Divergent evolution of developmental timing in the neocortex revealed by marsupial and eutherian transcriptomes.

Only mammals evolved a neocortex, which integrates sensory-motor and cognitive functions. Significant diversifications in the cellular composition and connectivity of the neocortex occurred between the two main therian groups: marsupials and eutherians. However, the developmental mechanisms underlying these diversifications are largely unknown. Here, we compared the neocortical transcriptomes of Sminthopsis crassicaudata, a mouse-sized marsupial, with those of eutherian mice at two developmentally equivalent time points corresponding to deeper and upper layer neuron generation. Enrichment analyses revealed more mature gene networks in marsupials at the early stage, which reverted at the later stage, suggesting a more precocious but protracted neuronal maturation program relative to birth timing of cortical layers. We ranked genes expressed in different species and identified important differences in gene expression rankings between species. For example, genes known to be enriched in upper-layer cortical projection neuron subtypes, such as Cux1, Lhx2 and Satb2, likely relate to corpus callosum emergence in eutherians. These results show molecular heterochronies of neocortical development in Theria, and highlight changes in gene expression and cell type composition that may underlie neocortical evolution and diversification. This article has an associated 'The people behind the papers' interview.

Polycomb repressive complex 2 is critical for mouse cortical glutamatergic neuron development.

The Polycomb Repressive Complex 2 (PRC2) regulates corticogenesis, yet the consequences of mutations to this epigenetic modifier in the mature brain are poorly defined. Importantly, PRC2 core genes are haploinsufficient and causative of several human neurodevelopmental disorders. To address the role of PRC2 in mature cortical structure and function, we conditionally deleted the PRC2 gene Eed from the developing mouse dorsal telencephalon. Adult homozygotes displayed smaller forebrain structures. Single-nucleus transcriptomics revealed that glutamatergic neurons were particularly affected, exhibiting dysregulated gene expression profiles, accompanied by aberrations in neuronal morphology and connectivity. Remarkably, homozygous mice performed well on challenging cognitive tasks. In contrast, while heterozygous mice did not exhibit clear anatomical or behavioral differences, they displayed dysregulation of neuronal genes and altered neuronal morphology that was strikingly different from homozygous phenotypes. Collectively, these data reveal how alterations to PRC2 function shape the mature brain and reveal a dose-specific role for PRC2 in determining glutamatergic neuron identity.

Differential timing of a conserved transcriptional network underlies divergent cortical projection routes across mammalian brain evolution.

A unique combination of transcription factor expression and projection neuron identity demarcates each layer of the cerebral cortex. During mouse and human cortical development, the transcription factor CTIP2 specifies neurons that project subcerebrally, while SATB2 specifies neuronal projections via the corpus callosum, a large axon tract connecting the two neocortical hemispheres that emerged exclusively in eutherian mammals. Marsupials comprise the sister taxon of eutherians but do not have a corpus callosum; their intercortical commissural neurons instead project via the anterior commissure, similar to egg-laying monotreme mammals. It remains unknown whether divergent transcriptional networks underlie these cortical wiring differences. Here, we combine birth-dating analysis, retrograde tracing, gene overexpression and knockdown, and axonal quantification to compare the functions of CTIP2 and SATB2 in neocortical development, between the eutherian mouse and the marsupial fat-tailed dunnart. We demonstrate a striking degree of structural and functional homology, whereby CTIP2 or SATB2 of either species is sufficient to promote a subcerebral or commissural fate, respectively. Remarkably, we reveal a substantial delay in the onset of developmental SATB2 expression in mice as compared to the equivalent stage in dunnarts, with premature SATB2 overexpression in mice to match that of dunnarts resulting in a marsupial-like projection fate via the anterior commissure. Our results suggest that small alterations in the timing of regulatory gene expression may underlie interspecies differences in neuronal projection fate specification.

A pan-mammalian map of interhemispheric brain connections predates the evolution of the corpus callosum.

