A Role for <i>Smoothened</i> during Murine Lens and Cornea Development

<div><p>Various studies suggest that Hedgehog (Hh) signalling plays roles in human and zebrafish ocular development. Recent studies (Kerr et al., <i>Invest Ophthalmol Vis Sci</i>. 2012; 53, 3316–30) showed that conditionally activating Hh signals promotes murine lens epithelial cell proliferation and disrupts fibre differentiation. In this study we examined the expression of the Hh pathway and the requirement for the <i>Smoothened</i> gene in murine lens development. Expression of Hh pathway components in developing lens was examined by RT-PCR, immunofluorescence and <i>in situ</i> hybridisation. The requirement of <i>Smo</i> in lens development was determined by conditional loss-of-function mutations, using LeCre and MLR10 Cre transgenic mice. The phenotype of mutant mice was examined by immunofluorescence for various markers of cell cycle, lens and cornea differentiation. Hh pathway components (<i>Ptch1, Smo, Gli2, Gli3</i>) were detected in lens epithelium from E12.5. Gli2 was particularly localised to mitotic nuclei and, at E13.5, Gli3 exhibited a shift from cytosol to nucleus, suggesting distinct roles for these transcription factors. Conditional deletion of <i>Smo</i>, from ∼E12.5 (MLR10 Cre) did not affect ocular development, whereas deletion from ∼E9.5 (LeCre) resulted in lens and corneal defects from E14.5. Mutant lenses were smaller and showed normal expression of p57Kip2, c-Maf, E-cadherin and Pax6, reduced expression of FoxE3 and Ptch1 and decreased nuclear Hes1. There was normal G1-S phase but decreased G2-M phase transition at E16.5 and epithelial cell death from E14.5-E16.5. Mutant corneas were thicker due to aberrant migration of Nrp2⁺ cells from the extraocular mesenchyme, resulting in delayed corneal endothelial but normal epithelial differentiation. These results indicate the Hh pathway is required during a discrete period (E9.5–E12.5) in lens development to regulate lens epithelial cell proliferation, survival and FoxE3 expression. Defective corneal development occurs secondary to defects in lens and appears to be due to defective migration of peri-ocular Nrp2⁺ neural crest/mesenchymal cells.</p></div

Ocular phenotype of <i>Smo</i> cKO mice.

<p>Images showing normal ocular phenotype in weanling (4 week old) wild-type (<b>A</b>) and Smox10 mice (<b>C</b>) but microphthalmia in LeSmox (<b>B</b>) mice. Smox10 mice lacked hair between whiskers (<b>C</b>, white arrow), due to ectopic Cre activity in these follicles. Histology confirmed microphthalmia in P21 LeSmox (<b>E</b>) mice compared to wild-type (<b>D</b>). The phenotype was characterized by abnormal lens development, reduction of the anterior chamber, overlapping irises and fusion of the iris to the lens (E, inset). <b>F.</b> RT-PCR showed no expression of <i>Smo</i> gene in two independent Smox10 lenses at P2, using primers specific for exons1 or 4, but normal expression of <i>Hprt</i>. Presence (+) or absence (−) of reverse transcriptase in reactions is indicated above the lanes. Scale bar <b>A–C</b>, 2 mm; <b>D–E</b>, 50 µm; inset, 90 µm.</p

RT-PCR Primers.

<p>RT-PCR Primers.</p

Cell death in LeSmox lenses.

<p>TUNEL reaction in wild-type (<b>A, C</b>) and LeSmox (<b>B, D</b>) lenses at E14.5 (<b>A, B</b>) and E16.5 (<b>C, D</b>). In wild-type lenses (<b>A, C</b>) no distinct TUNEL⁺ nuclei were detected. In E14.5 LeSmox lenses (<b>B</b>) numerous TUNEL⁺ nuclei were detected throughout the epithelium and occasionally in fibre cells in the transitional zone. At E16.5 (<b>D</b>), TUNEL⁺ nuclei were concentrated in the equatorial zone at or just anterior to the equator. Scale bar, <b>A–D</b>, 100 µm.</p

Expression of fibre cell markers in LeSmox lenses.

<p>Expression of c-Maf (<b>A–D</b>) and p57^Kip2 (<b>E–H</b>) proteins in Wt (<b>A, C, E, G</b>) and LeSmox (<b>B, D, F</b>, <b>H</b>) lenses at E14.5 (<b>A, B, E, F</b>) and at E16.5 (<b>C, D, G, H</b>). Reactivity for both c-Maf and p57^Kip2 proteins were detected in the nuclei of early differentiating fibre cells (arrowheads) below the lens equator (dashed line) in Wt and in LeSmox lenses in similar positions. Scale bar, <b>A–H</b> 100 µm.</p

Antibodies used for Immunofluorescence.

