Cell-by-cell reconstruction in reaggregates from neonatal gerbil retina begins from the inner retina and is promoted by retinal pigmented epithelium.

For future retinal tissue engineering, it is essential to understand formation of retinal tissue in a 'cell-by-cell' manner, as can be best studied in retinal reaggregates. In avians, complete laminar spheres can be produced, with ganglion cells internally and photoreceptors at the surface; a similar degree of retinal reconstruction has not been achieved for mammals. Here, we have studied self-organizing potencies of retinal cells from neonatal gerbil retinae to form histotypic spheroids up to 15 days in culture (R-spheres). Shortly after reaggregation, a first sign of tissue organization was detected by use of an amacrine cell (AC)-specific calretinin (CR) antibody. These cells sorted out into small clusters and sent unipolar processes towards the centre of each cluster. Thereby, inner cell-free spaces developed into inner plexiform layer (IPL)-like areas with extended parallel CR(+) fibres. Occasionally, IPL areas merged to combine an 'inner half retina', whereby ganglion cells (GCs) occupied the outer sphere surface. This tendency was much improved in the presence of supernatants from retinal pigmented cells (RPE-spheres), e.g. cell organization and proliferation was much increased, and cell death shortened. As shown by several markers, a perfect outer ring was formed by GCs and displaced ACs, followed by a distinct IPL and 1-2 rows of ACs internally. The inner core of RPE spheres consisted of horizontal and possibly bipolar cells, while immunostaining and RT-PCR analysis proved that photoreceptors were absent. This shows that (1) mammalian retinal histogenesis in reaggregates can be brought to a hitherto unknown high level, (2) retinal tissue self-organizes from the level of the IPL, and (3) RPE factors promote formation of almost complete retinal spheres, however, their polarity was opposite to that found in respective avian spheroids.

Lamina formation in the Mongolian gerbil retina (Meriones unguiculatus).

Retinae of nocturnal rodents, such as mice and rats, are almost exclusively rod-dominated. The gerbil, in contrast, shows active periods during day and night and uses both rod- and cone-based vision. However, its retina has not been studied in detail, except for one developmental study analysing its prenatal period (Wikler et al. 1989). Here, the formation of the laminar structure of the gerbil retina was studied from birth until late adult stages. At birth, the retina consisted of a wide neuroblastic layer, with 30% of cells still dividing, a rate decreasing to nearly zero by P6. Shortly after birth, segregation of a ganglion cell layer began. All retinal layers reached their final size around P20, as determined from DAPI-stained cryosections. Muller glial cells developed their typical structure from P1 onwards, e.g. announcing an outer plexiform layer (OPL) at P5, as analysed by the Ret-G7 and glutamine synthetase antibodies. The analyses of the inner retina were performed by antibodies to calretinin (CR) and calbindin (CB). CR is expressed in ganglion cells followed by amacrine cells from P1 onwards; their processes formed four subbands in the inner plexiform layer (IPL) and appeared sequentially after P5 until P20. CB stained a subtype of horizontal cells with their processes into the OPL from P14 onwards. The rod-specific antibody rho4D2 announced photoreceptors at P4, showing signs of outer segments from P10 onwards. The study shows that the formation of all retinal layers in the gerbil occurs postnatally. This and the fact that the gerbil retina is not exclusively rod-dominated could render the gerbil a valuable model for in vitro studies of retinogenesis in rodents.

Impaired formation of the inner retina in an AChE knockout mouse results in degeneration of all photoreceptors.

Blinding diseases can be assigned predominantly to genetic defects of the photoreceptor/pigmented epithelium complex. As an alternative, we show here for an acetylcholinesterase (AChE) knockout mouse that photoreceptor degeneration follows an impaired development of the inner retina. During the first 15 postnatal days of the AChE-/- retina, three major calretinin sublaminae of the inner plexiform layer (IPL) are disturbed. Thereby, processes of amacrine and ganglion cells diffusely criss-cross throughout the IPL. In contrast, parvalbumin cells present a nonlaminar IPL pattern in the wild-type, but in the AChE-/- mouse their processes become structured within two 'novel' sublaminae. During this early period, photoreceptors become arranged regularly and at a normal rate in the AChE-/- retina. However, during the following 75 days, first their outer segments, and then the entire photoreceptor layer completely degenerate by apoptosis. Eventually, cells of the inner retina also undergo apoptosis. As butyrylcholinesterase (BChE) is present at a normal level in the AChE-/- mouse, the observed effects must be solely due to the missing AChE. These are the first in vivo findings to show a decisive role for AChE in the formation of the inner retinal network, which, when absent, ultimately results in photoreceptor degeneration.