#sea urchin embryo

Specification Determination-l

What happens if a blastomere from a 4-cell stage sea urchin embryo is isolated?

Sea urchin blastomeres at four-cell stage possess extraordinary regulative potential because fate depends on conditional interactions rather than fixed determinants. Isolated blastomere initiates compensatory signaling through nuclear accumulation of beta-catenin, activation of Delta-Notch and Nodal pathways, respecifying missing micromere functions and re-establishing endomesoderm gene regulatory network including pmar1 and foxA. Resulting larva is proportionate and fully patterned with gut, skeleton and ciliated band despite originating from single cell, albeit smaller. This experiment by Hans Driesch challenged preformationism and demonstrated embryo as dynamic self-organizing system capable of regulation.

Ref: Gilbert, Developmental Biology, 12th ed., Chapter 8: Sea Urchin Regulative Development and Isolated Blastomeres.

View Details

Sea Urchin -ll

The double-negative gate in sea urchins involves the proteins:

Double-negative gate explains restricted skeletogenic gene regulatory network to large micromeres of sea urchin embryo. Maternal nuclear β-catenin activates transcription factor Pmar1 specifically in micromeres shortly after their birth. Pmar1 protein is a potent repressor that represses transcription of another global repressor, HesC, which is expressed ubiquitously otherwise. HesC normally represses many micromere fate genes including Alx1, Ets1, Tbr, Tel and signaling ligand Delta. By repressing HesC, Pmar1 derepresses those genes allowing skeletogenic differentiation only where Pmar1 present. This Pmar1-HesC tandem repression circuit exemplifies double-negative logic in developmental gene regulatory networks.

Ref: Oliveri et al., Development 2008; Revilla-i-Domingo et al., 2007 - Pmar1/HesC double-negative gate model.

View Details

Sea Urchin -ll

Inhibiting nuclear accumulation of β-catenin results in:

β-catenin nuclear entry is absolutely required for endomesoderm specification in vegetal hemisphere of sea urchin embryo. Vegetal Disheveled normally blocks GSK-3β mediated degradation, allowing TCF-dependent transcription of Pmar1 and downstream endodermal genes such as FoxA, GataE and Brachyury. If nuclear accumulation experimentally prevented by overexpressing GSK-3β, dominant-negative TCF, or sequestering β-catenin in cytoplasm, vegetal gene regulatory network never initiates. Embryos fail to gastrulate, form no gut or mesenchyme, and develop as hollow ciliated balls composed entirely of animal-like ectoderm, called animalized or dauerblastula phenotype. This demonstrates necessity of β-catenin.

Ref: Wikramanayake et al., PNAS 1998, β-catenin requirement; Gilbert 12th ed., Chapter 8: Animalization after β-catenin inhibition.

View Details

Sea Urchin -ll

Lithium chloride exposure in sea urchins leads to:

Lithium chloride is classic vegetalizing agent discovered by Herbst in sea urchin experiments. Mechanistically it directly inhibits glycogen synthase kinase-3β (GSK-3β), central component of Axin-APC destruction complex that phosphorylates β-catenin targeting it for ubiquitin-proteasome degradation. Inhibition stabilizes β-catenin, causing its cytoplasmic accumulation and translocation into nuclei even in animal blastomeres normally destined for ectoderm. Resulting ectopic activation of vegetal gene network expands endomesodermal domain at expense of ectoderm producing exogastrulae. Phenotype resembles Wnt overactivation. Therefore lithium exposure increases β-catenin nuclear localization, converting presumptive ectoderm to endoderm.

Ref: NCBI Bookshelf, Developmental Biology, Figure 8.15 Lithium vegetalization via GSK-3β inhibition and β-catenin.

View Details

Sea Urchin -ll

Driesch’s isolation experiments demonstrated:

Hans Driesch in 1891 separated blastomeres of two-cell and four-cell sea urchin embryos by vigorous shaking in calcium-free seawater, each surviving blastomere subsequently developed into small but complete pluteus larvae with gut, skeleton and ciliary band. This disproved preformationist mosaic theory of Roux and demonstrated each early cell retains totipotent developmental potential, forming any lineage depending on interactions and position. Driesch termed embryos harmonious equipotential systems where parts adjust to regenerate whole. Results established principle of conditional specification and regulative development, founding concepts of morphogenetic fields and embryonic regulation central to deuterostome embryology.

Ref: Driesch, 1891, Entwicklungsmechanik; Gilbert, Developmental Biology, 12th ed., Chapter 3: Driesch isolation and regulative development.

View Details

Sea Urchin -ll

Isolated large micromeres from a 16-cell embryo can independently form:

Classical isolation experiments by Sven Hörstadius cultured separated micromeres from 16-cell sea urchin embryos in isolation. Even without ectodermal cues, large micromeres undergo epithelial-mesenchymal transition, migrate within culture droplet and secrete calcium carbonate forming characteristic triradiate spicules typical of larval skeleton. They autonomously express Alx1, Sm50, Msp130 and other biomineralization genes under Pmar1-HesC circuit control. This proves skeletogenic program intrinsic to micromeres, not requiring inductive interactions. Other lineages lose ability to form gut or ectoderm alone. Skeletal spicule formation thus serves definitive functional assay for micromere autonomy and specification.

Ref: Hörstadius, Experimental Embryology of Echinoderms, 1973; Gilbert 12th ed., Chapter 8: Micromere autonomy tests.

View Details