<p>Antibodies used for Immunofluorescence.</p

Expression of epithelial cell markers in LeSmox lenses.

<p>Expression of E-cadherin (<b>A, B</b>), Pax6 (<b>C–D</b>), FoxE3 (<b>E–F</b>) and Ptch1 (<b>G, H</b>) proteins in Wt (<b>A, C, E, G</b>) and LeSmox (<b>B, D, F, H</b>) lenses at E16.5. <b>A–D.</b> Reactivity for both E-cadherin and Pax6 proteins is detected in epithelial cells (arrowheads) anterior to the lens equator (dashed line) in Wt and in LeSmox lenses. While the extent of the epithelium in LeSmox mice is decreased, the patterns of protein expression are similar to Wt. <b>E–F.</b> FoxE3 is normally detected as a nuclear reactivity in wild-type lens epithelium (arrowheads) but in LeSmox lenses the reactivity is reduced and is absent from many nuclei. The strongest FoxE3 reactivity is detected in equatorial nuclei. <b>G–H.</b> Ptch1 is predominantly detected in Wt epithelial cells and declines as cells commenced differentiation at the equator (<b>G</b>). In LeSmox lenses, reactivity for Ptch1 is greatly reduced and virtually absent, indicating reduced Hh signalling (<b>H</b>). Dashed line indicates position of the lens equator in each image. Scale bar, <b>A–H</b> 100 µm.</p

Exotic foods reveal contact between South Asia and the Near East during the second millennium BCE

Here we report the identification of staple and exotic food remains in Bronze and Early Iron Age dental calculus from the Southern Levant. The analysis of dietary plant microremains and proteins sheds new light on consumed exotic foods from South and East Asia during the second millennium BCE. We provide the earliest direct evidence in the Mediterranean to date for the consumption of sesame, soybean, probable banana, and turmeric. The recovery and identification of diverse foodstuffs using molecular and microscopic techniques enables a new understanding of the complexity of early trade routes and nascent globalization in the ancient Near East and raises questions about the long-term maintenance and continuity of this trade system into later periods.Although the key role of long-distance trade in the transformation of cuisines worldwide has been well-documented since at least the Roman era, the prehistory of the Eurasian food trade is less visible. In order to shed light on the transformation of Eastern Mediterranean cuisines during the Bronze Age and Early Iron Age, we analyzed microremains and proteins preserved in the dental calculus of individuals who lived during the second millennium BCE in the Southern Levant. Our results provide clear evidence for the consumption of expected staple foods, such as cereals (Triticeae), sesame (Sesamum), and dates (Phoenix). We additionally report evidence for the consumption of soybean (Glycine), probable banana (Musa), and turmeric (Curcuma), which pushes back the earliest evidence of these foods in the Mediterranean by centuries (turmeric) or even millennia (soybean). We find that, from the early second millennium onwards, at least some people in the Eastern Mediterranean had access to food from distant locations, including South Asia, and such goods were likely consumed as oils, dried fruits, and spices. These insights force us to rethink the complexity and intensity of Indo-Mediterranean trade during the Bronze Age as well as the degree of globalization in early Eastern Mediterranean cuisine.Protein spectra have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride) under the dataset identifier PXD021498

Localization of Hh pathway in lens.

<p>Immunolocalization of Ptch1 (<b>A, D, G</b>), Gli2 (<b>B, E, H</b>) and Gli3 (<b>C, F, I</b>) and non-immune IgG control (inset <b>D</b>), in developing lens at E12.5 (<b>A–C</b>), E13.5 (<b>D–F</b>) and E15.5 (<b>G–I</b>). In the lens vesicle at E12.5 cytoplasmic and membrane reactivity for Ptch1 is found in both epithelial (e) and primary lens fibre (lf) cells (<b>A</b>). Similar patterns are seen at E13.5 (<b>D</b>) and E15.5 (<b>G</b>), with reduced reactivity present in the more differentiated fibres. Predominantly nuclear reactivity for Gli2 is detected at all ages in the epithelial cells with some nuclear and cytoplasmic reactivity detected in fibre cells (<b>B, E, H</b>). Intense reactivity for Gli2 was associated with mitotic nuclei (arrowheads <b>B, E, H</b>; inset <b>H</b>). Inset in H shows a metaphase nucleus, with intense reactivity for Gli2, in the epithelium of an E15.5 lens. Diffuse cytoplasmic reactivity for Gli3 was detected in the lens vesicle at E12.5 (<b>C</b>) but distinct nuclear Gli3 reactivity was detected in epithelial and fibre cells at E13.5 and E15.5 (<b>F, I</b>). Hoechst dye fluorescence (blue) in <b>A–C, F</b> and <b>H</b> show presence of nuclei. Scale bar, 50 µm (<b>A–C</b>); 100 µm (<b>D–I</b>), 230 um (inset D), 25 µm (inset, <b>H</b>).</p