The brain of mammals differs from that of all other vertebrates, in having a six-layered neocortex that is extensively interconnected within and between hemispheres. Interhemispheric connections are conveyed through the anterior commissure in egg-laying monotremes and marsupials, whereas eutherians evolved a separate commissural tract, the corpus callosum. Although the pattern of interhemispheric connectivity via the corpus callosum is broadly shared across eutherian species, it is not known whether this pattern arose as a consequence of callosal evolution or instead corresponds to a more ancient feature of mammalian brain organization. Here we show that, despite cortical axons using an ancestral commissural route, monotremes and marsupials share features of interhemispheric connectivity with eutherians that likely predate the origin of the corpus callosum. Based on ex vivo magnetic resonance imaging and tractography, we found that connections through the anterior commissure in both fat-tailed dunnarts (Marsupialia) and duck-billed platypus (Monotremata) are spatially segregated according to cortical area topography. Moreover, cell-resolution retrograde and anterograde interhemispheric circuit mapping in dunnarts revealed several features shared with callosal circuits of eutherians. These include the layered organization of commissural neurons and terminals, a broad map of connections between similar (homotopic) regions of each hemisphere, and regions connected to different areas (heterotopic), including hyperconnected hubs along the medial and lateral borders of the cortex, such as the cingulate/motor cortex and claustrum/insula. We therefore propose that an interhemispheric connectome originated in early mammalian ancestors, predating the evolution of the corpus callosum. Because these features have been conserved throughout mammalian evolution, they likely represent key aspects of neocortical organization.

LINE-1 retrotransposons contribute to mouse PV interneuron development.

Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.

Bilateral visual projections exist in non-teleost bony fish and predate the emergence of tetrapods.

In most vertebrates, camera-style eyes contain retinal ganglion cell neurons that project to visual centers on both sides of the brain. However, in fish, ganglion cells were thought to innervate only the contralateral side, suggesting that bilateral visual projections appeared in tetrapods. Here we show that bilateral visual projections exist in non-teleost fishes and that the appearance of ipsilateral projections does not correlate with terrestrial transition or predatory behavior. We also report that the developmental program that specifies visual system laterality differs between fishes and mammals, as the Zic2 transcription factor, which specifies ipsilateral retinal ganglion cells in tetrapods, appears to be absent from fish ganglion cells. However, overexpression of human ZIC2 induces ipsilateral visual projections in zebrafish. Therefore, the existence of bilateral visual projections likely preceded the emergence of binocular vision in tetrapods.

DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation.

The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.

Alterations in ZENK and glucagon RNA transcript expression during increased ocular growth in chickens.

PURPOSE: To examine in detail the time-course of changes in Zif268, Egr-1, NGFI-A, and Krox-24 (ZENK) and pre-proglucagon (PPG) RNA transcript levels in the chick retina during periods of increased ocular growth induced by form-deprivation and negative-lens wear. To further elucidate the role of ZENK in the modulation of ocular growth, we investigated the effect of intravitreal injections of the muscarinic antagonist atropine and the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (ADTN), both of which block the development of experimental myopia, on the expression of ZENK in eyes fitted with negative-lenses. METHODS: Myopia was induced by fitting translucent diffusers or -10D polymethyl methacrylate (PMMA) lenses over one eye of the chicken. At times from 1 h to 10 days after fitting of the diffusers or negative lenses, retinal RNA transcript levels of the selected genes were determined by semi-quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). For the pharmacology experiments, -10D lenses were fitted over the left eye of chicks for a period of 1h. Intravitreal injections of atropine (10 mul-25 mM), ADTN (10 mul-10 mM), or a vehicle solution were made immediately before fitting of the lenses. RESULTS: ZENK RNA transcript levels were rapidly and persistently down-regulated following the attachment of the optical devices over the eye. With a delay relative to ZENK, PPG transcript levels were also down-regulated. Induced changes in gene expression were similar for both form-deprivation and negative-lens wear. When atropine or ADTN were administered immediately before lens attachment, the rapid down-regulation in ZENK RNA transcript levels normally seen following 1 h of negative-lens wear was not seen, and ZENK transcript levels rose above those values seen in control eyes. However, injection of atropine or ADTN into untreated eyes had no effect on ZENK transcript levels. CONCLUSIONS: Both form-deprivation and negative-lens wear modulated the retinal expression of ZENK and PPG RNA transcripts, with a similar time-course and strength of response. The ability of the tested drugs to prevent the down-regulation of ZENK in both lens-induced myopia (LIM) and form-deprivation myopia (FDM) suggests that atropine and ADTN act directly and rapidly on retinal circuits to enhance sensitivity early in the signaling process. These findings suggest that very similar molecular pathways are involved in the changes in eye growth in response to form-deprivation and negative lenses at 1 h after the fitting of optical devices.

