Development of a heat-stable alkaline phosphatase reporter system for cis-regulatory analysis and its application to 3D digital imaging of Xenopus embryonic tissues.

Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of ß-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.

PTEN regulates retinal interneuron morphogenesis and synaptic layer formation.

The lipid phosphatase PTEN is a critical negative regulator of extracellular signal-induced PI3K activities, yet the roles of PTEN in the neural retina remain poorly understood. Here, we investigate the function of PTEN during retinal development. Deletion of Pten at the onset of neurogenesis in retinal progenitors results in the reduction of retinal ganglion cells and rod photoreceptors, but increased Müller glial genesis. In addition, PTEN deficiency leads to elevated phosphorylation of Akt, especially in the developing inner plexiform layer, where high levels of PTEN are normally expressed. In Pten mutant retinas, various subtypes of amacrine cells show severe dendritic overgrowth, causing specific expansion of the inner plexiform layer. However, the outer plexiform layer remains relatively undisturbed in the Pten deficient retina. Physiological analysis detects reduced rod function and augmented oscillatory potentials originating from amacrine cells in Pten mutants. Furthermore, deleting Pten or elevating Akt activity in individual amacrine cells is sufficient to disrupt dendritic arborization, indicating that Pten activity is required cell autonomously to control neuronal morphology. Moreover, inhibiting endogenous Akt activity attenuates inner plexiform layer formation in vitro. Together, these findings demonstrate that suppression of PI3K/Akt signaling by PTEN is crucial for proper neuronal differentiation and normal retinal network formation.

Distinct effects of Hedgehog signaling on neuronal fate specification and cell cycle progression in the embryonic mouse retina.

Cell-extrinsic signals can profoundly influence the production of various neurons from common progenitors. Yet mechanisms by which extrinsic signals coordinate progenitor cell proliferation, cell cycle exit, and cell fate choices are not well understood. Here, we address whether Hedgehog (Hh) signals independently regulate progenitor proliferation and neuronal fate decisions in the embryonic mouse retina. Conditional ablation of the essential Hh signaling component Smoothened (Smo) in proliferating progenitors, rather than in nascent postmitotic neurons, leads to a dramatic increase of retinal ganglion cells (RGCs) and a mild increase of cone photoreceptor precursors without significantly affecting other early-born neuronal cell types. In addition, Smo-deficient progenitors exhibit aberrant expression of cell cycle regulators and delayed G(1)/S transition, especially during the late embryonic stages, resulting in a reduced progenitor pool by birth. Deficiency in Smo function also causes reduced expression of the basic helix-loop-helix transcription repressor Hes1 and preferential elevation of the proneural gene Math5. In Smo and Math5 double knock-out mutants, the enhanced RGC production observed in Smo-deficient retinas is abolished, whereas defects in the G(1)/S transition persist, suggesting that Math5 mediates the Hh effect on neuronal fate specification but not on cell proliferation. These findings demonstrate that Hh signals regulate progenitor pool expansion primarily by promoting cell cycle progression and influence cell cycle exit and neuronal fates by controlling specific proneural genes. Together, these distinct cellular effects of Hh signaling in neural progenitor cells coordinate a balanced production of diverse neuronal cell types.

Extraocular dorsal signal affects the developmental fate of the optic vesicle and patterns the optic neuroepithelium.

Dorsal-ventral (DV) specification in the early optic vesicle plays a crucial role in the proper development of the eye. To address the questions of how DV specification is determined and how it affects fate determination of the optic vesicle, isolated optic vesicles were cultured either in vitro or in ovo. The dorsal and ventral halves of the optic vesicle were fated to develop into retinal pigment epithelium (RPE) and neural retina, respectively, when they were separated from each other and cultured. In optic vesicles treated with collagenase to remove the surrounding tissues, the neuroepithelium gave rise to cRax expression but not Mitf, suggesting that surrounding tissues are necessary for RPE specification. This was also confirmed in in ovo explant cultures. Combination cultures of collagenase-treated optic vesicles with either the dorsal or ventral part of the head indicated that head-derived factors have an important role in the fate determination of the optic vesicle: in the optic vesicles co-cultured with the dorsal part of the head Mitf expression was induced in the neuroepithelium, while the ventral head portion did not have this effect. The dorsal head also suppressed Pax2 expression in the optic vesicle. These observations indicate that factors from the dorsal head portion have important roles in the establishment of DV polarity within the optic vesicle, which in turn induces the patterning and differentiation of the neural retina and pigment epithelium.

