Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster

Abstract

In Wolffian lens regeneration, lentectomized newt eye can produce a new lens from the dorsal marginal iris, but the ventral iris has never shown such capabilities. To investigate the difference of lens regenerating potency between dorsal and ventral iris epithelium at the cellular level, a transplantation system using cell reaggregates was developed. Two methods were devised for preparing the reaggregates from pigmented iris epithelial cells. One was rotating cells in an agar-coated multiplate on a gyratory shaker and the other was incubating cells in a microcentrifuge tube after slight centrifugation. Reaggregates made of dorsal iris cells that had been completely dissociated into single cells were phenotypically transformed into a lens when placed in the pupillary region of the lentectomized host eye. None of the ventral reaggregates produced a lens. Even dorsal reaggregates could not transdifferentiate into lens when they were placed away from the pupil. The produced lenses from the reaggregates were morphologically and immunohistochemically identified. To obtain evidence whether produced lenses really originated from singly dissociated cells, we labeled dissociated cells with a fluorescent dye (PKH26) before reaggregate formation and then traced it in the produced lens.

Determinative role of Wnt signals in dorsal iris-derived lens regeneration in newt eye

“…The iris pieces were treated with 1000 units/ ml Dispase (Godo Shusei) for 6 h at 27°C, and the sheets of pigmented epithelium were freed from iris stroma using fine forceps. Each piece of pigmented epithelial sheet was placed in a well of 1 cm diameter on type I collagen gel in 300 ll of culture medium consisting of 60%-strength of L-15 medium (Okamoto et al, 1998) plus 10% fetal bovine serum as described by Hayashi et al (2002), with various supplements indicated in the main text of this paper. FGF2 was added to the medium at 100 ng/ml, when indicated.…”

“…An important conclusion was the tissue autonomy of the pigmented epithelium of dorsal iris, namely even after grafting iris fragments in an eye chamber (Reyer, 1954), or placing pigmented epithelial explants under tissue culture conditions (Okamoto et al, 1998;Hayashi et al, 2002), only dorsally derived pigmented epithelial tissues develop lentoids. Due to this strict spatial restriction of the lens origin in the newt iris, it has often been assumed that all cellular processes to initiate lens regeneration occur only in the dorsal iris.…”

FGF2 triggers iris-derived lens regeneration in newt eye

“…Recombinant growth factors (human origin, except for mouse FGF8b) were from Sigma, and FGFR2(IIIc)/Fc and FGFR2(IIIb)/Fc from R&D. These proteins were dissolved in Hanks' saline of 80% strength optimized for use in newts (Okamoto et al, 1998), and injected into the anterior eye chamber through the cornea, or into the posterior chamber through the sclera using a fine glass pipette. Lentectomy was done first by making a horizontal incision through the cornea and by pulling out the whole lens with forceps through the incision.…”

Vertebrate Eye Development

“…The ventral iris cells were not able to form any lens after being cultured for 6 days (Okamoto et al 1998). When PEes are isolated from dorsal and ventral iris and placed in culture after dissociation they can both undergo trans differentiation.…”