Ryan Lecture 6 Flashcards
Name and describe the regions of a limb from proximal to distal
Stylopod = humerus
Zeugopod = ulna, radius
Autopod = carpals, digits
= blocks of tissue to form continuous structure
Name and describe 3 critical regions in developing limb bud
Progress zone
Zone of polarizing activity ZPA
Apical ectodermal ridge = AER
Describe AER
REGION of thickened ectoderm
Forms at boundary between dorsal and ventral sides limb bud
Required for patterning all 3 axes of limb
WHAT IS required for PD axis
AER
Removing AER = inhibits proximal distal growth of limb bud
Progress zone wont grow or do mitosis = wont form structures
Describe Ffg8 expression in AER
Specific patterned expression = thickened region ectoderm
What can replace AER activity
Fgf bead
If remove AER but implant fgf bead = normal wing develops, gets outgrowth
Bead replaces patterning signal
Describe gradient along limb P-D axis
opposing RA-FGF gradients
Proximal = retinoic acid
Distal = fgfs/wnts
Threshold patterns= retinoic acid —> fgf = stylopod meis, Zeugopod hox11, autopod hox13
Describe hox expression along trunk A-P axis and limb P-D axis
Homeotic selector genes
Come on later in dev - for limbs
Describe HOX expression along Limb P-D axis
Domains of expression correspond to subdomains of limb
Describe experiment to test RA effects on limb bud P-D patterning
Limb bud tips transplanted to bead region of embryo - tips, distal ends of progress zone
Limb bud tips were either transplanted directly or first treated with RA
Staging = number of somites present
Untreated = see distal structures of autopod
Ra treated = in early see more proximal structures, bones they form, but tissues less competent to respond at later dev
Explanation = effect of age of limb bud tip, effect of RA, older and more distal to RA signal = less likely to be responsive
Describe experiments to test how FGF/wnt effects on limb bud P-D patterning
Untreated stage 18 mesenchyme
Stage 18 mesenchyme incubated with fgf/wnt
= see less meis1 fgf repressing proximal gene expression
Not much effect in Zeugopod, hoxa10
In autopod = maybe enhancing, hoxa13
Opposite effects = suggests gradients opposing
What causes mice to miss Zeugopod
Hoxa11/Hoxd11 deficient mice
Loses whole structure if do not have tissue to pattern them
What casues polysyndactayly in humans - fused digits
Homozygous mutation in HOXD13
Autopod region
Lost patterning of mesenchyme, maybe loss of webbing early on
= misshaped did his but dorsal ventral axis fine (can see finger nails)
What does AER activity require
Limb bud mesenchyme
Replace limb bud mesenchyme with non limb mesenchyme = from diff part embryo
AER regresses = limbs dev stops, doesn’t support maintained of AER and low levels fgf8
Indicates that patterning molecules also exist in limb bud mesenchyme
What is P-D specification of limb correlated with
Age of progression zone mesenchyme
Young progression zone into old limb buds = still grow, duplicate Zeugopod and stylopod
Old progression zone onto young limb buds = lost midsection,retains info and dominates how it develops = lose most of stylopod and Zeugopod
Where does AER form
AT BOUDNARY OF DORSAL AND VENTRAL ECTODERM
How does AER FORM
Pushes out and get out pouching ectoderm
Turns around it
Dorsal = lmx1
Ventral = en-1
AER acts like local organizer = becomes limb bud
, also get patterning info in msenchyme
WHAT IS expressed in dorsal domains in limb bud
Wnt7a expressed in dorsal ectoderm
Lmx1 - lim1 expressed in dorsal mesoderm
What is expressed in ventral domains of limb bud
En-1 (engrailed) expressed in ventral ectoderm
BOUDNARY where engrailed Meets wnt7 = fgf turns on in AER
Describe D-V patterning pathway in limb bud
OPPOSE each other = keep d and v separate and keep ridge AER
Dorsal = Wnt7a, lmx1b
Ventral = en1, bmp
En1 inhibits r-fng (—>AER formation) and Wnt7a (—> lmx1—>dorsal pattern)
Describe Dorsal-ventral patterning by Wnt7a
Lmx1b dependent dorsal ventral patterning by Wnt7a
Foot pad=ventral
If lmx1 mutant = remove form dorsal mesoderm = lose dorsal structures on dorsal and ventral =footpads
What happens when engrailed KO
Loss of ventral patterning in limb