Differential expression of anti-angiogenic factors and guidance genes in the developing macula.

PURPOSE: The primate retina contains a specialized, cone-rich macula, which mediates high acuity and color vision. The spatial resolution provided by the neural retina at the macula is optimized by stereotyped retinal blood vessel and ganglion cell axon patterning, which radiate away from the macula and reduce shadowing of macular photoreceptors. However, the genes that mediate these specializations, and the reasons for the vulnerability of the macula to degenerative disease, remain obscure. The aim of this study was to identify novel genes that may influence retinal vascular patterning and definition of the foveal avascular area. METHODS: We used RNA from human fetal retinas at 19-20 weeks of gestation (WG; n=4) to measure differential gene expression in the macula, a region nasal to disc (nasal) and in the surrounding retina (surround) by hybridization to 12 GeneChip microarrays (HG-U133 Plus 2.0). The raw data was subjected to quality control assessment and preprocessing, using GC-RMA. We then used ANOVA analysis (Partek) Genomic Suite 6.3) and clustering (DAVID website) to identify the most highly represented genes clustered according to "biological process." The neural retina is fully differentiated at the macula at 19-20 WG, while neuronal progenitor cells are present throughout the rest of the retina. We therefore excluded genes associated with the cell cycle, and markers of differentiated neurons, from further analyses. Significantly regulated genes (p<0.01) were then identified in a second round of clustering according to molecular/reaction (KEGG) pathway. Genes of interest were verified by quantitative PCR (QRT-PCR), and 2 genes were localized by in situ hybridization. RESULTS: We generated two lists of differentially regulated genes: "macula versus surround" and "macula versus nasal." KEGG pathway clustering of the filtered gene lists identified 25 axon guidance-related genes that are differentially regulated in the macula. Furthermore, we found significant upregulation of three anti-angiogenic factors in the macula: pigment epithelium derived factor (PEDF), natriuretic peptide precurusor B (NPPB), and collagen type IValpha2. Differential expression of several members of the ephrin and semaphorin axon guidance gene families, PEDF, and NPPB was verified by QRT-PCR. Localization of PEDF and Eph-A6 mRNAs in sections of macaque retina shows expression of both genes concentrates in the ganglion cell layer (GCL) at the developing fovea, consistent with an involvement in definition of the foveal avascular area. CONCLUSIONS: Because the axons of macular ganglion cells exit the retina from around 8 WG, we suggest that the axon guidance genes highly expressed at the macula at 19-20 WG are also involved in vascular patterning, along with PEDF and NPPB. Localization of both PEDF and Eph-A6 mRNAs to the GCL of the developing fovea supports this idea. It is possible that specialization of the macular vessels, including definition of the foveal avascular area, is mediated by processes that piggyback on axon guidance mechanisms in effect earlier in development. These findings may be useful to understand the vulnerability of the macula to degeneration and to develop new therapeutic strategies to inhibit neovascularization.

Gradients of Eph-A6 expression in primate retina suggest roles in both vascular and axon guidance.

