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Keywords:

  • ADAM;
  • chicken development;
  • expression pattern;
  • retina

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The expression patterns of the seven members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23 were analyzed in the developing chicken retina by in situ hybridization and immunohistochemistry. Results show that each individual ADAM is expressed and regulated spatiotemporally in the developing retinal layers. ADAM9, ADAM10 and ADAM17 are widely expressed in the differential layers of the retina throughout the whole embryonic period, while ADAM12 and ADAM13 are mainly expressed in the ganglion cell layer at a later stage. ADAM22 and ADAM23 are restricted to the inner nuclear layer and the ganglion cell layer at a later stage. Furthermore, ADAM10 protein is co-expressed with the four members of the classic cadherins, N-cadherin, R-cadherin, cadherin-6B and cadherin-7 in distinct retinal layers. Therefore, the differential expression of the investigated ADAMs in the developing retina suggests the contribution of them to the retina development.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The chicken retina arises from lateral protrusions of the prosencephalon at embryonic stage 9. As the primary optic vesicles contact the surrounding ectoderm, they are induced to be invaginated to form a two-layered structure of the optic cup (Patten 1971). The thickened inner layer of the optic cup differentiates into the neural epithelium of the retina, and the outer layer into the retinal pigment layer (Mey & Thanos 2000). The mature neural retina mainly consists of: (i) the photoreceptors (rods and cones) located in the outer nuclear layer (ONL); (ii) the short projection neurons (bipolar cells); (iii) the local circuit neurons (horizontal and amacrine cells) in the inner nuclear layer (INL); and (iv) the long projection neurons (ganglion cells) in the ganglion cell layer (GCL). These different types of cells form a characteristic morphology, location and connectivity in the retina (Kaneko 1979).

Retinal development involves the processes of cellular proliferation, migration and differentiation. Many transcription factors, for example, activating protein 2 (AP-2), paired box gene 6 (Pax6), cone-rod homeobox gene (Crx), neurogenic differentiation factor (NeuroD), and the POU family transcription factor Brn3, are involved in the process of the retinal formation (Furukawa et al. 1997; Morrow et al. 1999; Liu et al. 2000; Li et al. 2008; Riesenberg et al. 2009). Sonic hedgehog (Shh) is required for eye morphogenesis and regulates the dorsal-ventral patterning of the eye by antagonizing the bone morphogenetic protein 4 (BMP4; Zhang & Yang 2001). Members of the cadherin family are also expressed in the embryonic chicken retina, suggesting that cadherins may provide adhesive cues for the specific formation of developing retinal circuits (Wöhrn et al. 1998).

ADAMs are comprised of multiple domains including a metalloprotease domain and a integrin domain with multiple functions involved in cell–cell and cell–matrix interactions, in proteolytic shedding of other membrane proteins, and in the intracellular signaling transduction (Wolfsberg et al. 1995; White 2003; Blobel 2005; Edwards et al. 2008). In the nervous system, ADAMs play roles in cell proliferation, migration, differentiation, and in axon outgrowth during embryonic development (Yang et al. 2006; Edwards et al. 2008). For example, knockdown of ADAM19 in Xenopus decreases the numbers of neurons and neural crest cells (Neuner et al. 2009); ADAM2 contributes to the migration of neuroblasts to the olfactory bulb (Murase et al. 2008); ADAM10 is essential for the correct projection of the axons of retinal ganglion cells to the target region in the tectum (Chen et al. 2007).

Individual ADAMs show variable expression patterns regulated spatiotemporally during embryonic development (Edwards et al. 2008; Lin et al. 2008, 2010; Alfandari et al. 2009; Yan et al. 2010). For example, several members of the ADAM family show spatio-temporal expression patterns in the brain, the spinal cord, and in the cochlea during chicken embryonic development (Lin et al. 2008, 2010; Yan et al. 2010). Chicken ADAM10 is widely expressed in epidermis, somites, and gut (Hall & Erickson 2003), while the quail ADAM12 and ADAM19 are found in the embryonic limb, gut, and kidney, respectively (Lewis et al. 2004; Neuner et al. 2009).

