Sepúlveda-Ramírez SP et al. (2018), Cdc42 controls primary mesenchyme cell morphoge...

ECB-ART-46193

Dev Biol 2018 May 15;4372:140-151. doi: 10.1016/j.ydbio.2018.03.015.

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Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo.

Sepúlveda-Ramírez SP , Toledo-Jacobo L , Henson JH , Shuster CB .

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In the sea urchin embryo, gastrulation is characterized by the ingression and directed cell migration of primary mesenchyme cells (PMCs), as well as the primary invagination and convergent extension of the endomesoderm. Like all cell shape changes, individual and collective cell motility is orchestrated by Rho family GTPases and their modulation of the actomyosin cytoskeleton. And while endomesoderm specification has been intensively studied in echinoids, much less is known about the proximate regulators driving cell motility. Toward these ends, we employed anti-sense morpholinos, mutant alleles and pharmacological inhibitors to assess the role of Cdc42 during sea urchin gastrulation. While inhibition of Cdc42 expression or activity had only mild effects on PMC ingression, PMC migration, alignment and skeletogenesis were disrupted in the absence of Cdc42, as well as elongation of the archenteron. PMC migration and patterning of the larval skeleton relies on the extension of filopodia, and Cdc42 was required for filopodia in vivo as well as in cultured PMCs. Lastly, filopodial extension required both Arp2/3 and formin actin-nucleating factors, supporting models of filopodial nucleation observed in other systems. Together, these results suggest that Cdc42 plays essential roles during PMC cell motility and organogenesis.