Ventral missing so dorsal no longer repressed so takes on dorsal phenotype
Nail replaces food pad
Repress other one so drives ventral and prevents dorsal
What can AER also do
Pattern a-p axis
Addition of AER tissue cases digit duplication
Has ability to pattern and duplicate But not so much overall polarizing region
In autopod region, depends on time too
Same orientation
Describe ZPA
Not morphologically visible
Located in posterior margin fo limb bud
Found via transplantation exp then molecularly
Post = shh concentrated in ZPA
Describe ZPA transplantation experiments
Front post to anterior = 2 zpa, transplant one
Now = digits face each other - mirror image
Change in dose shh patterns digits
Mouse zpa to chick also works - source does not matter, digits look like chick digits
Where is Shh expressed
In ZPA
What can replace ZPA activity
Shh
Transplantation exp = transplanting zpa or shh beads causes mirror image duplication - different from digit duplicated caused by transplanting AER
Describe how digit 1 response to shh signalling
Thumb, shh independent
Further away from zpa
Describe how digits 2 and. 3 respond to shh signaling
Differentiation dependent on shh concentration - paracrine signalling
Lower dose but still need it
Describe how digits 4 and 5 response to shh signaling
Shh dependent, time dependent - auto rise
Highest exposure, most dependent
Describe signalling from ZPA
Dose dependent
Long range
ZPA MUST be in contact with AER = feedback loop
Describe timing of expression of fgf8 and shh
Fgf8 expressed in AER before shh expressed in zpa
Aeschylus stage = formation 3 somites, = stage 16, so 4.5 hrs
Shh= comes on aprox 12 hrs after fgf8 in AER
What regulates outgrowth of progress zone
Feedback loops between AER and limb mesenchyme regulate outgrowth of the progress zone
Fgf8 = + effect on shh in ZPA, also inhibits gremlin =antagonist bmp
Complicated feedback between AER ad zpa, and molecules expressed in limb bud mesenchyme
Describe feedback loops between AER AND LIMB MESENCHYME
Low fgf = turns on shh —> grem1 (repress bmps)
Then HIGH fgf = represses grem1 so bmps increases and
Goes back to low fgf, repeat
Control timing and level of expression = gives differences
What determines limb identity
Position of fgf bead
Along a-p flank
Limb mesenchyme
What can change identity of limb
Replacing forelimb mesenchyme with hindlimb mesenchyme changes identity of limb
Cells contain patterning info = how we shape bones or how muscle attaches
Describe what is specific to forelimb or hindlimb bud
Tbx5 = in wing bud uniquely
Tbx4 = hind limb bud
Pitx1 = hind limb bud, close relative of pitx2
Describe Tbx5 and tbx4 expression in ectopic limb buds
Also pressed in other regions
Tbx5 = top half expresses,
Tbx4 =bottom, not top, like post limb bud
GIVES CHIMERA
What do Tbx5 knockout mice tell us = EARLY KO
Forelimbs do not Develop
Express recombinase in this region of limb - prx1cre, Tbx5 lox/lox
What do Tbx5 knockout mice tell us = LATER KO
Delete only after E9.5 = forelimbs patterned normally
Treat at diff days, some delay for effect tho
After limb buds already formed
Earlier knockout = much severe phenotype
What do Tbx5 knockout mice tell us =DESCRIBE EXP METHOD
Tbx flaked by lox sides
Cre recombinase clips lox site and repair so tissue specific knockout
Tbx5 flanked by loxP sites
Cre recombinase cuts dna at loxP sites
Expression of Cre is driven by prx1 promoter..limb specific, drives expression for limb protein
Cre fused to estrogen receptor (piece of) ligand binding domain, so only enters nucleus when estrogen ligand present, acts as tf
Introduce estrogen by gavaging pregnant moms at different days, via tamoxifen and will delect tbx5
DESCRIBE Exp where early ko Tbx5 and then express tbx4 or pitx1 in forelimb
If add ectopic tbx4 in forelimb bud = can rescue forelimb
If add ectopic pitx1 in forelimb bud = cannot compensate,cannot rescue
Tbx4 and tbx5 activate same set of target genes in developing limb bud
Date suggests that tbx4/5 required for fore/hind limb bud initiation but not identity
Describe exp = ectopic misexpression of pitx1 in wing bud
Retrovirus expression pitx1 is injected into flank at position of forelimb bud