Our previous study showed that the ADAMs are expressed in the developing chicken brain, especially in the visual system, e.g., in the nucleus of the basal optic root, the isthmo-optic nucleus, the visual nidopallial nucleus, and in the optic tectum (Lin et al. 2008). These findings lead to a question, whether the ADAMs are involved in the development of the retina, too. Little is known about the expression of ADAMs during the retina development. Therefore, in the present study, we continue to analyze the expression patterns of the ADAMs, ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22 and ADAM23 in the developing chicken retina. Our results show that each individual ADAM is expressed in different layers of the developing retina, but with a partial overlap. Furthermore, ADAM10 protein is co-expressed partially with four members of the classic cadherins including N-cadherin (Ncad), R-cadherin (Rcad), cadherin-6B (Cad6B), and cadherin-7 (Cad7) in the different anatomical layers of the retina.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Chicken embryos, RNA probes and antibodies

Fertilized eggs from white Leghorn chicken (Gallus domesticus) were incubated in a forced-draft egg incubator (BSS160, Ehret, Germany) at 37°C with 60% humidity. Chicken embryos were staged according to Hamburger & Hamilton (1951). After the eggs were deeply anesthetized by cooling on ice, embryos were removed from the shell and the eyes were collected at E5, 7, 9, 12, 14, 16 and E18 (stages 27, 31, 35, 38, 40, 42 and 44, respectively; at least five eyes for each stage) for study.

For in situ hybridization, digoxigenin (DIG)-labeled sense and antisense cRNA probes were synthesized in vitro using plasmids containing previously cloned ADAM sequences (Lin et al. 2007, 2008) as cDNA templates according to the manufacturer’s instructions (Roche, Mannheim, Germany). Sense cRNA probes were used as a negative control.

For immunohistochemistry, primary rabbit polyclonal antibody against ADAM10 (Chemicon, Hampshire, UK; Hall & Erickson 2003), and primary mouse or rat monoclonal antibodies against Cad6B (CCD6B-1) and Cad7 (CCD7-1; Nakagawa & Takeichi 1998), Ncad (NCD-2; Hatta & Takeichi 1986), Rcad (RCD-2; Redies et al. 1992), and Islet-1 (39.4D5) were used. NCD-2, and RCD-2 antibodies were kind gifts of Dr S. Nakagawa and Dr M. Takeichi (RIKEN Center for Developmental Biology, Kobe, Japan). CCD6B-1, CCD7-1 and 39.4D5 were obtained from the Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa, USA. Alexa 488-labeled (Molecular Probes, Eugene, USA) and Cy3-labeled (Dianova, Hamburg, Germany) secondary antibodies against rabbit, mouse or rat IgG were used.

In situ hybridization

In situ hybridization on cryosections was performed according to the protocol described previously (Luo et al. 2004). In brief, after postfixation with 4% formaldehyde in phosphate-buffered saline (PBS), cryostat sections were pretreated with proteinase K and acetic anhydride. Then sections were hybridized with cRNA probe at a concentration of about 1–5 ng/μL overnight at 70°C in hybridization solution (50% formamide, 3× standard saline citrate [SSC], 1× Denhardt’s solution, 250 μg/mL yeast RNA and 250 μg/mL salmon sperm DNA). Alkaline phosphatase-coupled anti-DIG Fab fragments (Roche, Mannheim, Germany) were added to bind to the cRNA probe. After the unbound cRNA was removed by the RNase, the sections were incubated with alkaline phosphatase-conjugated anti-DIG Fab fragments (Roche) at 4°C overnight. For visualization of the labeled mRNA, a substrate solution of nitroblue tetrazolium salt (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP) was added. The color reaction on sections were viewed and photographed under a transmission microscope (BX40; Olympus, Hamburg, Germany) equipped with a digital camera (DP70; Olympus). Photographs were adjusted in contrast and brightness by the Photoshop software (Adobe, Mountain View, CA, USA).

In order to overview the spatial relationship of the cell types expressing the different ADAMs, a colorcoded overlay of the in situ hybridization images of the ADAMs at the same position in adjacent sections were merged using Photoshop software (Adobe; Arndt et al. 1998).

