Developmental changes of Islet-1 and its co-localization with pituitary hormones in the pituitary gland of chick embryo by immunohistochemistry

Although Islet-1 expression in the pituitary gland of early mouse embryo has been previously described, there are no reports concerning the correlation of Islet-1 expression with lineage restrictions in cell types at the later stages of pituitary development. The role of Islet-1 in chickens is also unknown. The purpose of this study was to follow, by using immunohistochemistry, the ontogeny of pituitary Islet-1 and the various cell types that contain Islet-1 throughout chick embryo development. A few Islet-1-immunopositive (Islet-1⁺) cells were first detected in the pituitary primordium in two out of six embryos at embryonic day 5.5 (E5.5), most of the Islet-1⁺ cells being ventrally located. As development progressed, many more Islet-1⁺ cells were observed throughout the pars distalis. The relative percentage of Islet-1⁺ cells amongst the total Rathke’s pouch cells was 4.4% at E6.5. This increased significantly, reaching 11.1% by E10.5, followed by no significant change until hatching. Dual immunohistochemistry showed that adrenocorticotrophs, somatotrophs and lactotrophs did not express Islet-1. The cellular types expressing Islet-1 included luteinizing-hormone-positive (LH⁺) gonadotrophs and thyroid-stimulating-hormone-positive (TSH⁺) thyrotrophs. The cells co-expressing LH and Islet-1 were initially detected at E6.5, the proportion of LH⁺ cells possessing Islet-1 being about 4%; this increased to 63% at E14.5, followed by no significant changes until hatching. TSH and Islet-1 co-localized cells were first observed at E10.5, with about 37% TSH⁺ cell expressing Islet-1; this increased to about 50% by E16.5, after which there was no evident change until hatching. These results suggest that Islet-1 is involved in determining the cell lineages, proliferation, differentiation and maintenance of hormone-secreting functions of pituitary gonadotrophs and thyrotrophs of chick embryo. J. Liu and Y. He contributed equally to this article. This work was supported by grants from Beijing Natural Science Foundation (6042013) and the Natural Science Foundation of China (30471264, 30325034).     

The chicken RaxL gene plays a role in the initiation of photoreceptor differentiation

The paired type homeodomain gene, Rax, was previously identified as a key molecule in early eye formation in mice and humans. We report the expression patterns of two Rax family members from chicken, Rax and RaxL, and on the function of RaxL in photoreceptor development. Both Rax and RaxL are expressed in early retinal progenitor cells, with Rax being expressed at a significantly higher level than RaxL. At the time that photoreceptors begin to form, RaxL appears at a relatively high level in a subset of cells within the zone of proliferating progenitor cells. Subsequently, it is expressed in cells migrating to the photoreceptor layer, where it is highly expressed during the initial, but not late, stages of photoreceptor differentiation. To test the function of RaxL, a putative dominant-negative allele of RaxL comprising a fusion of the engrailed repressor domain and a region of RaxL (EnRaxLDeltaC) was introduced in vivo into the early chick eye using a retroviral vector. EnRaxLDeltaC, but not the dominant negative Rax (EnRaxDeltaC), caused a significant reduction in expression of early markers of photoreceptor cells. Examination of the transactivation activity of RaxL on a reporter construct bearing a canonical photoreceptor-specific enhancer element showed that RaxL exhibited significant activation activity, and that this activity was severely diminished in the presence of EnRaxLDeltaC. The effect on photoreceptor gene expression in vivo was specific in that other cell types were unaffected, as was general proliferation in the retina. The reduction in numbers of cells expressing photoreceptor markers was probably due to decreased survival of developing photoreceptor cells, as there was increased apoptosis among cells of the retina expressing dominant-negative RaxL. We propose that RaxL plays a role in the initiation of differentiation, and also possibly commitment, of photoreceptor cells in the chicken retina.     

