#experiment

Stem Cell -l

Which experiment first demonstrated the existence of pluripotent stem cells?

Pluripotency concept evolved from teratocarcinoma studies showing embryonal carcinoma cells could contribute to tissues. The definitive demonstration came with isolation of mouse embryonic stem cells in 1981 by Evans and Kaufman and independently by Martin. Cultured inner cell mass cells on feeder layers with LIF remained undifferentiated, expressed alkaline phosphatase, formed teratomas containing three germ layers, and contributed to chimeras including germline. This proved stable pluripotent lines exist in culture. Cloning Dolly and mesenchymal stem cell discovery addressed totipotency reversal and multipotency, not primary pluripotency proof.

Ref: Evans & Kaufman, Nature 1981; Gilbert, 12th ed., Chapter 6: Mouse ES isolation.

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Stem Cell -lll

The Gurdon and Yamanaka experiment demonstrated:

John Gurdon's nuclear transfer experiments in Xenopus and Yamanaka's transcription factor reprogramming collectively proved that differentiation does not entail irreversible genetic alteration or loss of genomic information. Gurdon demonstrated that intestinal nucleus could support cloned tadpoles developing to adult frogs, while Yamanaka showed differentiated fibroblasts return to pluripotency with defined factors alone. Both experiments established epigenetic plasticity as central to development and reversed Waddington landscape conceptually. Recognition transformed concepts of cell fate reversibility, culminating in Nobel Prize 2012, and enabled patient-specific induced pluripotent stem cells.

Ref: Gurdon 1962, Yamanaka 2006, Nobel 2012; Gilbert, Chapter 6: Reversibility of differentiation and pluripotency induction.

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Specification Determination-ll

Which experiment supports conditional development in early frog gastrula and autonomous development in late frog gastrul

Early frog gastrula cells remain conditionally specified and highly plastic. When presumptive neural ectoderm from early gastrula is transplanted to ventral epidermal region, it adopts epidermal fate under influence of high BMP signaling from host, revealing environmental instruction. Equivalent transplants performed using late gastrula donor tissue fail to switch, continuing to form neural tissue despite epidermal cues due to stabilized expression of factors like Sox2 and epigenetic lockdown. This demonstrates progressive loss of competence and acquisition of autonomous determination with age, where early flexibility gives way to irreversible commitment after sustained exposure to inductive signals.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 11: Neural Induction and Competence Windows.

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Specification Determination-ll

Which experiment demonstrated that cells of an early newt gastrula exhibit conditional development while late gastrula c

The transition from conditional to autonomous development during gastrulation was elegantly shown by transplantation experiments in newt embryos. Prospective ectoderm fragments from early gastrula transplanted into regions fated to become endoderm changed fate to match host location, indicating continued responsiveness to surrounding inductive cues and regulative potential. Identical grafts taken from late gastrula, however, differentiated according to donor origin, forming epidermis even within gut environment, demonstrating acquired autonomous determination. This temporal progression reveals how cumulative signaling, community effects and chromatin restriction progressively narrow potency, converting plastic specification into locked determination.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 10: Gastrulation and Fate Mapping in Amphibians.

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Eye Lens Induction -l

Which experiment demonstrated that Pax6 is required in surface ectoderm for lens formation?

Competence requirement was proven by tissue recombination. Fujiwara et al. used rSey rats carrying Pax6 mutation. Wild-type optic vesicle grafted onto wild-type head ectoderm induces lens efficiently. When same wild-type vesicle is apposed to Pax6 mutant ectoderm, no lens forms despite normal inductive signals. Reverse combination, mutant optic vesicle with wild-type ectoderm, still induces lens, showing Pax6 needed in responding tissue not inducer. This demonstrates transcription factor acts cell-autonomously in ectoderm to confer ability to interpret BMP and FGF cues, rather than affecting signaling source or neural crest components.

Ref: Fujiwara et al. 1994; NCBI Bookshelf, Developmental Biology Chapter: Induction and Competence experiment - Pax6 in surface ectoderm.

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Eye Lens Induction -ll

Which experiment demonstrated the role of Shh in separating the eye field?

Role of Sonic hedgehog in splitting single eye field into bilateral domains was demonstrated by knockout resulting in cyclopia. Shh-null mice, zebrafish cyclops mutants deficient in Nodal upstream of Shh, and pharmacologic cyclopamine inhibition of Smoothened all exhibit failure to suppress midline Pax6, retaining fused eye field leading to single median eye cup, absent hypothalamus, and holoprosencephaly. Overexpression of Pax6 in midline does not split field, optic vesicle graft to trunk tests lens competence not midline, L-Maf overexpression drives crystallin. Cyclopia after SHH loss directly illustrates midline inhibitory function and organizer role.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 12: Shh knockout cyclopia demonstrating midline split function.

