Shu-Zhen Wang

bHLH Genes and Retinal Cell Fate Specification

The various cell types in the vertebrate retina arise from a pool of common progenitors. The way that the cell types are specified has been a long-standing issue. Decades of research have yielded a large body of information regarding the involvement of extrinsic factors, and only recently has the function of intrinsic factors begun to emerge. This article reviews recent studies addressing the role of basic helix-loop-helix (bHLH) factors in specifying retinal cell types, with an emphasis on bHLH hierarchies leading to photoreceptor production. Photoreceptor genesis appears to employ two transcriptional pathways: ngn2→neuroD→raxL and ath5→neuroD→raxL. ngn2 and ath5 function in progenitors, which can potentially develop into different cell types. neuroD represents one of the central steps in photoreceptor specification. Ath5 is also essential for ganglion cell development. It remains to be demonstrated whether a bHLH gene functions as a key player in specifying the other types of retinal cells. Genetic knockout studies have indicated intricate cross-regulation among bHLH genes. Future studies are expected to unveil the mechanism by which bHLH factors network with intrinsic factors and communicate with extrinsic factors to ensure a balanced production of the various types of retinal cells.

A Role of ath5 in Inducing neuroD and the Photoreceptor Pathway

Photoreceptors in the vertebrate retina are light-sensitive neurons, and their degeneration results in irreversible visual loss. Understanding how photoreceptor fate is determined is a prerequisite for developing photoreceptor replacement therapies. Previous studies identified two basic helix-loop-helix genes, neurogenin2 (ngn2) and neuroD, participating in a genetic pathway leading to photoreceptor genesis. Here we present experimental data suggesting that ath5, which is known for its critical role in retinal ganglion cell development, may also lead to photoreceptor production. In the developing retina, ath5 expression was detected in two zones of cells, and coexpression with neuroD was observed in the zone adjacent to young photoreceptor cells accumulating on the retinal pigment epithelial side. Retroviral-driven misexpression of ath5 in retinal cells increased the population of photoreceptor cells, as well as ganglion cells, in a developmental stage-dependent manner that is consistent with ath5 being involved in the development of multiple types of retinal neurons. Ectopic ath5 expression in cultures of non-neural retinal pigment epithelial cells elicited transdifferentiation into cells that expressed photoreceptor-specific genes and displayed photoreceptor-like morphologies. Gene expression analysis showed that ngn2 did not induce ath5, and ath5 did not induce ngn2, but both induced neuroD and RaxL. These data suggest a pathway of “ath5 → neuroD → photoreceptor genes” separate from yet convergent with the ngn2 pathway.

Neurogenin3 promotes early retinal neurogenesis.

The transcriptional regulatory network governing the establishment of retinal neuron diversity is not well delineated. We report experimental results suggesting proneural gene neurogenin3 (ngn3) participating in this regulatory network. Retinal expression of chick ngn3 was confined to early neurogenesis. Overexpression of ngn3 in chick retina reduced cell proliferation and expanded the population of ganglion cells into the territory normally occupied by amacrine cells. Ngn3 overexpression altered the expression of a number of regulatory genes, including ash1, ath3, ath5, chx10, neuroD, ngn1, ngn2, and NSCL1. Early gene ngn1 was induced, but ash1, ngn2, ath3, and chx10, whose expressions persist through later phases of neurogenesis, were down-regulated. Expression of ath5 was up-regulated at the locale corresponding to young ganglion cells, but was down-regulated at the locale corresponding to progenitor cells. These results suggest that ngn3 regulates retinal neurogenesis by inducing regulatory genes for early-born neurons and repressing those for later-born cells.

NeuroD induces the expression of visinin and calretinin by proliferating cells derived from toxin‐damaged chicken retina

Müller glia have been shown to be a potential source of neural regeneration in the avian retina. In response to acute damage Müller glia de‐differentiate, proliferate, express transcription factors found in embryonic retinal progenitors, and some of the progeny differentiate into neurons and glia (Fischer and Reh [ 2001a ] Nat. Neurosci. 4:247–252). However, most of the cells produced by proliferating Müller cells appear to remain undifferentiated. The purpose of this study was to test whether the neurogenic gene NeuroD can promote the differentiation of proliferating cells derived from the postnatal chick retina. We used recombinant avian retroviruses to transfect green fluorescent protein (GFP) or NeuroD. The majority of cells transfected with GFP remained undifferentiated, with a few cells differentiating into calretinin‐immunoreactive neurons. Many cells transfected with the NeuroD‐virus expressed calretinin, neurofilament, or visinin, while most cells remained undifferentiated. The number of calretinin‐expressing cells that were generated was increased approximately 20‐fold with forced expression of NeuroD. In addition, we found that cells transfected with NeuroD never expressed glutamine synthetase, a marker of mature Müller glia, suggesting that NeuroD suppresses glial differentiation. We conclude that NeuroD stimulates cells from the toxin‐damaged chicken retina to acquire some neuronal phenotypes. We propose that most of these cells were derived from Müller glia. Developmental Dynamics 229:555–563, 2004.