Immunohistochemistry

Fluorescent immunostaining was performed on sections through mid-regions of the cochlea using the method described previously (Luo & Redies 2004). In brief, after postfixation in 4% formaldehyde, cryostat sections of 20 μm thickness were preincubated with blocking solution (5% skimmed milk and 0.3% Triton X-100 in TBS) at room temperature for 60 min. Then sections were incubated overnight at 4°C with the primary antibody, followed by the secondary antibody at room temperature for one hour. Finally, cell nuclei were stained with 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma). Fluorescence was imaged under a fluorescent microscope (BZ-8000; Keyence Deutschland GmbH, Neu-Isenburg, Germany). Digital images were adjusted in contrast and brightness with the Photoshop software (Adobe Systems).

For double-labeled fluorescent immunohistochemistry, sections were first immunostained with antibody against ADAM10 using the method described above. Subsequently, immunostaining for cadherins or Islet-1 was performed.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Morphological structures of the developing retina in chicken embryos can be distinguished easily by nuclear staining (Nu) with DAPI. For example, at embryonic incubation day 5 (E5), the retina consists of the retinal pigment epithelium (RPE) and the neuroblast layer (NBL) (Fig. 1A). At E7, the ganglion cell layer (GCL) becomes apparent in the inner side of the NBL (Fig. 1B). From E9, the multi-layered retina is formed. For example, the outer nuclear layer (ONL) appears beneath the RPE; the inner nuclear layer (INL) separates the plexiform layer into the inner and outer plexiform layers (Fig. 1C). From E12, the different retinal layers gradually appear more clear (Figs 1D, 3).

image

Figure 1.  Differential expressions of ADAMs in central regions of the developing chicken retina as detected by in situ hybridization. (A–D) Retinal tissue sections at the embryonic day (E) 5 (A), E7 (B), E9 (C), and E12 (D) are hybridized with digoxygenin (DIG)-labeled ADAM9, ADAM10, and ADAM17 antisense cRNA probes. In C, computer-generated overlay (merge; arrowheads and yellow color) of ADAM9 (red) and ADAM10 (green) are shown. (E) Sense RNA probes (sense) of ADAM9 (A9), ADAM10 (A10), ADAM17 (A17) and ADAM22 (A22) are used as negative control and anti-sense RNA proble of N-cadherin (Ncad) is used as a positive control. Arrow in A points to the outer zone of the neuroblast layer (NBL), in B to the ganglion cell layer (GCL), in C to the outer nuclear layer (ONL), and in D to amacrine cells of the inner nuclear layer (INL). Arrowheads in C indicate the expression overlap of ADAM9 and ADAM10 in bipolar cells and GCL (yellow color), in E indicate the retina pigment layer. IPL, inner plexiform layer; Nu, nuclear staining; OPL, outer plexiform layer; RPE, retinal pigment epithelium. Scale bar: 50 μm in A, and in B for (C, D and E).

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image

Figure 3.  Differential expressions of ADAMs in central regions of the developing chick retina as detected by in situ hybridization. (A–C) Retinal tissue sections at the embryonic day (E) 14 (A), E16 (B), and E18 (C) are hybridized with digoxygenin (DIG)-labeled ADAM9, ADAM10 and ADAM17 antisense RNA probes. Arrowheads point to the expression of ADAMs in the photoreceptor cells of the outer nuclear layer (ONL). GCL, ganglion cell layer; INL, inner part of inner nuclear layer; IPL, inner plexiform layer; Nu, nuclear staining; OPL, outer plexiform layer. Scale bar: 50 μm in A for all panels.

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In the present study, we analyzed the expression patterns of seven members of the ADAM family in adjacent transverse sections through mid-regions of the retina from E5 (stage 27) to E18 (stage 44). Expression patterns on transcription level were considered separately for early stages as shown in Figures 1 and 2 (E5–E12), and for later stages as shown in Figures 3 and 4 (E14–E18). Figures 5 and 6 demonstrate double-labeled immunostaining with antibody against ADAM10 protein in combination with the antibodies against Islet-1 and the four members of the cadherins at E9 and E16, respectively. For in situ hybridization, Ncad anti-sense cRNA probe was used as a positive control (e.g., Fig. 1E) and ADAM sense cRNA probes were used as negative controls (e.g., Fig. 1E). In general, each of the ADAMs investigated demonstrates a spatial and temporal expression pattern in the distinct layers and different cell types of the retina, with partial overlap between each other (Table 1 and Figs 1C, 4B). For example, ADAM9, ADAM10 and ADAM17 show similar expression patterns (Figs 1, 3), while ADAM22 and ADAM23 share also similar expression characteristics (Figs 2, 4), which are, however, distinct from the first group. Therefore, in the text we describe the expression patterns of the ADAM mRNAs separately for different groups and point out the similarities and differences between them in detail during retinal development.