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Eye Lens Induction -ll

Which experiment demonstrated that Pax6 is required in surface ectoderm for lens formation?

Requirement of Pax6 in surface ectoderm for lens formation was demonstrated by tissue recombination: grafting wild-type optic vesicle onto Pax6-deficient head ectoderm from Small eye mutant fails to induce lens placode or crystallin expression, whereas Pax6-deficient optic vesicle grafted onto wild-type ectoderm still induces lens. Knockout of Pax6 in optic vesicle alone does not block lens, overexpression in cup does not address ectodermal necessity, neural crest knockout irrelevant. Mosaic recombinant experiment proves cell-autonomous competence role of Pax6 within responding surface ectoderm, not inducing tissue itself.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 12: Pax6 tissue recombination – requirement in surface ectoderm.

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Limb Development -l

Which experiment demonstrated that mesodermal signals induce limb bud formation?

Instructive capacity for limb formation resides in lateral plate mesoderm, demonstrated by classical transplantation in chick embryos. Excision of forelimb-destined lateral plate mesoderm and grafting to ectopic flank induced complete supernumerary limb, including an apical ridge newly induced from host flank ectoderm. Ectoderm grafts alone were non-inductive, Hox gene knockouts altered axial position without testing induction, and ridge removal truncates rather than initiates. Molecular mechanism centers on FGF10 secreted by lateral plate mesoderm activating Wnt3a-FGF8 signaling cascade in overlying ectoderm, initiating budding and pattern.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 19: Classical LPM transplantation inducing ectopic limb buds.

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Limb Development -ll

Which experiment demonstrated that AER function can be replaced?

Chemical nature of AER was proven by replacement experiments restoring outgrowth without ridge tissue. After surgical removal of AER from chick wing bud causing predictable proximal-distal truncation, implantation of heparin acrylic beads soaked in recombinant FGF8 or FGF4 into distal mesenchyme restored progress zone proliferation, maintained FGF10 and Gremlin expression, and allowed otherwise normal limb development including complete digits and pattern. This demonstrated AER acts primarily via secreting diffusible FGFs and that purified FGF can substitute fully for its structure and function, unlike mesoderm removal, SHH overexpression or TBX5 inhibition which do not directly address AER roles.

Ref: Niswander et al., Cell 1993, Gilbert Chapter 20: FGF bead replaces AER function.

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Limb Development -ll

Which experiment demonstrated that mesodermal signals induce limb bud formation?

Proof that mesodermal FGF10 initiates limb budding came from gain-of-function implantation experiments. Implanting FGF10-soaked heparin beads beneath flank ectoderm of chick embryos outside normal limb fields induced ectopic Wnt3a, beta-catenin activation and AER formation expressing FGF8, followed by complete extra limb containing stylopod zeugopod autopod and digits with muscle and cartilage. Conversely, AER removal causes truncation, TBX5 knockout prevents forelimb, AER graft from another species still supports outgrowth due to permissive nature. Thus FGF10 bead experiment proved mesodermal signals sufficient to induce full limb formation program de novo.

Ref: Ohuchi et al., Development 1997, Gilbert Chapter 20: Ectopic FGF10 induces additional limbs.

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Limb Development -lll

Which experiment demonstrated the importance of BMP signaling in digit formation?

Requirement of BMP in digit patterning was revealed by manipulating BMP antagonists experimentally. Overexpressing Gremlin1 or Noggin throughout digit plate in chick via RCAS retrovirus blocked BMP2-4-7 signaling, preventing both apoptosis in interdigital regions and chondrogenic condensation of phalanges, resulting in webbed, fused and truncated digits with reduced phalanges and syndactyly. Conversely, ectopic BMP application induces apoptosis and digit loss. Wnt7a knockout ventralizes dorsal limb, ZPA graft duplicates AP pattern, early AER removal truncates PD axis. Hence Gremlin overexpression experiment proved BMP necessity for digit formation and separation.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 20: Gremlin and BMP in digit morphogenesis.

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Limb Development -lll

Which experiment demonstrated the role of the ZPA in anterior-posterior axis formation?

John Saunders and Mary Gasseling established ZPA function by transplanting posterior margin mesenchyme to anterior border of chick wing bud. Recipient buds developed mirror-image digit duplications with pattern 65432 alongside normal 23456, revealing a diffusible morphogen controlling anterior-posterior polarity and concentration-dependent digit specification. Later work identified morphogen as SHH acting through Gli3 processing and Hoxd activation. AER removal truncates proximo-distally, retinoic acid inhibition proximalizes, FGF8 overexpression extends outgrowth but does not duplicate AP pattern. Only anterior ZPA graft reproduces posterior digits.

Ref: Saunders & Gasseling 1968, Gilbert Developmental Biology Chapter 20: ZPA graft experiment.

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