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First published online 7 January 2004
doi: 10.1242/dev.00950

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The role of actin cables in directing the morphogenesis of the pharyngeal pouches

Robyn Quinlan¹, Paul Martin^2,* and Anthony Graham^1,

¹ MRC Centre for Developmental Neurobiology, 4th Floor New Hunts House, Guys Campus, Kings College London, London SE1 1UL, UK
² Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (97K): [in a new window]   Fig. 1. The pharyngeal pouch endoderm supports a two-dimensional web of supra-cellular actin cables. Side views of embryos (A-F) where Bmp7 expression (A-C) within the pharyngeal pouch endoderm has been used to highlight the elaboration of pouches along the proximodistal axis and phalloidin staining (D-F) has been used to visualise f-actin within the pouch endoderm. (A) At stage 14, two pharyngeal pouches (1pp and 2pp) have formed and begun to elongate along the proximodistal axis. (B) At stage 15, the third pharyngeal pouch (3pp) is evident. (C) At stage 18, all three pouches (1pp, 2pp and 3pp) have further elongated and display typical narrow slit-like pouch morphology. (D-F) Localised accumulation of actin is seen within the endoderm of each pouch as they undergo proximodistal elongation. (G) High magnification view of the third pharyngeal pouch (3pp) shows actin organised into a supracellular actin cable (red arrowhead), assembled along the apical margin of the endodermal cells (basal margin indicated by an asterisk). (H) Longitudinal section through the pharyngeal region, (at level indicated in F), showing pouches as distinct outpocketings of endoderm. The pharyngeal endoderm is described as pouch endoderm (PE) or interpouch endoderm (IPE). A two-dimensional web of supracellular cables, which follows the lumenal contours of the pouch endoderm, appears to be localised to regions where pouches are forming (red arrowhead) or have just formed, but at lower abundance in the interpouch endoderm (IPE). OV, otic vesicle; aa, arch artery. Anterior is towards the left.

View larger version (57K): [in a new window]   Fig. 2. The actin cables are networked within pouch endoderm via insertion into N-cadherin adherens junctions. The cellular junctions connecting the actin fibres into supracellular cables were characterised using (A) TEM, (B-D) N-cadherin in situ hybridisation, and (E-I) confocal analysis of phalloidin-stained f-actin (green) and anti-N-Cadherin antibody (red). (A) TEM image of the apical margin of cells within pouch endoderm (stage 17), showing a bundle of filaments (red arrowhead) running across the cells, just below the plasma membrane, and connecting via adherens junctions (*). (B,C) Side views showing N-cadherin expression within pouch endoderm of 1pp and 2pp at stage 14 (B) and 1pp, 2pp and 3pp at stage 17 (C). (D) Transverse section through second pouch (2pp) (stage 14); N-cadherin expression is localised to the apical surface of the pouch endoderm (red arrow). (E) Side view of a stage 18 embryo showing co-localisation (yellow) of actin (green) and N-cadherin protein (red) at the apical margin of each pouch (white arrow). (F) High magnification view of pouch endoderm showing N-cadherin protein (red) localised to the cellular junctions (white arrow) that support the actin cable (green). (G) Longitudinal section through a stage 16 embryo, showing an abundance of N-cadherin protein (red) in the last-to-form pouch (3pp) endoderm (white arrow). (H,I) Transverse confocal sections through a stage 18 embryo at the level of (H) the third pouch, indicating the pouch endoderm (PE) and (I) at lower magnification at the level of the second arch and therefore the interpouch endoderm (IPE). N-cadherin protein is found in the lateral endoderm of the pouch (PE in H), but is not apparent in the ventral pharyngeal endoderm (asterisk in H), or the interpouch endoderm (IPE in I). OV, otic vesicle; en, endoderm; nc, notochord; aa, arch artery; nt, neural tube; am, arch mesenchyme; ec, ectoderm. Anterior is towards the left.

View larger version (123K): [in a new window]   Fig. 3. Cytochalasin D results in disorganized fibres that fail to form a coherent actin cable. Confocal analysis of phalloidin-stained f-actin (green) and propridium iodide (red) was used to assess the effects of cytochalasin D on the organisation of the actin structures within the pouch endoderm (A-D) compared with an untreated specimen (E), all at equivalent stages. (A) Side view of an embryo that was treated by introducing cytochalasin D-soaked bead into the pharyngeal cavity at stage 14– and incubated for a further 5 hours; already there is evidence of aberrant pouch morphology, where pouches are contorted (1pp) or `relaxed' (2pp and 3pp). (B) High magnification of the dorsal tip of a second pouch shows that actin fibres fail to form a supracellular cable when treated with cytochalasin D; this embryo was treated by introduction of a bead into the pharyngeal cavity and incubated for a further 6 hours. (C,D) Mitotic cells (white arrows) are clearly evident within the pouch endoderm of embryos treated with cytochalasin D, either via a bead (C) or injection (D), as they are seen in the pouch endoderm of untreated embryos (E). CD, cytochalasin D. Anterior is towards the left.

View larger version (140K): [in a new window]   Fig. 4. Disruption of actin cable assembly results in pouches with aberrant morphology, owing to a failure in proper proximodistal elongation. Effects of cytochalasin D treatment on pouch morphogenesis analysed through (A-C) Bmp7; (D,F) Pax1; or (G,H) Dlx2 in situ hybridization. (A,B) Left- and right-hand side views of the same embryo that had cytochalasin D injected into the pharyngeal cavity at stage 11+, prior to pouch formation, and the embryo allowed to develop to stage 15–. As the chick embryo turns, gravity causes cytochalasin D to accumulate over the left-hand side (LHS) tissue, which results in aberrant pouch morphology on that side (A) but not on the right-hand side (RHS) (B). LHS pouches have an open diamond shape compared with the narrow, slit-like pouches on the RHS or in the DMSO control (C) embryo treated in the same manner. (D) Side view of an embryo where cytochalasin D was injected into the vicinity of the first pouch (1pp) at stage 15–, and allowed to develop to stage 19. (E) High magnification of the contralateral first pouch, which did not receive an injection of cytochalasin D, displaying normal slit-like morphology. The dots outline the contours of the pharyngeal endoderm. (F) High magnification of a first pouch that did receive a cytochalasin D injection showing a contorted morphology of the pharyngeal endoderm along the proximodistal axis. Again, the dots outline the contours of the pharyngeal endoderm. (G) Side view of an embryo, treated with cytochalasin D by injection into the pharyngeal cavity at stage 12 and allowed to develop to stage 18 and (H) an embryo similarly treated with DMSO at stage 13 and allowed to develop to stage 19. In both G and H, Dlx2 expression shows the arches have been properly populated with neural crest cells. OV, otic vesicle. Anterior is towards the left.