2/19, Lecture 10 Reading

Authors: Oscar A Tarazona, Davys H Lopez, Leslie A Slota, Martin J Cohn
Affiliations:
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, United States
- Department of Biology, UF Genetics Institute, University of Florida, Gainesville, United States

Cephalopod mollusks have evolved numerous anatomical novelties including arms and tentacles.
Developmental mechanisms underlying cephalopod limb evolution remain largely unexplored.
This research demonstrates that cuttlefish limb development follows signaling networks similar to those in vertebrates and arthropods.
Key findings include:
- Hedgehog expression located anteriorly in cuttlefish limb buds.
- Posterior transplantation of Hedgehog-expressing cells can induce mirror-image limb duplications.
- Bmp and Wnt signaling establish dorsoventral polarity, with similar polarization in cuttlefish limbs.
- Inhibition of Bmp2/4 leads to ectopic Notum expression and sucker development.
- Cuttlefish limbs exhibit regionalization of multiple developmental genes (Hth, Exd, Dll, Dac, Sp8/9, Wnt) along proximodistal axes.
Implications suggest cephalopod limbs evolved via activation of a genetic program for appendage development that is ancestral to bilaterians.

Animal appendages vary widely in morphology and function, crucial for locomotion, feeding, and reproduction.
Appendages have evolved multiple times across various lineages, suggesting independent origins due to the lack of shared precursors.
Divergent evolutionary histories of arthropods and vertebrates show retained developmental genetic similarities.
Debate exists on whether the genetic program for appendage development originated in the common ancestor of bilaterians early in the Cambrian period or if it arose through convergence.
A gap exists regarding limb development mechanisms in Spiralia, a superphylum of Bilateria apart from Ecdysozoa and Deuterostomia.
Mollusca, the largest Spiralian lineage, has a broad diversity of forms, including the cephalopod adaptations.
Cephalopod limbs have facilitated their evolutionary success as marine predators.

Limb structures:
- Arms: Short, wide appendages with suckers along the entire ventral surface.
- Tentacles: Longer, specialized retractable appendages with suckers only on a distal pad.
Tentacles are likely homologs of arms, found in decapods but absent in other cephalopods like nautilids and octopuses.
Cephalopod limbs likely evolved independently, without homologous precursors in other mollusk lineages.

Objective: To test if cephalopod limbs evolved via the recruitment of ancient gene regulatory networks conserved across Bilateria by examining the arm and tentacle development in the cuttlefish, Sepia officinalis.

Embryonic Development:
- Cuttlefish enter embryonic development through superficial cleavage at the animal pole.
- Initial limb formation is visible at stage 16, identifiable as small peripheral swellings on a flat embryo.
- Cell proliferation in limb primordia increases by stage 15, with clusters of phospho-histone H3 (PHH3)-positive cells indicating active mitotic activity.
- Limb buds become distinguishable at stage 17, and by stage 19, elongation along the proximodistal axis is noted.
Sucker Development:
- A sucker field primordium forms as a ridge visible by stage 21, later cleaving superficially into sucker buds.
- Mature sucker organization includes four parallel rows across the arms, with distinct patterns of sucker bud formation in tentacles.

Conservation of Axis Patterning:
- Objective: To ascertain if molecular mechanisms of limb development in cuttlefish mirror those in arthropods and vertebrates despite their independent evolution.
- Genes cloned include orthologs related to Wnt, Tcf/Lef, and others, indicating conservation across developmental networks.
- Phylogenetic reconstructions confirm interactions within gene families identified in cephalopods compared to known sequences from arthropods and vertebrates.
Gene Expression During Limb Development:
- Expression patterns along proximodistal, anteroposterior, and dorsoventral axes reveal polarization similar to that in arthropod and vertebrate limbs.
- Analysis includes significant findings such as:
- Proximodistal patterning involves Exd and Hth gene expression restricted proximally.
- Dll, Dac, Sp8/9a expression observed distally.
- Hedgehog (Hh) expression localized anteriorly, diverging from patterns observed in other Bilateria.

Bmp and Hedgehog Signaling in Limb Development:
- Implementation of Bmp inhibitors like Noggin leads to ectopic sucker formation.
- Graft experiments reveal Hedgehog signaling plays a critical role in anteroposterior patterning, with induced duplications in response to manipulated Hh signaling.
Conclusion of Functional Analysis:
- Bmp and Hh signaling pathways legitimately govern limb development in cuttlefish, further suggesting homologous roles across evolutionary independent origins.

Identified conserved molecular mechanisms emphasize that cephalopod limb evolution likely involved recruiting a shared genetic program for appendage formation.
Highlight the evolutionary implications of this finding, suggesting the genetic circuits were present in the bilaterian ancestor, perhaps influencing other morphological developments across diverse lineages.
Future explorations of other cephalopod-like structures (such as the funnel/siphon) might uncover deeper evolutionary connections regarding limb and appendage evolution.

Detailed descriptions of embryo collection, ex-ovo culture methods, gene cloning protocols, and molecular phylogenetic analysis techniques.
List of acknowledgments for contributions and support related to the research.