Spatial and temporal expression patterns of GDNF family receptor alpha4 in the developing chicken retina.

GDNF family receptor alpha (GFRalpha) receptors are involved in the regulation of different aspects of embryonic development such as proliferation, migration, differentiation and survival. To determine the possible role of GFRalpha4 in retinal development, we analysed its expression in the developing chicken retina. We found that GFRalpha4 is temporally co-expressed with c-ret. Both, the temporal and spatial expression of GFRalpha4 is developmentally regulated during retinogenesis and is first detected in cells of the ganglion cell layer at E6. As development of the retina proceeds, the expression of GFRalpha4 extends to cells of the inner half of the inner nuclear layer and to cells of the outermost cell row of the inner nuclear layer. Later on, GFRalpha4 expression is also found in additional cells of the outer half of the inner nuclear layer and in a subpopulation of photoreceptors. A central-to-peripheral gradient of retinal differentiation is evident, as the onset of GFRalpha4 expression is first detectable in the central retina, while it is delayed by two days in its periphery.

Light-dark and circadian melatonin rhythms are established de novo in re-aggregates of the embryonic chicken retina.

A central function of chicken photoreceptors is the rhythmic synthesis of the neurohormone melatonin; however, it is unclear how they can achieve--already before hatching (i.e. without light exposure)--a circadian pacemaker system. Here we studied melatonin synthesis and secretion in rosetted spheroids, which are de novo histotypic spheres, re-aggregated from dissociated retinal cells of chicken embryos. Spheroids were cultured during a 12-hour light/12-hour dark cycle. Shortly after the onset of photoreceptor differentiation in spheroids near day 5 in culture, melatonin secretion set in and, by day 8 in culture, showed distinct dark-light oscillations, with high values during the dark and lower ones during the light period. Concomitantly, expression of two key enzymes of melatonin synthesis, arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) was detected by northern blot analysis from day 6 onwards, increasing until day 8. Only HIOMT, but not AA-NAT levels were 2- to 3-fold higher at the end of the light compared with the dark phase. Even under permanent darkness, a slight melatonin oscillation was revealed after 6 days. In conclusion, photoreceptors in de novo regenerated retinal spheroids become light-responsive, establish appropriate pathways for melatonin synthesis and secrete more melatonin in the dark. The underlying circadian pacemaker seems to depend on photoreceptor differentiation, not on previous light-dark entrainment.