somites, limbs, disrupting embryonic development

proximal-distal axis

• proximal: closest to body (shoulder/pelvic girdle)

• distal: tips of digits

dorsal-ventral axis

• dorsal: back of limb/hand (top of foot)

• ventral: front of limb/hand (bottom of foot)

• nails form on dorsal side

anterior-posterior axis

• mostly looked at relative to hand/foot

• anterior: thumb/big toe (digit 1)

• posterior: pinky/little toe (digit 5)

limb bud morphogenesis

• begins as outpouching of cells → hindlimb and forelimb buds

• limb bud contains posterior lateral plate mesoderm (skeletal precursors), somite cells (muscle precursors), and overlying ectoderm

• at E11 (mouse), apical ectodermal ridge becomes clearly visible

• at E12: limb bud becomes paddle-shaped, indentations that will become the interdigit regions are visible

separation of digits (days 48-56 in humans)

• programmed cell death (apoptosis) in interdigit region important to prevent webbing

• failure → webbed digits or connected digits

limb bud formation

formed from outpouching of lateral plate mesoderm (epithelial-mesenchymal transition) at correct position along AP axis in parallel

experiments into limb bud induction and growth

• 1918, Detwiler and Harrison - showed removing groups of cells blocked limb bud growth

• 1925, Hertwig - transplanted groups of cells to new places and got a limb

• 1925/1933, Balinsky - showed that otic vesicle, pituitary, and olfactory sac could induce limb bud in interlimb region

• 1971, Rosenquist - marked groups of cells and watched were they went (fate mapping)

RA and FGF8 along AP axis

• Hoxc6 gene regulates expression of RA and FGF8 → act as antagonistic signals to induce Tbx5 expression

• Tbx5 (homeodomain protein) will determine position of forelimb bud

retinol dehydrogenase 10 knockout experiment

• RARE (RA response element): normally present in middle of embryo, absent in mutant

• FGF8: normally near heart and posterior end of embryo, regions expanded in mutant

• Tbx5: normally in somites and flank region, absent from flank region in mutant → no forelimb production

FGF10 transplantation experiment in chick

• forelimb bud → wing

• place FGF10 bead in interlimb region → ectopic chimeric limb bud

• hindlimb bud → leg

→ position along flank matters in determining limb identity

progress zone

zone of highly proliferative mesenchyme that fuels limb outgrowth (most distal end of limb bud)

zone of polarizing activity (ZPA)

• molecular, not visible

• located in posterior mesenchyme of limb bud

apical ectodermal ridge (AER)

• formed at boundary of dorsal and ventral ectoderm

• region of thickened ectoderm

• required for patterning all 3 axes of the limb

  • removing AER inhibits PD growth of limb bud

  • addition of AER tissue causes digit duplication (AP patterning)

• FGF8 expressed in sharp band pattern

  • AER activity can be replaced by FGF bead → normal wing develops

  • FGF8 signal from bead sufficient to result in proper growth

• activity requires limb bud mesenchyme

  • indicates that pattern molecules also exist in limb mesenchyme

regions of limb (proximal to distal)

1. stylopod

2. zeugopod

3. autopod

Hox expression along trunk AP and limb PD axes

• anterior → posterior = lower → higher Hox gene (3’ → 5’)

• proximal → distal = lower → higher Hox gene (3’ → 5’)

  • Hox13 → autopod

  • Hox11 → zeugopod

  • Hox10 → stylopod

Ra-FGF gradients along limb PD axis

• high RA proximal, high FGF distal

  • FGF → Cyp26b1 —| RA

  • RA → Meis1/2 —| Cyp26b1

• high RA → stylopod (Meis)

• high FGF/Wnts → autopod (Hox13)

• moderate RA and FGF/Wnts → zeugopod (Hox11)

experiments to test RA effects on limb bud PD patterning

• limb bud tips transplanted to head region, either directly or first treated with RA

• earlier stage: more potential to develop into different structures → potential changes with age of progress zone

• treated with RA: more structures form

experiments to text FGF/Wnt effects on limb bud PD patterning

• untreated: expression of all three zones

• incubated with FGF/Wnt:

  • Meis expressed inhibited (no RA)

  • slight different Hoxa11 pattern (no longer 2 distinct bands)

  • Hoxa13 highly expressed

Hoxa11/Hoxd11-deficient mice

loss of zeugopod structures

human homozygous HOXD13 mutation

patterns autopod region, causes polysyndactyly (fused digits)

importance of age of progress zone mesenchyme

• young PZ onto old limb bud → repetition of stylopod and zeugopod regions before autopod

  • young PZ has potential to pattern whole limb

• old PZ onto young limb bud → missing zeugopod region

  • old PZ can only pattern distal regions

formation of AER

pre-AER cells overlying where limb bud cells will grow out of the lateral plate mesoderm, dorsal and ventral ectoderm grow into future AER and express different genes

gene expression in dorsal and ventral domains of limb bud

• Wnt7a and radical fringe (Rfrg) expressed in dorsal ectoderm

• Lmx1b (Lim1) expressed in dorsal mesoderm/mesenchyme

• engrailed (En-1) expressed in ventral ectoderm

  • En-1 —| Rfrg → AER formation

  • En-1 —| Wnt7a → Lmx-1 → dorsal pattern

Lmx1b knockout mouse

• wildtype: footpad on ventral side, digits towards dorsal side

• mutant: ventralization (footpads on both sides), digits in middle

engrailed (En-1) knockout

• wildtype: nails on dorsal side

• mutant: dorsalization (nails forming on both sides)