image

Figure 2.  Differential expressions of ADAMs in central regions of the developing chicken retina as detected by in situ hybridization. (A–D) Retinal tissue sections at the embryonic day (E) 5 (A), E7 (B), E9 (C), and E12 (D) are hybridized with DIG-labeled ADAM12, ADAM13, ADAM22 and ADAM23 antisense cRNA probes. For positive and negative controls see Fig. 1. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NBL, neuroblastic layer of retina; Nu, nuclear staining; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium. Scale bar: 50 μm in A, and in B for (C and D).

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image

Figure 4.  Differential expressions of ADAMs in central regions of the developing chick retina as detected by in situ hybridization. (A–C) Retinal tissue sections at the embryonic day (E) 14 (A), E16 (B), and E18 (C) are hybridized with digoxygenin (DIG)-labeled ADAM12, ADAM13, ADAM22 and ADAM23 antisense RNA probes. In B, computer-generated overlay (merge; arrowheads and yellow color) of ADAM22 (red) and ADAM23 (green) are shown. (D–F) Schematic diagrams outline the expression patterns of the seven investigated ADAMs at E5 (D), E9 (E) and E18 (F), respectively. Different ADAMs are represented by distinct color cycles. Arrowheads in B indicate the expression overlap of ADAM22 (A22) and ADAM23 (A23) in amacrine cells and ganglion cell layer (GCL) (yellow color). IINL, inner part of inner nuclear layer; IPL, inner plexiform layer; MINL, middle part of inner nuclear layer; NBL, neuroblastic layer of retina; Nu, nuclear staining; OINL, outer part of inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PSL, photoreceptor segment layer; RPE, retinal pigment epithelium. Scale bar: 50 μm in A for all panels.

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image

Figure 5.  Double-labeled immunofluorescence analysis of ADAM10 with Islet-1 and different cadherins, respectively, in the central region of the developing chicken retina at E9 (marked). Nuclear staining (Nu; blue) and double-labeled immunostaining of ADAM10 (green) together with Islet-1 (red, A–D), Ncad (red, E–H), Rcad (red, I–L), Cad7 (red, M–P), and with Cad6B (red, Q–T) are shown. Small arrows in B–D indicate ADAM10-positive horizontal cells; in F–T the co-expressions of ADAM10 and cadherins in the outer limiting membrane (OLM). Small arrowheads in B–D indicate ADAM10-positive amacrine cells; in F–T the co-expressions of ADAM10 and cadherins in the outer plexiform layer (OPL). Large arrows in B–D point to ADAM10-positive photoreceptor cells; in G–H to the co-expression of ADAM10 and Ncad in the inner margin of the inner nuclear layer (INL). Large arrowheads in C–D represent ADAM10-positive bipolar cells; in N–T ganglion cells. Cad6B, cadherin6B; Cad7, cadherin7; GCL, ganglion cell layer; IPL, inner plexiform layer; Ncad, N-cadherin; ONL, outer nuclear layer; Rcad, R-cadherin. Scale bar: 50 μm in A for all panels.