PURPOSE: Recently we identified high levels of expression of Eph-A6 in the macula of developing human retina and showed localization of Eph-A6 to ganglion cells (GC). In the present study we investigated the expression of some members of the ephrin family in developing primate retina, including the topography of Eph-A6 expression, and its ligands, in developing macaque retinas. METHODS: We extracted RNA from human fetal retinas and probed for Eph-A5-A7, Eph-B1, ephrin-B2, and ephrin-A1-A5 by RT-PCR, then prepared riboprobes for Eph-A5-A7, Eph-B1 and ephrin-A1, -A4 and -B2. Paraffin sections of fetal macaque retinas were used to localize expression of Ephs and ephrins by in situ hybridization and immunohistochemistry. RESULTS: We identified prominent gradients of Eph-A6 mRNA expression in the ganglion cell layer (GCL) of fetal macaque retinas of different ages. The gradient of Eph-A6 expression was high near the optic disc and low at the developing macula at fetal day (Fd) 55. At Fd 70 and 80, the gradient of Eph-A6 expression was reversed, being higher temporal to the macula, and low at the disc. By Fd 110, when the fovea begins to form, a pattern of expression was established that persisted into the postnatal period, in which the highest levels of expression were detected at the developing fovea, and progressively lower levels of expression were detected at increasing distance from the fovea. Beginning at Fd 70, we also detected a gradient of Eph-A6 expression running perpendicular to the retinal surface within the GCL of central retina that was high in the inner GCL and low in the outer GCL. This second pattern persisted into the neonatal period. We found the two ligands for Eph-A6, ephrin-A1 and ephrin-A4, expressed by Pax2-immunoreactive astrocytes, in the optic nerve head and in the retina, by in situ hybridization and immunohistochemistry. We propose that during development of the retinal vasculature, migration of ligand-bearing astrocytes is slowed along this Eph-A6 expression gradient through repellent Eph-A6 - ephrin-A1 and -A4 signaling. CONCLUSIONS: Patterns of Eph-A6 expression in the developing macaque retina suggest that Eph-A6 - ephrin-A1 and -A4 repellent signaling has a role in retinal vascular patterning, and in the postnatal maintenance of projections from macular and foveal GC.

Effects of a head-mounted display on the oculomotor system of children.

PURPOSE: This study evaluated the effects of short term and extended viewing of virtual imagery using the Binocular Viewer (new generation bi-ocular viewer) on the visual system of children, and compared these effects with that of viewing a high definition television (HDTV) display. METHODS: Sixty children aged 5 to 16 years viewed 30 min of virtual imagery using the Binocular Viewer and a HDTV display on two occasions. Sixteen subjects, aged 13 to 16 years, completed a third session of extended viewing (80 min) with the Binocular Viewer. Oculomotor function and symptoms were assessed previewing, immediately postviewing, and 10 min postviewing. RESULTS: Thirty minutes of Binocular Viewer use resulted in symptom increases (p < 0.05) immediately postviewing ("feeling tired," "feeling sleepy," "difficulty concentrating," and "sore/aching eyes") however most symptoms had dissipated by 10-min postviewing. There were no significant symptom differences between viewing with the Binocular Viewer and the HDTV display at either time point. An increase in symptoms (p < 0.05) immediately postviewing was recorded after 80 min of Binocular Viewer use ("feeling tired," "feeling bored," "feeling sleepy," and "tired eyes"), however only "feeling tired" and "feeling bored" remained significantly increased (p < 0.05) 10-min postviewing. Near unaided visual acuity demonstrated a significant and consistent reduction immediately (p < 0.01) and at 10 min (p < 0.05) following 30 min of Binocular Viewer use and immediately following 80 min of use (p < 0.01). Near unaided VA was also significantly reduced (p < 0.01) immediately after 30 min of HDTV display use. CONCLUSIONS: Virtual imagery viewing with the Binocular Viewer in children aged 5 to 16 years had few additional adverse effects when compared to viewing a more conventional HDTV display. The Binocular Viewer was comfortable to wear for up to 80 min of viewing. The consistent reduction in near vision for both viewing durations with the Binocular Viewer requires further investigation.

Shared and differential features of Robo3 expression pattern in amniotes.

In Bilaterians, commissural neurons project their axons across the midline of the nervous system to target neurons on the opposite side. In mammals, midline crossing at the level of the hindbrain and spinal cord requires the Robo3 receptor which is transiently expressed by all commissural neurons. Unlike other Robo receptors, mammalian Robo3 receptors do not bind Slit ligands and promote midline crossing. Surprisingly, not much is known about Robo3 distribution and mechanism of action in other vertebrate species. Here, we have used whole-mount immunostaining, tissue clearing and light-sheet fluorescent microscopy to study Robo3 expression pattern in embryonic tissue from diverse representatives of amniotes at distinct stages, including squamate (African house snake), birds (chicken, duck, pigeon, ostrich, emu and zebra finch), early postnatal marsupial mammals (fat-tailed dunnart), and eutherian mammals (mouse and human). The analysis of this rich and unique repertoire of amniote specimens reveals conserved features of Robo3 expression in midbrain, hindbrain and spinal cord commissural circuits, which together with subtle but meaningful modifications could account for species-specific evolution of sensory-motor and cognitive capacities. Our results also highlight important differences of precerebellar nuclei development across amniotes.