=cause it to have more hindlimb identity, more important for hindlimb identity
Suggest = pitx1 dominantly drives expression of genes needed to give you hind limb identity
Describe species specific patterning of limb bud
Same set of genes needed for initiating patterning
Species specific events layered on top
Describe disrupting embryonic dev
2 haploid cells, genes and environment = lead to 1 trillion diploid cells + some haploid cells, germ cells
How often are babies born with birth defects
Every 4.5 mins there is a baby born in the US with a birth defect
What is leading cause of infant mortality
Birth defects
1 in 5 infant deaths due to birth defect - 2006 (in first 2 years life)
Among top 5 causes of death between 2 and 18 y/o
Describe some estimated stats for embryos
20-50% of human cleavage stage embryos implant
40% of embryos that implant survive to term
2.5% live births have a recognizable birth defect, could be minor or severe
What accounts for neonatal death
Congenital abnormalities account for ~9% of annual neonatal deaths
Preterm birth is a direct cause of 35% of all neonatal deaths
Cost = huge if severe birth defect
Describe embryo vs fetus = EMBRYO
Period of dev during which organism does not resemble adult
First 8 weeks in humans
All major organ systems established
Describe embryo vs fetus = FETUS
Period of dev where organism resembles adult
Weeks 9-40 in humans
Growth and refinement = cells differentiation
WHEN IS risk of defects highest
Organogenesis
3 weeks before gastrulation = wont notice but die
Formation of organs= period of max sensitivity to abnormal dev - 2-8 weeks, heart defects, spina bifida
Growth and maturation of organ systems = 8-38 weeks, affect differentiation of systems, fetal development
Describe critical times in prenatal development
Major congenital abnormalites
Highly sensitive period bc that is when they develop
Death of embryo and spontaneous abortion common during weeks 1-2 = periof of dividing zygote, implantation, bilaminar embryo
Describe period of sensitivity during dev
Heart = weeks 3.5 to 9
Eyes = weeks 4.5 to full term
Cns =weeks 3 to full term
Ears = 4.25 to 20
Teeth = 6.75 to full term
Palate = weeks 6.75 to 16
Upper limbs = 4.5 to 9
External genitalia = weeks 7 to full term
Lower limbs = weeks 4.5 to 9
Describe genetic heterogeneity
Similar phenotypes casues by diff genes/pathways
Mutations
Describe phenotypic heterogeneity
Same mutation cases diff phenotypes in diff people
One gene among many - also second hit
Also environment = mother gets disease of malnourished = can affect embryo, Matt ion makes embryo more susceptible
Name causes of congenital anomalies/birth defects
Genetic factors
Environmental factors
Stochastic
Most =dont know
Describe causes of congenital anomalies/birth defects = genetic factors
Chromosomal - numerical and structural
Gene mutations
Describe causes of congenital anomalies/birth defects = environmental factors
Teratogens - disruptions
Physical environment = deformations
Describe causes of congenital anomalies/birth defects = stochastic
Wild type genes, favourable environment
+ bad luck
Protein synthesis fluctuations
Random and cause differences
Name 4 structural changes in dev
Dysplasia
Deformations
Malformations
Disruptions
Describe dysplasia - structural changes in dev
Abnormal organizer of cells into tissues and its morphological result
Abnormal growth mostly benign
Describe deformations - structural changes in dev
Caused by mechanical forces = psychical environment, disrupt how formed
Describe malformations - structural changes in dev
Genes
Caused by intrinsically abnormal developmental process
Abnormal form beginning
Defect of a morphogenetic or whole developmental field, particular step
Something is off
Describe disruptions - structural changes in dev
Teratogens
Casues by interference of an originally normal process. - disrupted
Cannot be inherited, genotype may predispose response of embryo
But genotype not cause of defect
Describe an ex of limb deformation - clubfoot
Potentially caused by oligohydraminos - low levels of amniotic fluid
Not enough fluid for fetus to move around
Pushed into position, soft = casues bones to bend, can be corrected after birth