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Genes referenced: actr2 cdc42 LOC100887844 LOC100893746 LOC115919910 LOC590297
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	Fig. 2. Archenteron elongation and PMC organization are disrupted upon depletion of Cdc42. A–E) S. purpuratus eggs were injected with untargeting control or Cdc42 morpholinos, and embryos were scored for developmental progression 48 h post-fertilization, when embryos normally reach the gastrula stage (Bar, 50 μm). Embryos were scored as gastrulae (with archenterons extended at least 75% of total length and spicules present, 2A, blue); mid-gastrulae, with elongating archenteron and spicules (2B, green); embryos with a primary invagination and a lack of organized PMCs (2C, orange); embryos containing disorganized PMCs and lacking an archenteron (2D, pink); and embryos displaying both disrupted mesenchymal cells and epithelia (2E, gray). F) Quantification of phenotypes in embryos injected with control or Cdc42 antisense morpholinos at 48 h post-fertilization. While control embryos were in the gastrula or mid-gastrula stages, in embryos injected with 500 μM MASO showed defects in PMC organization and archenteron elongation (2H, pink bars). These defects were rescued with co-injection of human Cdc42. Each experimental condition represents at least three experimental replicates, with a minimum of 100 embryos per condition. *** p < 0.001; ns: p = 0.9765.
	Fig. 3. Cdc42 activity is required for correct PMC migration, filopodia formation and initiation of skeletogenesis. A) Cdc42 activity assay. Lysates from gastrulae treated with carrier control (0.1%) DMSO or the Cdc42 inhibitor ML141 were incubated with PAK beads, and Cdc42 bound to the beads or present in unfractionated lysates was detected by Western blotting. B–U) L. variegatus embryos were treated with 5 μM ML141 at the blastula stage, and then fixed and processed for immunolabeling with PMC-specific (6a9, green) and anti-actin (magenta) antibodies or analyzed live by polarization microscopy. Maximum intensity projections of Lateral (L) or Vegetal (V) views of control or ML141-treated embryos revealed differences in PMC morphology and migration. Control embryos exhibited PMCs organized in ventrolateral clusters (VLCs) (3B, arrows) and in a ring around the vegetal pole (3G, arrow), with extended filopodia contacting the ectoderm (3E and J, arrows). Inhibition of Cdc42 resulted in PMCs organized around VLCs and the vegetal ring, but lacked filopodia (3O and T, arrows), and the tight organization of controls (3L and Q). Imaging by polarization microscopy revealed the presence of birefringent, tri-radiate spicules in controls (3K) but not detectable in ML141-treated embryos (3U). Bar, 50 μm.
	Fig. 4. PMC syncytium formation and skeletogenesis requires Cdc42 activity. A–D) L. variegatus embryos were treated with ML141 at the gastula stage (A), and embryos were scored for developmental progression when controls reached the prism/early larval stage. D) Quantification of phenotypes represented in B and C for three experimental replicates, with an average of 250 embryos scored per condition per experiment. *, p < 0.0003; * *, p < 0.0001. E) L. variegatus embryos with treated with 5 μM ML141 at the gastrula stage, and then fixed and processed for immunolabeling with PMC-specific (6a9, green) and anti-actin (magenta) antibodies or analyzed live by polarization microscopy. Control embryos presented a well-formed common synctytium (panels a–d) and larval skeleton (panel f), whereas PMCs in embryos incubated with ML141 failed to form a common syncytium (panels g–j). Deposition of skeletal material was limited to what was generated at the time of treatment (panel l). Bar, 50 μm.
	Fig. 5. PMCs resume skeletogenesis upon reversal of Cdc42 inhibition. Viability of PMCs following ML141 treatment was confirmed by treating embryos at the blastula stage with 2.5 μM ML141 and then releasing embryos from Cdc42 inhibition. As viewed by polarization microscopy, ML141-treated embryos that failed to initiate spiculogenesis by 24 h were able to reinitiate skeletogenesis upon removal of ML141 (5 M, arrows). Arrowhead denotes the presence of an ectopic spicule. Bar, 50 μm.
	Fig. 6. Live-cell imaging of filopodia in the presence or absence of Cdc42 activity. L. pictus gastrulae expressing GFP-Lifeact were imaged by brightfield and confocal microscopy. A) Control embryos form dynamic filopodia along long the length of the spicule (pink and yellow arrowheads), at the distal tips (red arrowhead), and in contacts with non-skeletogenic mesenchymal cells (green arrowhead). Bar, 50 μm. B and C) Observation of ventro-lateral clusters (VLCs) at early maturation stages showed PMCs associated with small tri-radiate spicules (6B, panels d and e, black arrows) and later to the growing spicules (6B, panels c and f). In all stages abundant filopodia extended from the PMCs and spicule rods (6B, panels a–c, white arrows). Embryos incubated with 10 μ ML141 for 20 min exhibited PMCs associated with skeletal rods but with decreased filopodia (6 C, panels a–d) compared to controls (6B, panels a–c). Longer treatments resulted in stronger effects on PMCs organization and filopodia processes, and loss of syncytial cytoplasm associated with the spicule rods (6C, panels e and f). D) Control S. purpuratus gastrulae expressing GFP-Lifeact exhibited dynamic filopodia processes elaborated from PMCs migrating along the ectoderm (6D, panel a, arrows) as well as from the basal membrane of ectodermal cells (6D, panel a, red arrowheads). Treatment with ML141 for 2.5 h inhibited filopodia formation in PMCs (6D, panel b, arrows), whereas ectodermal filopodia were unaffected by Cdc42 inhibition (6D, panel b, red arrowheads). Bar, 25 μm.
	Fig. 7. Contribution of Cdc42, formins and Arp2/3 in sea urchin PMC filopodia formation. A) L. pictus embryos were treated at the blastula stage with either 0.1% DMSO, 100 μM Arp2/3 inhibitor (CK666, ) or 10 μM formin inhibitor (SMIFH2) for 24 h before being fixed and processed for immunolabeling with PMC-specific (6a9, green) and anti-actin (magenta) antibodies or analyzed live by polarization microscopy. Bar, 50 μm. B) L. pictus gastrulae expressing GFP-Lifeact were treated with 0.1% DMSO, 100 μM CK666 or 50 μM SMIFH2 and imaged by confocal microscopy. Live-cell imaging of PMCs in ventrolateral clusters from L. pictus gastrulae revealed that embryos treated with Arp2/3 and formin inhibitors did not exhibit the dynamic filopodia observed in controls. Bar, 25 μm. C–E) Isolated PMCs from L. variegatus embryos were treated with Cdc42 (10 μM ML141), Arp2/3 (100 μM CK666) and formin (5 μM SMIFH2) inhibitors, and cultures were fixed, probed with 6a9 and the number and length of filopodia were quantified for 30 cells/experiment, 3 experimental replicates. * p = 0.0001; * * p < 0.0001. Bar, 10 μm.

References [+] :

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