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image

Figure 6.  Double-labeled immunofluorescence analysis of ADAM10 with Islet-1 and different cadherins, respectively, in the central region of the developing chicken retina at E16 (marked). Nuclear staining (Nu; blue) and double-labeled immunostaining of ADAM10 (green) together with Islet-1 (red, A–D), Ncad (red, E–H), Rcad (red, I–L), Cad7 (red, M–P), and with Cad6B (red, Q–T) are demonstrated. Small arrows in B–D indicate ADAM10-positive horizontal cells; in F–L the co-expression of ADAM10 and cadherins by the photoreceptor cells; in N–T the co-expressions of ADAM10 and cadherins in the outer limiting membrane (OLM). Small arrowheads in B–H point to the co-expressions of ADAM10 and N-cadherin in the amacrine cells; in J–T to the co-expressions of ADAM10 and cadherins in the OPL. Large arrows in B–D represent co-expressions of ADAM10 and Islet-1 in the photoreceptor cells; in J–P co-expressions of ADAM10 and cadherins in the amacrine cells. Large arrowheads in L–T indicate ganglion cells. Cad6B, cadherin6B; Cad7, cadherin7; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; Ncad, N-cadherin; ONL, outer nuclear layer; OPL, outer plexiform layer; Rcad, R-cadherin. Scale bar: 50 μm in A for all panels.

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Table 1.   Summary of the ADAM expression in retinal layers as detected by in situ hybridization
 E5E7E9E12E14E16E18
  1. Assessment of expression levels: −, absent; +, weak; ++, moderate; +++, strong. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NBL, neuroblastic layer of retina; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium.

ADAM9
 RPE++++     
 NBL++++++     
 GCL +++++++++++++
 INL
  Inner  +++++++
  Middle  ++++++++
  Outer  +++++
 OPL  +
 ONL  ++++++
ADAM 10
 RPE++++     
 NBL++++++     
 GCL +++++++++++++++++
 INL
  Inner  +++++++++++++
  Middle  +++++++
  Outer  +++++++
 OPL  +++++
 ONL  +++++++
ADAM 12
 RPE+     
 NBL+     
 GCL+++++
 INL
  Inner  +
  Middle  
  Outer  
 OPL  
 ONL  
ADAM 13
 RPE+++     
 NBL++++     
 GCL +++
 INL
  Inner  +
  Middle  
  Outer  
 OPL  
 ONL  
ADAM 17
 RPE+     
 NBL++++++     
 GCL ++++++++
 INL
  Inner  +++++++
  Middle  ++++++++
  Outer  ++++++
 OPL  +++++
 ONL  +++++
ADAM 22
 RPE+     
 NBL+++++     
 GCL ++++++++++++++++++
 INL
  Inner  ++++++++++++
  Middle  +++++
  Outer  
 OPL  
 ONL  
ADAM 23
 RPE+     
 NBL+++++     
 GCL ++++++++++++++++++
 INL
  Inner  ++++++++++++
  Middle  ++++
  Outer  +
 OPL  
 ONL  +

ADAM9, ADAM10 and ADAM17

At E5, when the majority of retina cells is in proliferation (Prada et al. 1991), ADAM9, ADAM10 and ADAM17 mRNAs are expressed abundantly and strongly by the cells in the NBL (Fig. 1A). Remarkably, the outer and proliferative zones of the NBL express stronger ADAMs (arrow, Fig. 1A). In the RPE, signals for ADAM9 and ADAM10 are detectable strongly, but for ADAM17 weakly (Fig. 1A).

At E7, strong and homogenous ADAM9, ADAM10 and ADAM17 signals are found in the NBL (Fig. 1B). At this stage, the ganglion cells in the GCL express strong ADAM9 and ADAM10 signals (arrow, Fig. 1B), but weak ADAM17 signals.

At E9, in the GCL, ADAM9 and ADAM10 mRNAs are detected strongly, but ADAM17 mRNA only moderately (Fig. 1C). In the INL, ADAM9, ADAM10 and ADAM17 signals are strong in the middle part, but moderate in the outer and inner parts for ADAM9 and ADAM17 (Fig. 1C). To visualize the spatial relationships of the regions expressing ADAM9 and ADAM10, a color coded overlay of the in situ hybridization was generated and shown in Figure 1C (merge; red color for ADAM9 and green for ADAM10) and the partial overlay was found between ADAM9 and ADAM10 as indicated by yellow color (arrowheads in Fig. 1C). Furthermore, ADAM10 mRNA is expressed strongly in the outer plexiform layer (OPL) and the ONL (arrow in the insert box) (Fig. 1C), while ADAM9 and ADAM17 signals are weak or moderate in the OPL and the ONL (Fig. 1C).