Describe ex of limb malformation = holt oram syndrome - gen
Gene originally linked to 12q21.2-q22
Due to mutation tbx5
Upper limb anomalies, -/+ atrial septal defect in 75%, forelimb affected
Limb anomalies may be limited to thumb or more extensive
Describe ex of limb malformation = holt oram syndrome - acc mutations
Autosomal dominant disorder with complete penetrance - everyone will have same phenotype, but maybe some variations
Null alleles have more extensive skeletal and cardiac abnormalities - no protein
Missense mutations have moderate phenotypes = protein less functional, so less severe than no protein
Give ex of limb disruptions due to thalidomide
1956 - used to treat influenza, then as sedeative then to treat nausea vomiting during pregnancy
Classic thalidomide embryopathy = range of defects, depends on dose, include limb anomalies (phocomelia or amelia), congenital heart defects, ear abnormalities, dudodenal atresia, aplasia of thumbs
Give ex of limb disruptions due to thalidomide - timing
Limb disruptions from thalidomide occurs early during limb dev - tbx5 malformations= major congenital anomalies - weeks 1-8
Limb deformations - club foot = occur later during dev, functional defects and minor anomalies - weeks 9-38
Describe summary of limb congenital anomalies
Limb malformations can be caused by mutations in signaling molecules (SHH, FGFs and BMPs) or transcription factors (Hox, Tbx).
Limb development is sensitive to teratogens (e.g. thalidomide) that can cause limb disruptions.
Limb deformations (e.g. club foot) occur at later time points during fetal development.
What are teratogens
Greek origin
Teratos = monster or marvel
Disrupt normal processes
Agent to factor that affects morphogenesis, dev aand differentiation through cell death, failed cell interactions or alterations of cell movements - disrupt many things
Usually applied to environmental causes like drugs, chemicals and viruses
Different teratogens may produce common effects, ex=hot tubs linked to neural tube defects
Describe discovery of teratogens - history
<1940s = thought embryos were protected from environment by extra embryonic and fetal membranes, thought it was super safe
1941= rubella virus, 1st documented case that an evironental agent could produce disruptions
Silent spring by Rachel Carson 1962, DDT
Thalidomide - summer 1962
Effect of chemicals becomes clear
Name the 3 considerations - teratogens
Critical periods of development
Dosage of drug/chemical
Genotype of embryo
Describe consideration of teratogens = critical periods of dev
Early vs late effects - is embryo or fetus sensitive at that time
May extend into postnatal periods = teeth, brain
Describe consideration of teratogens = dosage of drug/chemical
Dose used in animals if often way»_space; than human exposures, mouse and rats have diff metabolism
Need to establish a dose response curve
Describe consideration of teratogens = genotype of embyro
Response varies based on genotype of embryo
Like variations of proteins that metabolize the products
Describe why/why not drugs are tested on pregnant women
In humans, unless a drug is intended to treat a condition unique to pregnancy - studies systematically attempt to exclude women and ensure that women of child bearing age do not become pregnant during period of drug exposure - so no risk
In humans = unethical to conduct trial to assess teratogenicity
Oh 172 fda approved drugs between 2000 and 2010 = 97.7% majority had an undetermined teratogen risk in human pregnancy
73.3% had no data available -regarding safety in pregnancy
Name main exs of known teratogens
Drugs = alcohol (tied to genotype, many diff genotypes and responses), isotretinoin (retinoic acid), thalidomide
Chemicals= endocrine disrupters, like brith control pills, polluted water makes fish change sex
Infections = rubella virus
Name all teratogens
Drugs = alcohol, androgens, busulfan, cocaine, DES, isotretinoin, lithium carbonate, methotrexate, phenytoin (dilantin), tetracycline, thalidomide, trimethadione, valproic acid, warfarin
Ionizing radiation
Hyperthermia
Chemicals = methyl mercury, PCBs, endocrine disrupters