Changes in progenitor populations and ongoing neurogenesis in the regenerating chick spinal cord

The chick spinal cord can regenerate following injury until advanced developmental stages. It is conceivable that changes in stem/progenitor cell plasticity contribute to the loss of this capacity, which occurs around E13. We investigated the contribution of proliferation, phenotypic changes in radial glia progenitors, and neurogenesis to spinal cord regeneration. There was no early up-regulation of markers of gliogenic radial glia after injury either at E11 or E15. In contrast, increased proliferation in the grey matter and up-regulation of transitin expression following injury at E11, but not E15, suggested high levels of plasticity within the E11 spinal cord progenitor population that are lost by later stages. Changes in neural progenitors with development were also supported by a higher neurosphere forming ability at E11 than at E15. Co-labelling with doublecortin and neuron-specific markers and BrdU in spinal cord sections and dissociated cells showed that neurogenesis is an ongoing process in E11 chick spinal cords. This neurogenesis appeared to be complete by E15. Our findings demonstrate that the regeneration-competent chick spinal cord is less mature and more plastic than previously believed, which may contribute to its favourable response to injury, and suggest a role for neurogenesis in maintaining regenerative capacity.     

Changing patterns of ganglion cell coupling and connexin expression during chick retinal development

We have used dye injection and immunolabeling to investigate the relationship between connexin (Cx) expression and dye coupling between ganglion cells (GCs) and other cells of the embryonic chick retina between embryonic days 5 and 14 (E5-14). At E5, GCs were usually coupled, via soma-somatic or dendro-somatic contacts, to only one or two other cells. Coupling increased with time until E11 when GCs were often coupled to more than a dozen other cells with somata in the ganglion cell layer (GCL) or inner nuclear layer (INL). These coupled clusters occupied large areas of the retina and coupling was via dendro-dendritic contacts. By E14, after the onset of synaptogenesis and at a time of marked cell death, dye coupling was markedly decreased with GCs coupled to three or four partners. At this time, coupling was usually to cells of the same morphology, whereas earlier coupling was heterogeneous. Between E5 and E11, GCs were sometimes coupled to cells of neuroepithelial morphology that spanned the thickness of the retina. The expression of Cx 26, 32, and 43 differed and their distribution changed during the period studied, showing correlation with events such as proliferation, migration, and synaptogenesis. These results suggest specific roles for gap junctions and Cx's during retinal development.     

Impaired retinal vascular development in anencephalic human fetus

This study was to investigate the effect of the absence of ganglion cells on the development of human retinal vasculature. Anencephaly (AnC) and age-matched control eyes derived from each three spontaneously aborted fetus (ranging from 15 to 20 weeks gestation) were subjected to immunofluorescence staining for HIF-1α, Thy-1, glial fibrillary acidic protein (GFAP) and platelet/endothelial cell adhesion molecule (PECAM) and apoptosis assay. In developing mouse retina, Western blotting for hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) was performed. Under hypoxic condition (O₂ < 1%), cellular proliferation and VEGF mRNA expression in astrocytes were measured. Apoptotic cells in AnC retina were primarily localized in the ganglion cell layer (GCL), whereas apoptotic cells in normal retina were distributed in the retinoblastic layer. With increase of apoptotic cells in GCL of AnC retina, HIF-1α expression were severely distinguished in avascular retina and GFAP expression in junctional area between avascular and vascular retina was much reduced, accompanied by decrease of PECAM expression compared to normal retina. In developing mouse retina, HIF-1α and VEGF expression were high in hypoxic retina of early stage with incomplete vascular development and then progressively decreased with regression to arborous pattern of matured vascular networks. In hypoxic condition, a significant increase in cellular proliferation and VEGF mRNA expression was observed in astrocytes. Therefore, our results suggest that vascular attenuation in AnC retina could be closely related to the absence of ganglion cells as the metabolic demander to induce retinal vascular development.     

