Lesson Plan

Spallanzani's Question

1768 to 1864, Pavia and Paris • Currie / Brown / Poss, PNAS, mid-April 2026: SP6 and SP8 transcription factors are conserved switches in the regenerating epidermis of axolotls, zebrafish and mice; gene-therapy activation partially restores digit regeneration in adult mice. • May 11, 2026

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Lesson Plan: Spallanzani's Question

Subject area

STEM: developmental biology, regenerative medicine, history of science, comparative biology, gene therapy.

Recommended audience

High-school biology (Years 11-13 / Grades 10-12)

First-year undergraduate biology, biochemistry, or pre-medical students

General STEM enrichment for ages 14+

Lesson length

60-75 minutes (one period plus optional follow-up).

Learning objectives

By the end of the lesson, students will be able to:

Describe Lazzaro Spallanzani's 1768 experiments demonstrating limb regeneration in salamanders, newts and tadpoles.

State the central comparative question of regenerative biology: why do amphibians and fish regrow limbs and mammals do not.

Identify the Mexican axolotl, the zebrafish, and the mouse digit tip as the three principal vertebrate model systems for limb regeneration.

Define a transcription factor and explain how SP6 and SP8 act as molecular switches in the regenerating epidermis.

Describe the conserved-toolkit hypothesis and explain what the April 2026 Currie/Brown/Poss paper adds to it.

Discuss the gap between a partial regeneration result in mice and a clinical therapy in humans.

Key terms

Regeneration (epimorphic regeneration vs morphallaxis)

Blastema, mesenchymal cells, epidermis

Transcription factor, gene expression, gene regulation

Conserved toolkit, gene knockout, gene therapy

Axolotl (Ambystoma mexicanum), zebrafish (Danio rerio), Mexican salamander

SP6, SP8 (transcription factors)

Spallanzani, Aristotle, Duméril

Pre-class preparation

A photograph of an axolotl (the iconic pale-pink-and-white "feathery gilled" Mexican salamander).

A periodic table or a printed schematic of vertebrate evolutionary relationships.

Optional: a short clip of zebrafish fin regeneration time-lapse, available on multiple research-laboratory websites.

Lesson outline

1. Hook (5 min): "the leg was back"

Read aloud the cold open. Pose: in 1768, an Italian priest watched a salamander grow back a leg. He asked a question that was perfectly clear but had no answer for 258 years: why does a salamander do this and a mammal does not?

2. The orthodoxy and the question (10 min)

Aristotle had observed lizard tails regrowing around 340 BC, in the Historia Animalium.

Spallanzani, working at the University of Pavia, performed the first systematic experimental study of limb regeneration in 1768. He amputated the legs and tails of salamanders, newts, frogs, and tadpoles, kept careful records, and observed that most animals regrew the missing parts within weeks.

Spallanzani's observation was unambiguous. The orthodoxy of the time, however, had no biological framework for explaining differences between species: the cell theory was 70 years away (Schwann and Schleiden, 1838-39), the gene was 100 years away (Mendel 1866, rediscovered 1900), and the molecular biology of development was 200 years away.

Spallanzani put the question on the page. The answer required a vocabulary that did not yet exist.

3. The model organisms (10 min)

The Mexican axolotl (Ambystoma mexicanum). Originally a salamander species native to Lake Xochimilco near Mexico City. Brought to Europe in 1864 by Auguste Duméril, who established a captive colony at the Paris Museum from a small shipment of live animals. Axolotls regrow limbs, tails, jaws, parts of the spinal cord, parts of the retina, parts of the heart. They do this as adults. They do it again, dozens of times, in the same individual.

The zebrafish (Danio rerio). Small striped tropical fish. Adult zebrafish regenerate fins, retina, kidney, parts of the heart and parts of the spinal cord. Major model organism since the 1980s. Cardiac regeneration in zebrafish was demonstrated by Kenneth Poss and colleagues in 2002.

The mouse. The standard mammalian model organism. Surprising finding: mice partially regrow the distal phalanx (tip) of their digits when amputated through the nail-matrix region. This was characterised in the 1970s and refined through 2011 work by the Lehoczky and Tabin laboratories.

The pattern: limb regeneration is broadly preserved across vertebrates, but mammals have largely turned it off.

4. Transcription factors and the conserved toolkit (10 min)

A gene encodes a protein. Genes can be turned on or off in different cells at different times.

The molecules that turn genes on or off are called transcription factors: DNA-binding proteins that recruit (or block) the cellular machinery that reads the gene.

The conserved-toolkit hypothesis: most of the genes required for regeneration are present in mammals; what differs is the pattern of which transcription factors are switched on after injury.