At E12, the expression of ADAM10 mRNA is strong in the GCL, but the expression of ADAM9 and ADAM17 signals is moderate or weak (Fig. 1D). In the INL and the ONL, the three ADAMs are expressed moderately (Fig. 1D), but ADAM10 shows strong signals at the inner margin of the INL (arrow, Fig. 1D). It should be noted that the ADAM signals in the RPE layer from E12 onward are difficult to be evaluated because of the huge amount of pigment, which may mask the signals (see the negative controls in Fig. 1E).

From E14 onward, the expression of ADAM9, ADAM10 and ADAM17 mRNAs becomes gradually restricted to the GCL, the INL and the ONL, but with different intensities (Fig. 3A–C). For example, ADAM9 mRNA is moderately expressed, ADAM10 strongly, and ADAM17 weakly (Fig. 3A–C).

ADAM12 and ADAM13

At E5, the ADAM12 gene is weakly transcribed in the NBL and RPE, but the ADAM13 mRNA is strongly expressed (Fig. 2A). Between E7 and E14, ADAM12 mRNA is not detected in the retinal layers (Figs 2B–D, 4A). ADAM12 signals in GCL are moderate at E16 (Fig. 4B) and strong at E18 (Fig. 4C). The cells in the inner part of the INL also express ADAM12 at E18 (Fig. 4C).

At E7, ADAM13 mRNA strongly decreases in the NBL (Fig. 2A,B), and no ADAM13 expression is detected between E9 and E16 (Figs 2C,D, 4A,B). At E18, strong ADAM13 signals are found in the GCL and are weak in the INL (Fig. 4C).

ADAM22 and ADAM23

At E5, ADAM22 and ADAM23 mRNAs are expressed strongly in the NBL with the strongest signal in the inner part (Fig. 2A) and a weaker signal in the RPE. From E7 onward, the strong signals move to the GCL, while in the INL, they gradually decrease and restrict their expressions to the inner margin as embryo develops (Figs 2C,D, 4A–C). The partial overlap between ADAM22 and ADAM23, for example, was also found (arrowheads and yellow color in Fig. 4B).

In summary, each of the seven investigated ADAMs is expressed in the developing retinal layers with different patterns between each other, while sharing a partial overlap (Figs 1C, 4B,D–F, Table 1).

Co-expression of ADAM10 protein and members of classic cadherin proteins

At E9 and E16, ADAM10 protein (green color in Figs 5, 6) is expressed in the different retinal layers, being consistent with the expression of ADAM10 at mRNA level (Figs 1C, 3B). Neuron-specific marker Islet-1 recognizes most ganglion cells (GCL in Figs 5C, 6C), some amacrine cells (small arrowheads in Figs 5C, 6C), bipolar cells (large arrowheads in Fig. 5C), horizontal cells (small arrows in Figs 5C, 6C) and some photoreceptor cells (large arrows in Figs 5C, 6C) in the chicken retina (red color in Figs 5C, 6C; Galli-Resta et al. 1997; Fischer et al. 2002). Double immunostaining reveals that some ADAM10-positive cells (green color in Figs 5, 6) in the different retinal layers co-express Islet-1 (yellow color in Figs 5D, 6D).

Of interest, ADAM10 can shed the ectodomains of Ncad and E-cad, modulating the cell–cell adhesion and signal transduction (Maretzky et al. 2005; Reiss et al. 2005). Several members of the classic cadherin family, including Ncad, Rcad, Cad6B and Cad7, have been shown to be expressed in distinct layers and different types of cells in the developing retina (Matsunaga et al. 1988; Inuzuka et al. 1991; Wöhrn et al. 1998; Etzrodt et al. 2009). Therefore, we further investigated whether ADAM10 is co-expressed with these classic cadherins.