Neuronal differentiation from postmitotic precursors in the ciliary ganglion

In the chick ciliary ganglion, neuronal number is kept constant between St. 29 and St. 34 (E6-E8) despite a large amount of cell death. Here, we characterize the source of neurogenic cells in the ganglion as undifferentiated neural crest-derived cells. At St. 29, neurons and nonneuronal cells in the ciliary ganglion expressed the neural crest markers HNK-1 and p75(NTR). Over 50% of the cells were neurons at St. 29; of the nonneuronal cells, a small population expressed glial markers, whereas the majority was undifferentiated. When placed in culture, nonneuronal cells acquired immunoreactivity for HuD, suggesting that they had commenced neuronal differentiation. The newly differentiated neurons arose from precursors that did not incorporate bromodeoxyuridine. To test whether these precursors could undergo neural differentiation in vivo, purified nonneuronal cells from St. 29 quail ganglia were transplanted into chick embryos at St. 9-14. Subsequently, quail cells expressing neuronal markers were found in the chick ciliary ganglion. The existence of this precursor pool was transient because nonneuronal cells isolated from St. 38 ganglia failed to form neurons. Since all ciliary ganglion neurons are born prior to St. 29, these results demonstrate that there are postmitotic neural crest-derived precursors in the developing ciliary ganglion that can differentiate into neurons in the appropriate environment.     

bHLH genes cath5 and cNSCL1 promote bFGF-stimulated RPE cells to transdifferentiate toward retinal ganglion cells

The molecular mechanism of retinal ganglion cell (RGC) genesis and development is not well understood. Published data suggest that the process may involve two bHLH genes, ath5 and NSCL1. Gain-of-function studies show that ath5 increases RGC production in the developing retina. We examined whether two chick genes, cath5 and cNSCL1, can guide retinal pigment epithelial (RPE) cells to transdifferentiate toward RGCs. Ectopic expression of cath5 and cNSCL1 in cultured chick RPE cells was achieved through retroviral transduction. cath5 alone was unable to induce de novo expression of early RGC markers, such as RA4 antigen, neurofilament (160 kDa), and a neurofilament-associated antigen. However, cath5 induced the expression of these proteins when the RPE cells were cultured with medium supplemented with bFGF. Since bFGF alone can induce only RA4 antigen, the expression of the additional RGC markers reflects a synergism between cath5 and bFGF in promoting RPE transdifferentiation toward RGCs. Morphologically, the RA4(+) cells in bFGF + cath5 cultures appeared more neuron-like than those generated by bFGF alone. cNSCL1 also promoted bFGF-stimulated RPE cells to transdifferentiate toward RGCs that expressed RA4 antigen, N-CAM, Islet-1, neurofilament, and neurofilament-associated antigen. We found that cath5 induced cNSCL1 expression, but not vice versa. Our data suggest that cath5 or cNSCL1 alone was insufficient to induce RPE transdifferentiation into RGCs, but could further neural differentiation initiated by bFGF. We propose that intrinsic factors act synergistically with extrinsic factors during RGC genesis and development.     

不同胚龄北京鸭视网膜节细胞层细胞大小、数量及密度

采用视网膜铺片Nissl染色法和Scion Image图像分析法,研究北京鸭胚胎期11（E11）、14、117、20、23和26日龄视网膜节细胞层（Ganglion cell layer,GCL）细胞大小、数量及密度分布变化。结果表明：E11-E14,GCL细胞小,均呈圆形,E17开始出现大细胞;E11至E26视网膜中央区（CA）细胞大小增长1．97倍,而颞侧周边区（TP）增长3．1倍,相邻胚龄间细胞大小差异显著。GCL细胞总数在E17达到最大值2．03&#215;10^6个,随后细胞数量显著下降。视网膜中央-周边密度梯度在E11出现。CA细胞密度在E17达到最大值2．54&#215;10^4个/mm^2,随后密度下降;周边区细胞密度随胚龄增长而持续下降,且TP细胞密度下降最显著。结果提示,北京鸭胚胎发育过程中,GCL细胞大小、数量及密度均发生显著变化,而E17是其中的一个重要转折点[动物学报54（6）：1082—1088,2008]。     