If the hypothesis is correct, then identifying the transcription factors that are activated in regenerating tissues but not in scar-forming tissues should reveal what the field has been looking for since 1768.

5. The April 2026 paper (15 min)

A team led by Josh Currie (Wake Forest, axolotl), David A. Brown (Duke, mouse digit), and Kenneth D. Poss (University of Wisconsin-Madison, zebrafish) catalogued the genes activated at the wound site in their respective animals immediately after amputation.

They laid the lists alongside one another. Two genes appeared in all three species. The genes are SP6 and SP8, both transcription factors of the Sp/KLF family.

Crucially, SP6 and SP8 were already known to regulate digit and limb development in vertebrate embryos. They had not been known to play a role in adult regeneration.

The team performed knockout experiments:

They then performed a "gain-of-function" experiment: they used a gene therapy inspired by a zebrafish regulatory pattern to put SP6 and SP8 into a slightly different on-state in adult mouse fingertips. Partial regeneration returned: not a full finger, not a complete bone, but more than the mouse would have done unaided.

Published in PNAS, mid-April 2026. Wake Forest news release: 16 April 2026.

6. What this is and is not (5 min)

What it is: the first identification of two transcription factors that are required for vertebrate limb-bone regeneration in both amphibians and mammals, and the first demonstration that activating them in mammals can partially restore regeneration.

What it is not: a clinical therapy. The mouse experiment is at proof-of-principle scale. Translation to humans would require addressing safety (cancer risk: SP factors regulate cell proliferation), delivery (how to get the gene therapy to the right cells at the right time), and dose-response (how much regeneration can be safely induced).

The next steps the field is now planning: identify the upstream signals that activate SP6/SP8 in salamanders and zebrafish; ask whether mammalian fibroblasts (the scar-forming cells) can be reprogrammed into regenerative epidermal cells; pursue larger-scale digit regeneration in mice and other mammals.

7. The 258-year arc (5 min)

Spallanzani: 1768. The question.

Aristotle: 340 BC. The original observation in lizards.

Duméril: 1864. The axolotl arrives in Europe as the model organism.

2018: axolotl genome sequenced, ten times the size of the human genome.

2026: SP6/SP8 identified as conserved switches; partial regeneration restored in mice by gene therapy.

Discussion: 258 years from question to first molecular ingredient is long. What does this say about how scientific questions get answered? Compare with: cell theory waited 170 years from Hooke's microscope, the structure of DNA waited 100 years from Mendel.

8. Wrap and assignment (5 min)

Pose: "Identify a contemporary biological mystery that you think will require new vocabulary or new technology to answer. What technology would you bet on?"

Optional homework: write a 500-word memo from the perspective of a 2080 graduate student looking back at the 2026 paper, describing what subsequent decades have revealed about safe activation of SP6/SP8 in adult human tissues.

Discussion questions

Spallanzani performed his salamander experiments in 1768, before the cell theory, before genetics, before molecular biology. What did he do that made his observations scientific even without that vocabulary?

Why is the axolotl a more useful regeneration model than the human? Identify three properties of the axolotl that make it experimentally tractable.

What does it mean for a transcription factor to be "conserved" across species separated by hundreds of millions of years of evolution? Why is conservation strong evidence for biological importance?

The 2026 paper restored partial regeneration in mice by gene therapy. What ethical, safety, and dose-response questions would have to be answered before this approach could be considered for human clinical trials?

Mammals have the SP6 and SP8 genes but apparently do not activate them in the regenerating-epidermis pattern. What evolutionary logic might have caused mammals to suppress regeneration during their development?

Compare the 258-year arc from Spallanzani's question to the 2026 first molecular ingredient with three other long-running biological questions: the structure of DNA, the cause of stomach ulcers, and the function of the appendix. Identify what each case has in common and what differs.

Extension activities

Lab demonstration (where facilities allow): observe planarian regeneration. Schmidtea mediterranea regenerates entire individuals from small fragments and is widely used in classroom kits.

Reading exercise: read the abstract of Spallanzani's 1768 Prodromo (in modern English translation, available via the Wellcome Collection or in Comfort's biography of Spallanzani) and the abstract of the 2026 PNAS paper. Identify what each claims and how each was received.

Modelling exercise: using Boolean network logic, model how a single transcription factor (SP8) can switch a tissue between scar-forming and regenerative response. Discuss the limits of binary models.

Writing assignment: write a 1000-word essay on what makes a scientific question "answerable in principle." Use Spallanzani's question as the central case study. Address the role of new vocabulary, new technology, and new model organisms.

Time Rhymes