Multiple events of gene manipulation via in pouch electroporation in a marsupial model of mammalian forebrain development.

BACKGROUND: The technique of in utero electroporation has been widely used in eutherians, such as mice and rats, to investigate brain development by selectively manipulating gene expression in specific neuronal populations. A major challenge, however, is that surgery is required to access the embryos, affecting animal survival and limiting the number of times it can be performed within the same litter. NEW METHOD: Marsupials are born at an early stage of brain development as compared to eutherians. Forebrain neurogenesis occurs mostly postnatally, allowing electroporation to be performed while joeys develop attached to the teat. Here we describe the method of in pouch electroporation using the Australian marsupial fat-tailed dunnart (Sminthopsis crassicaudata, Dasyuridae). RESULTS: In pouch electroporation is minimally invasive, quick, successful and anatomically precise. Moreover, as no surgery is required, it can be performed several times in the same individual, and littermates can undergo independent treatments. COMPARISON WITH EXISTING METHOD: As compared to in utero electroporation in rodents, in pouch electroporation in marsupials offers unprecedented opportunities to study brain development in a minimally invasive manner. Continuous access to developing joeys during a protracted period of cortical development allows multiple and independent genetic manipulations to study the interaction of different systems during brain development. CONCLUSIONS: In pouch electroporation in marsupials offers an excellent in vivo assay to study forebrain development and evolution. By combining developmental, functional and comparative approaches, this system offers new avenues to investigate questions of biological and medical relevance, such as the precise mechanisms of brain wiring and the organismic and environmental influences on neural circuit formation.

Astroglial-mediated remodeling of the interhemispheric midline during telencephalic development is exclusive to eutherian mammals.

The corpus callosum forms the major interhemispheric connection in the human brain and is unique to eutherian (or placental) mammals. The developmental events associated with the evolutionary emergence of this structure, however, remain poorly understood. A key step in callosal formation is the prior remodeling of the interhemispheric fissure by embryonic astroglial cells, which then subsequently act as a permissive substrate for callosal axons, enabling them to cross the interhemispheric midline. However, whether astroglial-mediated interhemispheric remodeling is unique to eutherian mammals, and thus possibly associated with the phylogenetic origin of the corpus callosum, or instead is a general feature of mammalian brain development, is not yet known. To investigate this, we performed a comparative analysis of interhemispheric remodeling in eutherian and non-eutherian mammals, whose lineages branched off before the evolution of the corpus callosum. Whole brain MRI analyses revealed that the interhemispheric fissure is retained into adulthood in marsupials and monotremes, in contrast to eutherians (mice), in which the fissure is significantly remodeled throughout development. Histological analyses further demonstrated that, while midline astroglia are present in developing marsupials, these cells do not intercalate with one another through the intervening interhemispheric fissure, as they do in developing mice. Thus, developing marsupials do not undergo astroglial-mediated interhemispheric remodeling. As remodeling of the interhemispheric fissure is essential for the subsequent formation of the corpus callosum in eutherians, our data highlight the role of astroglial-mediated interhemispheric remodeling in the evolutionary origin of the corpus callosum.

Development of body, head and brain features in the Australian fat-tailed dunnart (Sminthopsis crassicaudata; Marsupialia: Dasyuridae); A postnatal model of forebrain formation.

Most of our understanding of forebrain development comes from research of eutherian mammals, such as rodents, primates, and carnivores. However, as the cerebral cortex forms largely prenatally, observation and manipulation of its development has required invasive and/or ex vivo procedures. Marsupials, on the other hand, are born at comparatively earlier stages of development and most events of forebrain formation occur once attached to the teat, thereby permitting continuous and non-invasive experimental access. Here, we take advantage of this aspect of marsupial biology to establish and characterise a resourceful laboratory model of forebrain development: the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-sized carnivorous Australian marsupial. We present an anatomical description of the postnatal development of the body, head and brain in dunnarts, and provide a staging system compatible with human and mouse developmental stages. As compared to eutherians, the orofacial region develops earlier in dunnarts, while forebrain development is largely protracted, extending for more than 40 days versus ca. 15 days in mice. We discuss the benefits of fat-tailed dunnarts as laboratory animals in studies of developmental biology, with an emphasis on how their accessibility in the pouch can help address new experimental questions, especially regarding mechanisms of brain development and evolution.