Misexpression of a bHLH gene, cNSCL1, results in abnormal brain development

NSCL1 is a basic helix‐loop‐helix transcription factor involved in the development of the nervous system. To elucidate its role in neurogenesis, we cloned chick NSCL1 (cNSCL1) and examined its expression pattern and the effect of its misexpression on brain development. cNSCL1 was predominantly expressed during active neurogenesis. Double‐labeling experiments showed that proliferating neuroblasts in the ventricular zone lacked cNSCL1 expression and cells expressing cNSCL1 were located just outside the ventricular zone. Retroviral misexpression of cNSCL1 in chick embryos produced a brain with abnormal structure. While the forebrain of the embryonic day‐12 (E12) brain appeared normal, the tectum was enlarged. The enlargement was likely due to an increase in cell proliferation, since more radioactivity was detected in this region of the brain after [3H]thymidine labeling at E9. The cerebellum, on the other hand, was reduced in size. Fewer cells were labeled with BrdU in the external granule layer (a secondary germinal layer required for cerebellum development) in experimental embryos than in the controls, suggesting that misexpression of cNSCL1 might interfere with cell proliferation in the external granular layer. Our data indicate that regulated expression of cNSCL1 is required for normal brain development. They also imply that cNSCL1 might be involved in preventing some postmitotic cells from reentering the cell cycle during neurogenesis. Dev Dyn 1999;215:238–247.

Neurogenin1 effectively reprograms cultured chick retinal pigment epithelial cells to differentiate toward photoreceptors.

Photoreceptors are highly specialized sensory neurons in the retina, and their degeneration results in blindness. Replacement with developing photoreceptor cells promises to be an effective therapy, but it requires a supply of new photoreceptors, because the neural retina in human eyes lacks regeneration capability. We report efficient generation of differentiating, photoreceptor‐like neurons from chick retinal pigment epithelial (RPE) cells propagated in culture through reprogramming with neurogenin1 (ngn1). In reprogrammed culture, a large number of the cells (85.0% ± 5.9%) began to differentiate toward photoreceptors. Reprogrammed cells expressed transcription factors that set in motion photoreceptor differentiation, including Crx, Nr2E3, NeuroD, and RXRγ, and phototransduction pathway components, including transducin, cGMP‐gated channel, and red opsin of cone photoreceptors (equivalent to rhodopsin of rod photoreceptors). They developed inner segments rich in mitochondria. Furthermore, they responded to light by decreasing their cellular free calcium (Ca2+) levels and responded to 9‐cis‐retinal by increasing their Ca2+ levels after photobleaching, hallmarks of photoreceptor physiology. The high efficiency and the advanced photoreceptor differentiation indicate ngn1 as a gene of choice to reprogram RPE progeny cells to differentiate into photoreceptor neurons in future cell replacement studies. J. Comp. Neurol. 518:526–546, 2010.

Using neurogenin to Reprogram Chick RPE to Produce Photoreceptor-like Neurons

PURPOSE One potential therapy for vision loss from photoreceptor degeneration is cell replacement, but this approach presents a need for photoreceptor cells. This study explores whether the retinal pigment epithelium (RPE) could be a convenient source of developing photoreceptors. METHODS The RPE of chick embryos was subjected to reprogramming by proneural genes neurogenin (ngn)1 and ngn3. The genes were introduced into the RPE through retrovirus RCAS-mediated transduction, with the virus microinjected into the eye or added to retinal pigment epithelial explant culture. The retinal pigment epithelia were then analyzed for photoreceptor traits. RESULTS In chick embryos infected with retrovirus RCAS-expressing ngn3 (RCAS-ngn3), the photoreceptor gene visinin (the equivalent of mammalian recoverin) was expressed in cells of the retinal pigment epithelial layer. When isolated and cultured as explants, retinal pigment epithelial tissues from embryos infected with RCAS-ngn3 or RCAS-ngn1 gave rise to layers of visinin-positive cells. These reprogrammed cells expressed genes of phototransduction and synapses, such as red opsin, the alpha-subunit of cone transducin, SNAP-25, and PSD-95. Reprogramming occurred with retinal pigment epithelial explants derived from virally infected embryos and with retinal pigment epithelial explants derived from normal embryos, with the recombinant viruses added at the onset of the explant culture. In addition, reprogramming took place in retinal pigment epithelial explants from both young and old embryos, from embryonic day (E)6 to E18, when the visual system becomes functional in the chick. CONCLUSIONS The results support the prospect of exploring the RPE as a convenient source of developing photoreceptors for in situ cell replacement.

