2/17, Lecture 9 Reading

Many animals have left–right (L–R) asymmetry in internal and external features.
In vertebrates, Nodal is a key molecular pathway involved in determining this asymmetry.
- Nodal: Signalling molecule of the transforming growth factor-beta (TGF-β) superfamily.
- Expression: Occurs in the left lateral plate mesoderm.
- Function: Loss of Nodal function causes randomization of L–R asymmetry of visceral organs.
Nodal orthologues detected in some deutrostomes (e.g., ascidians, sea urchins); not found in Ecdysozoa (flies, nematodes) or Lophotrochozoa (snails, annelids).
This study presents the first evidence of a nodal orthologue in a non-deuterostome: snails.

Identification of Nodal and Pitx in two snail species:
- Dextral (right-handed): Lottia gigantea.
- Sinistral (left-handed): Biomphalaria glabrata.

Nodal and Pitx Expression:
- In Lottia gigantea, both are expressed on the right side.
- In Biomphalaria glabrata, both are expressed on the left side.
Pharmacological inhibition of the Nodal pathway indicated that Nodal acts upstream of Pitx.
- Loss of shell chirality observed in some treated animals.

The Nodal pathway's involvement in L–R asymmetry may represent an ancestral feature of Bilateria.
Asymmetric Nodal expression is conserved in deuterostomes:
- Chordates: Nodal and Pitx act on the left side of the embryo.
- Echinoderms (sea urchins): Act on the right side.

Snail Chirality: Body handedness and direction of shell coiling; prevalent forms are dextral and sinistral.
- Developmental determination linked to a maternal locus controlling chirality.
Previous gene analysis indicated critical roles of certain genes in L–R asymmetry but Nodal signaling previously unexamined.

Study involved two snail species differing in chirality:
- B. glabrata: Intermediate host for schistosomiasis.
- L. gigantea: Complete genome sequenced.
Isolated and characterized genes related to Nodal and Pitx.

Bayesian analyses confirmed Nodal orthologues closely related to deuterostome Nodal, not other TGF-β family members.
Identification of a potential Nodal orthologue in the annelid Capitella species I further supports evolutionary continuity.

Snail embryos exhibit indirect development: Trochophore -> Veliger larva stages.
Trochophore Stage Expression:
- L. gigantea: Nodal expressed in two ectodermal domains on the right side.
- B. glabrata: Nodal expressed on the left ectoderm near shell formation site.
Pitx Expression:
- In L. gigantea, expressed on the right side, adjacent to Nodal expression.
- In B. glabrata, Pitx seen in the left ectoderm, close to nodal-expressing cells.

Asymmetry in nodal transcripts first detected at the 32-cell stage.
- Bilateral Axis: Established between 32-64 cell stage. - Nodal clearly left–right asymmetric beyond 32-cell stage.
- Pitx expression initiated at the 64-cell stage in proximity to nodal-expressing cells.

Inhibition Method: SB-431542 used to target specific TGF-β receptors affecting Nodal signalling only.
Observations Post-Treatment:
- At low concentrations (5 mM), 8% of gastrulating embryos showed non-coiling shells; at 10 mM, likelihood increased to 43%.
- Most severe defects occurred when drug administered at the 1-16 cell stage.
Treatment at later stages (trochophore or later) led to normal shell coiling.
Drug exposure before blastula stage is crucial for L–R asymmetry implications on shell formation.

Employing rapamycin did not yield non-coiled shells, indicating a specific Nodal role.
Pitx expression in drug-treated embryos showed significant reduction in asymmetric regions but maintained standard symmetric expression.

Nodal's asymmetric expression is posited to be an ancestral trait within Bilateria.
Evidence suggests L. gigantea and B. glabrata share a common ancestor with dextral traits.

Nodal and Pitx's relationship could reveal new insights into development and morphology of snails.
Future studies needed to further delineate how Nodal operates and regulates chirality and asymmetry during early embryonic development.

Search for TGF-β family members in L. gigantea's genome via NCBI.
Phylogenetic analysis employed for relation assessments among identified Nodal genes. - In Situ Hybridization: Techniques for gene expression verification. - Chemical Treatments: Specific inhibitors to test the effects on development.