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

AbstractAlthough 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

Fourth Regiment Armory, Jersey City, New Jersey, circa 1907-1910

Fourth Regiment Armory, Jersey City, New Jersey, circa 1907-1910. Postmark date: February 11, 1910; Message included

Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens

Abstract Background Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous. Result Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses. Conclusions Although P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies

Pax6 Regulates Gene Expression in the Vertebrate Lens through miR-204

<div><p>During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene <i>miR-204</i> are co-regulated by Pax6 during eye development. <i>miR-204</i> is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of <i>miR-204</i> targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of <i>miR-204</i> were revealed, among them <i>Sox11</i>, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of <i>Trpm/miR-204</i> and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of <i>miR-204</i>.</p> </div

<i>Trpm3/miR-204</i> expression is dependent on Pax6 activity during eye development.

<p>Paraffin sections of control (A–E,G,I,K), <i>Pax6^lacZ/lacZ</i> (F,J) and <i>Pax6^loxP/loxP;Mlr10-cre</i> (H,L) stained for <i>Trpm3</i> mRNA (B,D,E–H), Pax6 protein (A,C, red) and <i>miR-204</i> (I–L, green). <i>Trpm3</i> expression begins after E9.5 in the developing eye (B,D), although it is already active in the otic vesicle on E9.5 (B′ inset). <i>Trpm3</i> and <i>miR-204</i> are lost from the optic rudiment of <i>Pax6^lacZ/lacZ</i> embryos (F,J; optic cup rudiment is traced with a line) and in the lens of <i>Pax6^loxP/loxP;Mlr10-cre</i> mutants (H,L). Di, diencephalon; LP, lens placode; LV, lens vesicle; OV, optic vesicle; NT, neural tube; OC, optic cup; OtV, otic vesicle; RPE, retinal pigmented epithelium; Re, developing retina. Scale bar = 100 µM.</p

<i>miR-204</i> affects expression of multiple genes and mediates Pax6 suppression of gene expression.

<p>(A) H36CE cells transfected with <i>hsa-miR-204 mimic</i> or control miRNA (<i>cel-miR-67 mimic</i>) or <i>hsa-miR-204 inhibitor</i> or control miRNA inhibitor (<i>cel-miR-67 inhibitor</i>). Significant reduction following over-expression of <i>hsa-miR-204 mimic</i> and significant elevation following transfection with <i>hsa-miR-204 inhibitor</i> was detected by qPCR (expressed as 2-ΔΔCt values) for transcripts of <i>Sox11</i>, <i>Cpn8</i>, <i>Nfia</i>, <i>Myo10</i> and <i>Fbn2</i>. Error bars are SD (**P<0.001 and ***P<0.0001, n = 3). (B) Fold-change (expressed as 2-ΔΔCt values) in mRNA levels of indicated medaka genes quantified by qRT-PCR, from stage 24 embryos injected with <i>hsa-miR-204 mimic</i> compared to control <i>cel-miR-67mimic</i>, morpholino against miR-204 (<i>Mo-miR-204</i>) and mismatched morpholino (<i>mm-Mo-miR-204</i>). 400 embryos were pooled for each assay, and technical triplicate experiments were independently executed at least three times. (C) Fold-change in mRNA levels of the indicated genes quantified by qRT-PCR from embryos injected with <i>Pax6</i>, <i>Pax6/Mo-miR-204</i> and pGFP-expressing plasmid as a control. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003357#s2" target="_blank">Results</a> are shown as means ± SD, 250 embryos pooled for each assay. Technical triplicate experiments were independently executed at least three times, n = 3, **<i>P</i><0.001, ***<i>P</i><0.0001.</p