Ncad signals at E9 are strong in the outer limiting membrane (OLM, small arrow in Fig. 5G) and the OPL (small arrowhead in Fig. 5G; Matsunaga et al. 1988), but weak in the INL, where ADAM10 is co-expressed (small arrow and small arrowhead, yellow color in Fig. 5H). The Ncad-positive cells in the IPL do not co-express ADAM10 (Fig. 5F–H). Remarkably, the co-expression of ADAM10 and Ncad in the INL shows a reverse manner, i.e. Ncad expression is stronger in the inner margin of the INL (large arrow, Fig. 5G,H), where ADAM10 expression is weaker. At E16, Ncad is expressed by the photoreceptor cells (small arrows in Fig. 6G) in the ONL and by the amacrine cells (small arrowheads in Fig. 6G) in the INL (Wöhrn et al. 1998), where ADAM10 is also co-expressed (yellow color in Fig. 6H).

As expected, Rcad expression at E9 is found to be strong in the OLM (small arrows in Fig. 5K,L) and the OPL (small arrowheads in Fig. 5K,L), but weak in the INL, where ADAM10 is co-expressed (yellow, Fig. 5L). At E16, Rcad is expressed by some amacrine cells of the INL (large arrow in Fig. 6K; Inuzuka et al. 1991; Wöhrn et al. 1998) and by the photoreceptor cells in the ONL (small arrow in Fig. 6K), which also co-express ADAM10 (yellow color, Fig. 6L). However, Rcad in the ganglion cells of the GCL is only partially co-expressed with ADAM10 (large arrowheads, Fig. 6L).

Cad7 is at E9 expressed in the entire neural retina, especially a strong signal in the OLM (small arrow in Fig. 5O), the OPL (small arrowhead in Fig. 5O), the INL, and in the GCL (large arrowhead in Fig. 5O), where ADAM10 is co-expressed (yellow color in Fig. 5P). At E16, Cad7 is expressed by the ganglion cells of the GCL (larger arrowhead in Fig. 6O), by the amacrine cells of the INL (large arrow in Fig. 6O), in the OPL (small arrowhead in Fig. 6O), and the OLM (small arrow in Fig. 6O; Wöhrn et al. 1998), where ADAM10 is also co-expressed (yellow color in Fig. 6P).

Furthermore, Cad6B and ADAM10 are co-expressed in the OLM (small arrows in Figs 5R–T, 6R–T) and the OPL (small arrowheads in Figs 5R–T, 6R–T), and partially by ganglion cells in the GCL (large arrowheads in Figs 5R–T, 6R–T; Wöhrn et al. 1998).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Recent studies have shown that ADAMs are expressed in the optic cup at early embryonic stages and in the visual system at later embryonic stages (Chen et al. 2007; Lin et al. 2007, 2010; Xie et al. 2008). In the present work, we further analyze the ADAM expression profiles in the developing retina. Our results show that each of the ADAMs investigated here demonstrates a distinct spatial and temporal expression pattern in different layers and different cell types of the retina, with partial overlap between each other (Figs 1–4).

Expression of ADAMs in RPE

The RPE is a single layer of cuboidal cells interposed between the choroid and the neural retina (Bok 1993) and plays a central role in retinal physiology by forming the outer blood-retinal barrier and supporting the function of the photoreceptors (Boulton & Dayhaw-Barker 2001). Growth factors, for example, epidermal growth factor (EGF) and tumor-necrosis factor-α (TNF-α), have been found to be expressed in the RPE and to regulate the growth of RPE cells (Inuzuka et al. 1991; Mey & Thanos 2000; Martínez-Morales et al. 2004; Hollborn et al. 2006). In the present study, we show that ADAM9, ADAM10, and ADAM17 mRNAs are expressed in the RPE between E5 and E7 (Fig. 1A,B), but at later stages, their signals are difficult to be visualized because of the huge amount of the darker pigment (see arrowheads in Fig. 1E). Of interest, it is known that ADAM10 and ADAM17 are the principal proteases involved in the activation of TNF-α and EGF receptor pathways by shedding of pro-TNF-α and EGF receptor ligands (Sahin et al. 2004; Edwards et al. 2008). In addition, loss of ADAM9 function results in disorganization of RPE cells, resulting in the interruption of photoreceptor cell functions (Parry et al. 2009). Therefore, the ADAMs may play a role in the development of retinal pigment epithelium, possibly by shedding growth factors or remodeling the extracellular matrix between the RPE and photoreceptor outer segments.

