Tyrosinase expression during neuroblast divisions affects later pathfinding by retinal ganglion cells

Abstract

Occulocutaneous albinism is caused by mutations in the gene encoding the enzyme tyrosinase. Individuals with this disorder are predisposed to visual system deficits. We determined the critical period during development when tyrosinase expression is essential for the appropriate pathfinding of ganglion cell axons from the retina to the dorsal lateral geniculate nucleus. We used a line of mice with a Tyrosinase transgene, the expression of which is regulatable with the lac operator-repressor system, to restrict tyrosinase activity to discrete periods of embryogenesis. When tyrosinase was expressed throughout the period of neuroblast divisions that produce the ipsilaterally projecting ganglion cells, axonal projections innervated the same volume of the ipsilateral dorsal lateral geniculate nucleus of the thalamus as in normal mice. If tyrosinase expression ceased before the end of neuroblast divisions, or was not initiated until after they had begun, the degree of ipsilateral innervation was smaller, as in albino mice. Tyrosinase expression was not required during the entire period of pathfinding itself or during final maturation of the retinogeniculate pathway. Thus, tyrosinase appears to set up a signal early in visual system development that determines the pathway taken later by ganglion cell axons.

Figure 1.

Induction of RPE pigmentation by treatment with the lactose analog IPTG in mouse embryos doubly transgenic for a regulatable Tyrosinase transgene and the lac repressor. A, Photomicrographs of the RPE from E16.5 embryos show that Tyrosinase transgenic animals are highly pigmented relative to albino control animals. B, For Tyr, LacI double-transgenic animals, treatment with IPTG during development allows for differential control of the level of pigmentation achieved. C, The percentage of area containing detectable pigment (as assessed by densitometry; see Materials and Methods) was determined from images of the RPE from E16.5 animals. Repression of the Tyrosinase transgene in the Tyr, LacI double-transgenic animals results in an essentially unpigmented RPE. Treatment of double-transgenic animals with IPTG derepresses the Tyrosinase transgene, leading to pigmentation of the RPE (beginning at E11.5, as in wild-type animals). Treatment up to the time of the assay (E0 -E16.5) and a delayed treatment (E12.5-E16.5) both lead to increased pigmentation, whereas termination of treatment at E13.5 yields a level of pigmentation close to that of the untreated animals. Asterisk indicates significantly different from both the albino baseline and the untreated Tyr, LacI group. Scale bar, 20 μm.

Figure 2.

Anterograde labeling of the dLGN after unilateral eye injection of peroxidase-conjugated wheat germ agglutinin (WGA-HRP). A, A model of the retinogeniculate pathway is shown, highlighting ganglion cells from one eye labeled after injection of the neuronal tracer WGA-HRP into the eye. B, A brain section showing the pattern of labeling in the ipsilateral and contralateral dLGN after the injection of WGA-HRP into one eye. Brains were sectioned and reacted for peroxidase activity to determine the volume of the dLGN containing projections on the ipsilateral side. This section is from approximately the middle of the nucleus of a wild-type pigmented mouse.

Figure 3.

Derepression of tyrosinase expression at specific time points in development rescues a defect in ipsilateral dLGN projection. The ipsilateral projection volume of the dLGN is graphed. A, Albino mice have a deficit in ipsilateral projection volume in the dLGN. The Tyrosinase transgene rescues this deficit, and the rescue is reversed in the Tyr, LacI double-transgenic animals, in which expression of the Tyrosinase transgene is repressed. B, IPTG treatment throughout embryogenesis and until weaning at P21 rescues the albino deficit, as does treatment E0 -E16.5. Treatment E0 -E13.5 and E12.5-P21 fail to rescue the deficit. Asterisk indicates significantly different from both the albino and the Tyr, LacI groups.

Figure 4.

Tyrosinase expression is necessary during the early development of the ipsilateral retinogeniculate pathway. Tyrosinase is first expressed at E10.5. By E11.5, there is graded pigmentation in the RPE, and the first ipsilateral ganglion cells are produced from the neuroblasts. At E12.5 these first cells begin sending out axons. At E16.5 the last ipsilateral ganglion cells are produced. By E18.5 all of the ipsilaterally projecting axons have traversed the optic chiasm. E18-P5 is the period of refinement of connections and ganglion cell death, which produces the final form of the retinogeniculate pathway. Periods of IPTG treatment in Tyr, LacI double-transgenic animals that rescue the albino abnormality are represented by shaded bars. Tyrosinase is necessary throughout the period of neuroblast divisions that produce ipsilaterally projecting ganglion cells.