Proneural gene ash1 promotes amacrine cell production in the chick retina

The diverse types of neurons and Müller glia in the vertebrate retina are believed to arise from common progenitor cells. To better understand how neural diversity is achieved during retinal neurogenesis, we examined the function of ash1, a proneural bHLH gene expressed in progenitor cells throughout retinal neurogenesis. Published studies using retinal explant culture derived from knockout mice concluded that ash1 is required for the production of late‐born neurons, including bipolar cells. In this study, gain‐of‐function experiments were carried out in ovo in embryonic chick retina. In the developing chick retina, expression of ash1 temporally overlapped with, but spatially differed from, the expression of ngn2, also a proneural gene expressed in progenitor cells throughout retinal neurogenesis. Retrovirus‐driven overexpression of ash1 in the developing chick retina decreased the progenitor population (BrdU+ or expressing ngn2), expanded the amacrine population (AP2α+ or Pax6+), and reduced bipolar (chx10 mRNA+) and Müller glial (vimentin+) populations. Photoreceptor deficiency occurred after the completion of neurogenesis. The number of ganglion cells, which are born first during retinal neurogenesis, remained unchanged. Similar overexpression of ngn2 did not produce discernible changes in retinal neurogenesis, nor in ash1 expression. These results suggest that ash1 promotes the production of amacrine cells and thus may participate in a regulatory network governing neural diversity in the chick retina.

Pro-Photoreceptor Activity of Chick neurogenin1

PURPOSE Better understanding of photoreceptor fate specification may lead to efficient production of photoreceptors for cell replacement studies. The authors investigated the role of proneural bHLH gene neurogenin1 (ngn1) in photoreceptor genesis using the chick retina. METHODS In situ hybridization was used to delineate the spatial and temporal pattern of ngn1 expression. RCAS retrovirus was used to drive overexpression of ngn1 in retinal cells, and siRNA was used to reduce ngn1 expression in loss-of-function experiments. RESULTS Chick ngn1 was transiently expressed during early phases of retinal neurogenesis, from embryonic day (E)3 to E6, with cells expressing ngn1 confined to the apical side of the retinal neuroepithelium. The time window and the anatomic location of ngn1 expression coincided with photoreceptor genesis and differed from those of other transiently expressed proneural bHLH genes, such as ash1, ath3, ath5, and ngn2. Most ngn1-expressing cells lacked BrdU incorporation and lacked phosphorylated histone H3. In low-density cell culture, ngn1 overexpression increased neuroD expression and expanded the photoreceptor population but reduced the ganglion population. Treatment of dissociated retinal cells with siRNA against ngn1 mRNA specifically reduced the photoreceptor population. Overexpression of ngn1 in the retina reduced the expression of ash1, ath5, chx10, and ngn2. CONCLUSIONS The data suggest that ngn1 participates in a complex transcriptional network and may play a role in guiding a progenitor cell to the photoreceptor pathway.

Reprogramming Progeny Cells of Embryonic RPE to Produce Photoreceptors: Development of Advanced Photoreceptor Traits under the Induction of neuroD

PURPOSE In examining the prospect of producing functional photoreceptors by reprogramming the differentiation of RPE progeny cells, this study was conducted to investigate whether reprogrammed cells can develop highly specialized ultrastructural and physiological traits that characterize retinal photoreceptors. METHODS Cultured chick RPE cells were reprogrammed to differentiate along the photoreceptor pathway by ectopic expression of neuroD. Cellular ultrastructure was examined with electron microscopy. Cellular physiology was studied by monitoring cellular free calcium (Ca(2+)) levels in dark-adapted cells in response to light and in light-bleached cells in response to 9-cis-retinal. RESULTS Reprogrammed cells were found to localize red opsin protein appropriately to the apex. These cells developed inner segments rich in mitochondria, and while in culture, some formed rudimentary outer segments, analogous to those of developing photoreceptors in the retina. In response to light, reprogrammed cells reduced their Ca(2+) levels, as observed with developing retinal photoreceptors in culture. Further, on exposure to 9-cis-retinal, the light-bleached, reprogrammed cells increased their Ca(2+) levels, reminiscent of visual cycle recovery. CONCLUSIONS These results indicate the potential of reprogrammed cells to develop advanced ultrastructural and physiological traits of photoreceptors and point to reprogramming progeny cells of embryonic RPE as a possible alternative in producing developing photoreceptors.