The mechanism of lens placode formation: A case of matrix-mediated morphogenesis

Abstract

Although placodes are ubiquitous precursors of tissue invagination, the mechanism of placode formation has not been established and the requirement of placode formation for subsequent invagination has not been tested. Earlier measurements in chicken embryos supported the view that lens placode formation occurs because the extracellular matrix (ECM) between the optic vesicle and the surface ectoderm prevents the prospective lens cells from spreading. Continued cell proliferation within this restricted area was proposed to cause cell crowding, leading to cell elongation (placode formation). This view suggested that continued cell proliferation and adhesion to the ECM between the optic vesicle and the surface ectoderm was sufficient to explain lens placode formation. To test the predictions of this “restricted expansion hypothesis,” we first confirmed that the cellular events that accompany lens placode formation in chicken embryos also occur in mouse embryos. We then showed that the failure of lens placode formation when the transcription factor, Pax6 was conditionally deleted in the surface ectoderm was associated with greatly diminished accumulation of ECM between the optic vesicle and ectoderm and reduced levels of transcripts encoding components of the ECM. In accord with the “restricted expansion hypothesis,” the Pax6-deleted ectoderm expanded, rather than being constrained to a constant area. As a further test, we disrupted the ECM by deleting Fn1, which is required for matrix assembly and cell-matrix adhesion. As in Pax6CKO embryos, the Fn1CKO lens ectoderm expanded, rather than being constrained to a fixed area and the lens placode did not form. Ectoderm cells in Fn1CKO embryos expressed markers of lens induction and reorganized their cytoskeleton as in wild type ectoderm, but did not invaginate, suggesting that placode formation establishes the minimal mechanical requirements for invagination.

FGF signaling activates a Sox9-Sox10 pathway for the formation and branching morphogenesis of mouse ocular glands

“…Laser microdissection was performed as described previously (Huang et al, 2011). Briefly, E14.5 embryos were embedded in OCT compound (Sakura Finetek) and snap frozen on dry ice.…”

Mechanisms of cranial placode assembly

“…Knocking-out the fibronectin gene disrupts cell crowding in the lens placode, which appears larger than in controls (Huang et al, 2011). This result suggests that fibronectin works as a retention/anchoring cue for lens cells located above the retina (as proposed by the authors).…”

“…This suggests a capacity of Pax6-positive and Pax6-negative populations to segregate from each other, although loss of Pax6 -/-cells by apoptosis in the lens tissue has not been ruled out. It has been hypothesised that Pax6 regulates the expression of intercellular adhesion molecules responsible for this sorting out process (Collinson et al, 2000), but a more recent study in mouse rather suggests that Pax6 controls lens formation by regulating the expression of extracellular matrix (ECM) components, including fribronectin (Huang et al, 2011). In zebrafish, forced expression of Dlx5, normally present in a domain anterior and adjacent to the Pax6 + region (the presumptive olfactory placode domain), leads to the formation of clusters of Dlx5-overexpressing cells and their exclusion from the lens placode.…”

“…During this process, lens cells change their shape from cuboidal to columnar, but do not proliferate more or become smaller in volume than pre-placodal ectodermal cells (Huang et al, 2011). Huang and co-workers attributed this local increase in cell density to continued proliferation of lens cells combined with their anchoring to the underlying ECM, a retention mechanism that would work against the expansion of the placodal ectoderm.…”

“…In mouse embryos, the increase in cell density observed during lens formation correlates with the accumulation of fibronectin, a major ECM component, between the retina and the overlying placodal ectoderm (Huang et al, 2011). Knocking-out the fibronectin gene disrupts cell crowding in the lens placode, which appears larger than in controls (Huang et al, 2011).…”