ZPA transplantation experiment

• ZPA transplanted from posterior to host anterior

• ectopic digits at mirror angle

• distance from ZPA helps determine digit fate (type of digit)

SHH signalling in limb bud

• expressed in ZPA

• can replace ZPA activity

  • cell pellet secreting SHH → mirror image duplication of digits (same as transplanting entire ZPA)

• digit precursors respond differently

  • digit 1 (thumb): SHH independent

  • digits 2 and 3: differentiation dependent on SHH concentration (paracrine)

  • digits 4 and 5: SHH-dependent, time-dependent (autocrine), highest exposure

signalling from ZPA

• dose-dependent and long-range

• must be in contact with AER for feedback loop

• expresses SHH after FGF8 is expressed in AER

AER-limb mesenchyme feedback loops

• regulate outgrowth of progress zone

• regulating downstream targets that are important for giving rise to patterned phenotype

• act to refine signalling domains of other signalling molecules

importance of limb mesenchyme identity

• determines limb identity

• replacing forelimb mesenchyme with hindlimb mesenchyme changes identity of limb (wing → chimeric limb)

Tbx4 and Tbx5 expression in ectopic limb buds

• Tbx4: posterior

• Tbx5: anterior

early Tbx5 knockout (mouse)

forelimbs don’t develop

late Tbx5 knockout (mouse, after E9.5) experiment

• forelimbs are patterned normally

• Cre recombinase cuts DNA at loxP sites flanking Tbx5

• expression of Cre driven by Prx1 promotor (limb specific)

• Cre fused to estrogen receptor ligand binding domain, so only enters nucleus when estrogen ligand is present

• introduce estrogen by gavaging pregnant moms at different days

• → Tbx5 required for initial growth and outpouching, doesn’t seem to be important for later patterning

Tbx5 knockout with Tbx4 or Pitx1 expression in forelimb

• Tbx5 knockout + ectopic Tbx4: rescue (normal forelimb)

• Tbx5 knockout + ectopic Pitx1: no rescue (no forelimb)

• → Tbx5/4 required for forelimb/hindlimb bud initiation but not identity

• → Pitx1 not equivalent to Tbx4/5

ectopic Pitx1 misexpression in wing bud

• retrovirus expressing Pitx1 injected into flank at position

• expression of some hindlimb features → Pitx1 has some hindlimb patterning features

birth defects statistics

• 1/5 infant deaths due to birth defects (2006)

• among top 5 causes of deaths between 2-18 years

• 2.5% live births with recognizable birth defects

embryonic period

• period of development during which the organism does not resemble the adult

• first 8 weeks in humans

• all major organ systems are established

  • highest risk of defects

  • errors can have more fatal consequences

fetal period

• period of development during which the organism resembles the adult

• weeks 9-40 in humans

• growth and refinement

genetic heterogeneity

similar phenotypes caused by different genes/pathways

phenotypic heterogeneity

same mutation causes different phenotypes in different individuals

genetic factors of congenital anomalies/birth defects

• chromosomal (numerical and structural)

• gene mutations

environmental factors of congenital anomalies/birth defects

• teratogens (disruptions)

• physical environment (deformations)

stochastic causes of congenital anomalies/birth defects

• wild type genes, favourable environment, bad luck

• protein synthesis fluctuations

dysplasia

abnormal organization of cells into tissue(s) and its morphological result(s)

deformations

• caused by mechanical forces, physical environment

• occur at later time points during fetal development

malformations

• caused by an intrinsically abnormal developmental process

• abnormal from the beginning

• defect of a morphogenetic or developmental field

• can be caused by mutations in signalling molecules (SHH, FGFs, BMPs) or transcription factors (Hox, Tbx)

disruptions

• caused by interference of an originally normal process

• cannot be inherited, genotype may predispose response of embryo

• can be caused by teratogens (thalidomide)

clubfoot

• limb deformation

• potentially caused by oligohydramnios (low levels of amniotic fluid)

  • affected by environment

  • cramped environment due to low amniotic fluid → deformation

Holt-Oram syndrome

• autosomal dominant disorder due to mutation in Tbx5, complete penetrance

• upper limb anomalies, atrial septal defects in 75% of individuals

• limb anomalies may be limited to thumb or more extensive

• null alleles → more extensive skeletal and cardiac anomalies

• missense mutations → more moderate phenotypes

thalidomide

• used to treat influenza → sedative → used to treat nausea and vomiting during pregnancy

• embryopathy includes limb anomalies (phocomelia or amelia), congenital heart defects, ear abnormalities, duodenal atresia, aplasia of the thumbs

• effect depends on phase during which thalidomide was taken

  • limb disruptions occur early during limb development

  • limb deformations occur later during limb development