人胎视网膜神经肽Y免疫反应神经元的发育

本文用免疫细胞化学ＡＢＣ法,研究１５—３８周龄人胎视网膜神经肽Ｙ免疫反应（ＮｅｕｒｏｐｅｐｔｉｄｅＹｉｍｍｕｎｏｒｅｃｔｉｖｅ,ＮＰＹ－ＩＲ）神经元（以下称ＮＰＹ－ＩＲ细胞）的发育。结果表明：①胎龄１５周视网膜中央部已出现不同类型的ＮＰＹ－ＩＲ细胞：位于黄斑及其周围外核层的为ＮＰＹ－ＩＲ视锥细胞;位于内核层最内一列的为ＮＰＹ－ＩＲ无长突细胞位于节细胞层的可能为ＮＰＹ－ＩＲ移位无长突细胞或节细胞;内核层和节细胞层的ＮＰＹ－ＩＲ细胞的突起均分布在内网层的第１亚层。②胎龄２４周后,ＮＰＹ－ＩＲ视锥细胞完全消失。③随着视网膜的发育,内核层和节细胞层的ＮＰＹ－ＩＲ细胞数量增多,突起增粗增长,胞体分布由中央部扩展到周边部,其中内核层ＮＰＹ－ＩＲ细胞的密度呈现从中央部向周边部逐渐降低的分布方式,节细胞层ＮＰＹ－ＩＲ细胞则多数集中分布在视网膜的边缘和黄斑之间,形成较高密度的环状区。     

The neuronal EGF-related gene Nell2 interacts with Macf1 and supports survival of retinal ganglion cells after optic nerve injury

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).     

Dlx1 and Dlx2 function is necessary for terminal differentiation and survival of late-born retinal ganglion cells in the developing mouse retina

Dlx homeobox genes, the vertebrate homologs of Distal-less, play important roles in the development of the vertebrate forebrain, craniofacial structures and limbs. Members of the Dlx gene family are also expressed in retinal ganglion cells (RGC), amacrine and horizontal cells of the developing and postnatal retina. Expression begins at embryonic day 12.5 and is maintained until late embryogenesis for Dlx1, while Dlx2 expression extends to adulthood. We have assessed the retinal phenotype of the Dlx1/Dlx2 double knockout mouse, which dies at birth. The Dlx1/2 null retina displays a reduced ganglion cell layer (GCL), with loss of differentiated RGCs due to increased apoptosis, and corresponding thinning of the optic nerve. Ectopic expression of Crx, the cone and rod photoreceptor homeobox gene, in the GCL and neuroblastic layers of the mutants may signify altered cell fate of uncommitted RGC progenitors. However, amacrine and horizontal cell differentiation is relatively unaffected in the Dlx1/2 null retina. Herein, we propose a model whereby early-born RGCs are Dlx1 and Dlx2 independent, but Dlx function is necessary for terminal differentiation of late-born RGC progenitors.     

Analysis of glia cell differentiation in the developing chick peripheral nervous system: sensory and sympathetic satellite cells express different cell surface antigens.

To identify and analyse precursor cells of neuronal and glial cell lineages during the early development of the chick peripheral nervous system, monoclonal antibodies were raised against a population of undifferentiated cells of E6 dorsal root ganglia (DRG). Non-neuronal cells of E6 DRG express surface antigens that are recognized by four monoclonal antibodies, G1, G2, GLI 1 and GLI 2. The proportion of non-neuronal cells in DRG that express the GLI 1 antigen is very high during ganglion formation (80% at E4) and decreases during later development (15% at E14). GLI 2 antigen is expressed only on a minority of the cells at E6 and increases with development. The G1 and G2 antigens are expressed on about 60-80% of the cells between E6 and E14. All cells that express the established glia marker O4 are also positive for the new antigens. In addition, it was demonstrated that GLI 1-positive cells from early DRG, which are devoid of O4 antigen, could be induced in vitro to express the O4 antigen. Thus, the antigen-positive cells are considered as glial cells or glial precursor cells. Surprisingly, the antigen expression by satellite cells of peripheral ganglia is dependent on the type of ganglion: antigens G1, G2 and GLI 1 were not detectable on glial cells of lumbosacral sympathetic ganglia and GLI 2 was expressed only by a small subpopulation. These results demonstrate an early immunological difference between satellite cells of sensory DRG and sympathetic ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibiting Matrix Metalloproteinase 3 Ameliorates Neuronal Loss in the Ganglion Cell Layer of Rats in Retinal Ischemia/Reperfusion