Infections = cytomegalovirus, herpes simplex virus, human immunodeficiency virus, human parovirus B19, rubella virus, toxoplasma gondii, treponema palladium, varicella virus
Describe thalidomide - conclusions
Teratogenic effects casued withdrawn from use 1961-1962
Now being used to treat hiv and cancer
Sensitive periof between 27 and 40 days after conception
Maybe damage neural crest cells
What become mandatory after thalidomide incident
Testing of drugs in pregnant experimental animals
What can ethanol do
Induce congenital abnormalities
Alcohol is most devastating teratogen with respect to frequency and cost to society
Prevalent, many defects
Neural tube defects = 1st trimester pregnancy
Fetal alcohol syndrome = hAPPENS LATER, period of dev
Describe symptoms of fetal alcohol syndrome
A baby with fetal alcohol syndrome may have the following symptoms: Poor growth while the baby is in the womb and after birth Decreased muscle tone and poor coordination
Delayed development and problems in three or more major areas:
thinking, speech, movement, or social skills
Heart defects such as ventricular septal defect/atrial septal defects
Characteristic craniofacial features
Describe ethanol induced dysmorphology
Treated on gestational day 7 =
Embryos lose midline structures and eyes move towards Center
Get proboscis - almost Cyclopia
Animal study
Describe FAS - fetal alc syndrome stats
Craniofacial structural defects
2001 estimate = 1out of evert 650
Current estimates higher = up to >1%
Describe ethanol induced dysmorphology in human babies
Phenotypes range from relative mid fetal alcohol syndrome, cognitive effects and affects ability learn to holoprosencephaly = HPE, since nostril TO HPE + CLEFT LIP (V SERIOUS)
What does too much vit a do
TERATOGENIC
Retinol = morphogen
Effects = very bad, need to be on 2 forms of birth control before can get higher dose retinol = iPLEDGE PROGRAM
What can also induce holoprosencephaly
Retinoic acid, cylopia = lost central structures
Describe congenital anomalies casues by isotretinoin
Increased risk of spontaneous abortions
Hydrocephaly (enlargement of the fluid-filled spaces in the brain)
Microcephaly (small head and brain)
Mental retardation
Ear and eye abnormalities
Cleft palate and other craniofacial abnormalities
Describe iPLEDGE PROGRAM
Roche Pharmaceuticals had shown birth defects in animals. FDA approved Accutane in 1982 with implementation of voluntary risk management to prevent pregnant women from using drug – not successful!
Lammer et al., 1985 followed 59 women who elected to remain pregnant after RA exposure: 12 spontaneous abortions; 21 major observable anomalies
In 2005 FDA started iPLEDGE a MANDATORY risk-management program that all patients, including men, who take isotretinoin, must participate to achieve two goals (2):
To ensure that no pregnant woman starts taking isotretinoin
To ensure that no woman taking isotretinoin becomes pregnant
Must use at least 2 forms of birth control for 1 month before, during and 1 month after [multiple pregnancy tests].
DESCRIBE ethical considerations
Animal models
Stem cells
Organoids
Impact of research on planetary health
What is next?
Describe ethics of embryology - Gen conc
7 YEARS AGO = one health WHO ORGANIZATION = integrated, unifying approach to balance and optimize the health of people, animals and the environment. It is particularly important to prevent, predict, detect, and respond to global health threats such as the COVID-19 pandemic.
Could only culture to 14 days = just before gastrulation
On may 4th 2016 = 2 groups recorded that they had sustained human embryos in vitro for 12-13 days
What are responsibilities, when does life begin
Organoids = take induced pluripotent stem cells and grow tissues
Future considerations
DEFINE PROGRESS ZONE
Progress zone = zone of highly proliferative mesenchyme that fuels limb outgrowth, progresses outwards, underlines ectoderm
DEFINE ZONE OF POLARIZING ACTIVITY
Zone of polarizing activity (ZPA)= located in posterior mesenchyme of limb bud - important for pattering a-p or limb digit
DEFINE APICAL ECTODERMAL RIDGE
Apical ectodermal ride (AER)= formed at boundary of dorsal and ventral ectoderm, thickened
