymmetric organogenesis - nodal and lefty present in cell species
Describe lr patterning cascade summary - species specific
Axis of LR symmetric oriented and info spread throughout multicellular fields, H+/K+ atpase and membrane potential, polycystin-2 and calcium signalling, gap junctions
Describe lr patterning cascade summary - evolutionarily conserved
Asymmetric pattern amplified and propagated by cascades of gene espreccion = lefty1, lefty2, nodal
Proteins that influence morphogenesis are induced in target tissues = Pitx2c, morphogenetic proteins