It has been demonstrated that matrix metalloproteinase 3 (MMP3) is integrally involved in the neuronal degeneration of the central nervous system by promoting glial activation, neuronal apoptosis and damage to the brain–blood barrier. However, whether MMP3 also contributes to the neuronal degeneration induced by retinal ischemia/reperfusion is still uncertain. In the present study, we detected the cellular localization of MMP3 in adult rat retinae and explored the relationship of its expression with neuronal loss in the ganglion cell layer (GCL) in retinal ischemia/reperfusion. We found that MMP3 was widely expressed in many cells throughout the layers of the rat retinae, including Vertebrate neuron-specific nuclear protein (NeuN)-, parvalbumin-, calbindin-, protein kinase C-α-, glial fibrillary acidic protein-, glutamine synthetase- and CD11b-positive cells. Furthermore, all rats were treated with high intraocular pressure (HIOP) for 1 h (h) and sacrificed at 6 h, 1 day (d), 3 d, and 7 d after HIOP. Compared to the normal control, the expression of both proenzyme MMP3 and active MMP3 were significantly up-regulated after HIOP treatment without alteration of the laminar distribution pattern. Moreover, inhibiting MMP3 ameliorated the loss of NeuN-positive cells in the GCL following HIOP. In summary, our data demonstrates that MMP3 is expressed in multiple types of neurons and glial cells in normal rat retinae. Simultaneously, the up-regulation of its expression and activity are closely involved in neuronal loss in the GCL in retinal ischemia/reperfusion.     

Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid

We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.     

Expression of Lhx6 in the adult and developing mouse retina

PurposeTo investigate the expression patterns of LIM Homeobox 6 (Lhx6) in the adult and developing mouse retina.MethodsThe Lhx6-GFP knock-in allele was used to activate constitutive expression of a GFP reporter in Lhx6 expressing cells. Double labeling with GFP and retinal markers in the mouse retina at postnatal day 56 (P56) was performed to identify the cell types expressing Lhx6. To determine the neuronal cell types that express Lhx6, double labeling with GFP and various retinal markers was employed in the differentiating retina at P7 and P15.ResultsGFP + Lhx6 lineage cells were determined in Brn3a + retinal ganglion cells (RGCs), ChAT + amacrine cells (ACs), and Islet-class LIM-homeodomain 1 (Isl1+) ACs in the mouse retina at P56. In the ganglion cell layer (GCL), Lhx6 was expressed in Brn3a + RGCs but not Brn3b + RGCs at P15. Moreover, in the inner nuclear layer (INL), Lhx6 was not expressed in Bhlhb5+ ACs at P15. However, Lhx6 was weakly expressed in Glyt1+ ACs and Pax6+ ACs, and strongly expressed in Isl1+ and ChAT + ACs at P15.ConclusionLhx6 was expressed in RGCs and ACs in both the adult and developing mouse retina.     

Expression patterns of chick Musashi-1 in the developing nervous system

Vertebrate homologues of musashi have recently been referred to as neural stem cell markers because of their expression patterns and RNA-binding interactions. In the context of the notch signaling pathway, Musashi-1 (Msi-1) is a regulator of neural cell generation, cooperating with notch to maintain mitosis. In an effort to identify definitive stem cell markers of the neural retina, a portion of the Msi-1 cDNA was cloned, and the expression of Msi-1 in the chick eye was analyzed. Using an Msi-1-specific antibody and RNA probe, we show that expression of Msi-1 in the early neural tube is consistent with neural stem identity. In the neural retina, expression starts shortly before embryonic day 3 (E3) and continues up to and including E18. A BrdU incorporation assay shows Msi-1 to be found in both proliferating and differentiating cells of E5 neural retina. At E8 (when proliferation is complete in the fundus of the retina) and E18 (mature retina) Msi-1 expression was found in the ciliary marginal zone (CMZ) as well as in a subpopulation of differentiated cells, including photoreceptors and ganglion cells.