Analysis of complement expression in light-induced retinal degeneration: synthesis and deposition of C3 by microglia/macrophages is associated with focal photoreceptor degeneration.

PURPOSE: To investigate the expression and localization of complement system mRNA and protein in a light-induced model of progressive retinal degeneration. METHODS: Sprague-Dawley (SD) rats were exposed to 1000 lux of bright continuous light (BCL) for up to 24 hours. At time points during (1-24 hours) and after (3 and 7 days) exposure, the animals were euthanatized and the retinas processed. Differential expression of complement genes at 24 hours of exposure was assessed using microarray analysis. Expression of complement genes was validated by quantitative PCR, and expression of selected genes was investigated during and after BCL exposure. Photoreceptor apoptosis was assessed using TUNEL and C3 was further investigated by spatiotemporal analysis using in situ hybridization and immunohistochemistry. RESULTS: Exposure to 24 hours of BCL induced differential expression of a suite of complement system genes, including classic and lectin components, regulators, and receptors. C1qr1, MCP, Daf1, and C1qTNF6 all modulated in concert with photoreceptor death and AP-1 expression, which reached a peak at 24 hours exposure. C1s and C4a reached peak expression at 3 days after exposure, while expression of C3, C3ar1, and C5r1 were maximum at 7 days after exposure. C3 mRNA was detected in ED1- and IBA1-positive microglia/macrophages, in the retinal vessels and optic nerve head and in the subretinal space, particularly at the margins of the emerging lesion. CONCLUSIONS: The data indicate that BCL induces the prolonged expression of a range of complement genes and show that microglia/macrophages synthesize C3 and deposit it in the ONL after BCL injury. These findings have relevance to the role of complement in progressive retinal degeneration, including atrophic AMD.

The cellular expression of antiangiogenic factors in fetal primate macula.

PURPOSE: To characterize the cellular expression patterns of antiangiogenic factors differentially regulated in the fetal human macula. METHODS: RNA was extracted from macular, nasal, and surround biopsies of three human fetal retinas at midgestation. Relative levels of expression of pigment epithelium-derived factor (PEDF), brain natriuretic peptide (BNP), collagen type IValpha2 (COL4A2), and natriuretic peptide receptors A and C (NPRA and NPRC) were determined with quantitative PCR. Cellular expression of PEDF and BNP was investigated by in situ hybridization on retinal sections from monkeys aged between fetal day 55 and 11 years. BNP, COL4A2, and NPRA proteins were localized by immunohistochemistry. Labeling was imaged and quantified by confocal microscopy and optical densitometry. RESULTS: Quantitative PCR confirmed higher levels of PEDF and BNP and lower levels of COL4A2 in the macula at midgestation. PEDF mRNA was detected in ganglion cells (GCs) and the pigment epithelium (RPE). BNP mRNA was detected in GCs and macroglia, although BNP immunoreactivity (IR) was predominantly perivascular. COL4A2-IR was detected in large blood vessels and NPRA-IR on the retinal vascular endothelium, GC axons in fetal retinas, and cone axons at all ages. Optical densitometry showed a graded expression of PEDF and BNP at all ages, with highest levels of expression in GCs in the developing fovea. CONCLUSIONS: Because the retinal vessels initially form in the GC layer, it is likely that PEDF has a key role in defining and maintaining the foveal avascular area. The precise role of BNP is unclear, but it may include both antiangiogenic and natriuretic functions.

Differential gene expression in the developing human macula: microarray analysis using rare tissue samples.

The macula is a unique and important region in the primate retina that achieves high resolution and color vision in the central visual field. We recently reported data obtained from microarray analysis of gene expression in the macula of the human fetal retina (Kozulin et al., Mol Vis 15:45-59, 1). In this paper, we describe the preliminary analyses undertaken to visualize differences and verify comparability of the replicates used in that study, report the differential expression of other gene families obtained from the analysis, and show the reproducibility of our findings in several gene families by quantitative real-time PCR.