Temporal expression of L-Maf and RaxL in developing chicken retina are arranged into mosaic pattern.

Members of the maf gene family encode basic/leucine zipper transcription factors and play important roles during cell differentiation in vertebrate and Drosophila development. To investigate the possible roles of chicken lens-specific Maf (L-Maf)/MafA in retinal development, we analyzed its expression in the developing chicken retina. We first determined that L-Maf is expressed in a subset of ganglion cells, amacrine cells and rod cells during retinal development. To characterize further the L-Maf expressing cells during photoreceptor development, we compared the expression patterns of L-Maf and RaxL, a marker of cone cells. We found the L-Maf proteins are detectable from E10 between RaxL positive cone cells in outer nuclear layer (ONL). Subsequently, the expression of L-Maf is restricted to the innermost nuclear cells in the ONL during rod differentiation. In contrast, RaxL positive cone cells are distributed in the outermost layer of ONL. Furthermore, tangential retinal sections showed that L-Maf positive rods and RaxL positive cones form a mosaic pattern during photoreceptor development.

RaxL regulates chick ganglion cell development.

RaxL is a paired-like homeobox gene involved in vertebrate eye morphogenesis. We examined RaxL protein expression patterns during chick retinal development in combination with ganglion cell markers including the RA4 antigen, cBrn-3, Islet-1 and neuronal type III beta-tubulin. Double-immunostaining demonstrated that downregulation of RaxL protein correlates with upregulation of ganglion cell markers in the ganglion cell layer (GCL). To explore this correlation in vivo, we performed gain- and loss-of-function experiments by electroporating retroviral vectors encoding wild-type and dominant-negative-RaxL into the optic vesicles of stage 10 chick embryos. Infection with virus expressing RaxL led to a 35% decrease in Islet-1-positive ganglion cells at E5.0 and a complete loss of ganglion cells at E15, with no effect on displaced amacrine cells in the GCL. When dominant-negative RaxL was expressed, the total number of cells in the GCL increased by approximately 40% at E5.0 but was reduced to 40% at E15, due to ectopic apoptosis in the GCL from E9 to E15. These results suggest that RaxL gives an inhibitory effect on ganglion cell development and that the loss of RaxL expression is required for maintenance of ganglion cells.

Development of dorsal-ventral polarity in the optic vesicle and its presumptive role in eye morphogenesis as shown by embryonic transplantation and in ovo explant culturing.

Dorsal and ventral specification in the early optic vesicle appears to play a crucial role in the proper development of the eye. In the present study, we performed embryonic transplantation and organ culturing of the chick optic vesicle in order to investigate how the dorsal-ventral (D-V) polarity is established in the optic vesicle and what role this polarity plays in proper eye development. The left optic vesicle was cut and transplanted inversely in the right eye cavity of host chick embryos. This method ensured that the D-V polarity was reversed while the anteroposterior axis remained normal. The results showed that the location of the choroid fissure was altered from the normal (ventral) to ectopic positions as the embryonic stage of transplantation progressed from 6 to 18 somites. At the same time, the shape of the optic vesicle and the expression patterns of Pax2 and Tbx5, marker genes for ventral and dorsal regions of the optic vesicle, respectively, changed concomitantly in a similar way. The crucial period was between the 8- and 14-somite stages, and during this period the polarity seemed to be gradually determined. In ovo explant culturing of the optic vesicle showed that the D-V polarity and choroid fissure formation were already specified by the 10-somite stage. These results indicate that the D-V polarity of the optic vesicle is established gradually between 8- and 14-somite stages under the influence of signals derived from the midline portion of the forebrain. The presumptive signal(s) appeared to be transmitted from proximal to distal regions within the optic vesicle. A severe anomaly was observed in the development of optic vesicles reversely transplanted around the 10-somite stage: the optic cup formation was disturbed and subsequently the neural retina and pigment epithelium did not develop normally. We concluded that establishment of the D-V polarity in the optic vesicle plays an essential role in the patterning and differentiation of the neural retina and pigment epithelium.