ADAMs in retinal NBL and cell proliferation

In contrast to the future RPE, where the cell proliferation ceases early, stem cells continue to divide in the presumptive neural retina (Mey & Thanos 2000). At E5, the majority (>80%) of cells in the NBL are proliferating. As the embryo develops, about 60% of cells in the NBL are still in proliferation at E7 (Prada et al. 1991). ADAMs are involved in cells proliferation during embryonic development (Gschwind et al. 2003; Schäfer et al. 2004; Itoh et al. 2005). For example, loss of ADAM17 function results in the reduction of epithelial cell proliferation and the inhibition of epithelial cell differentiation in fetal mouse lungs. In the present study, ADAM9, ADAM10, ADAM13, ADAM17, ADAM22 and ADAM23 are strongly expressed in the NBL at E5 (Figs 1A, 2A), although some of them decrease gradually from E7 (Figs 1B, 2B). Further studies should be performed to investigate whether the ADAMs are involved in the regulation of the neuroblast cell proliferation during early retinal development.

Expression of ADAMs in GCL

Retinal ganglion cells are located in the innermost layer of the retina. After their final mitosis from E3 onward, the neuroblasts of chicken ganglion cells detach from the outer ventricular zone and migrate vertically towards the inner limiting membrane, where they create the GCL (Prada et al. 1981; Watanabe et al. 1991; Snow & Robson 1994). There, these cells send out their axonal projections through the optic nerve to the optic tectum, and a few to the thalamus (Mey & Thanos 2000).

The present study reveals that the seven ADAM mRNAs are strongly expressed by the ganglion cells of GCL during development (Figs 1–4), and ADAM10 protein is also expressed in retina ganglion cells, where Cad6B and Cad7 are co-expressed (Figs 5N–T, 6N–T). Studies have shown that the ADAMs are involved in retinal axon guidance (Webber et al. 2002; McFarlane 2003). Loss of ADAM10 function results in the wrong targeting of retinal ganglion cell axon in the tectum (Chen et al. 2007). ADAM17 can shed the ectodomain of neural cell adhesion molecule (NCAM), modulating the neurite outgrowth (Kalus et al. 2006). Furthermore, Cad6B and Cad7 expressing in retinal ganglion cells are associated with a specific retinofugal pathway for the axon target recognition (Müller et al. 2004). Therefore, it is of interest whether or not the ADAMs play a role in the guidance of the ganglion axon targeting during chicken retinal development.

Expression of ADAMs in INL

The INL consists of a number of closely packed cells with three varieties including amacrine cells, bipolar cells and horizontal cells (Kaneko 1979). The amacrine cells locate in the innermost region of the INL, while the bipolar cells with the round shape nucleus locate in the middle part of the INL. The horizontal cells place in the outermost margin of the INL.

Amacrine cells are responsible for mediating lateral interactions between adjacent groups of photoreceptors, bipolar cells and retina ganglion cells, enabling adjacent regions within the retina to compare the intensity of light arising from contiguous regions of the visual field (Blanks 2001). During chicken embryonic development, the retinal amacrine cells are born between E3 and E10 (Prada et al. 1991), and show an organized dendritic tree by E14 (Ríos et al. 1997). At E16, the number of differentiated amacrine cells reaches the largest increase (Doh et al. 2010). Of interest, ADAM9, ADAM10, ADAM17, ADAM22 and ADAM23 mRNAs are temporally expressed in the innermost part of the INL, in which developing amacrine cells can be identified on the basis of their location. Specially, as the embryo develops, ADAM22 and ADAM23 mRNAs are strongly restricted to the amacrine cells (Figs 3, 4). The changes of ADAM mRNA levels in the amacrine cells coincide with the development of most amacrine cells from differentiation to maturation. Which roles the ADAMs play in the development of retinal amacrine cells should be further investigated.

Bipolar cells oriented parallel to photoreceptor cells are primarily responsible for transmitting signals from photoreceptors to ganglion cells (Kolb et al. 2001), and are generated from E5 to E12 (Prada et al. 1991). In the present study, ADAM9, ADAM10, ADAM17 and ADAM23 are temporally expressed in the middle part of the INL (Figs 1C,D, 2C,D), where developing bipolar cells can be identified on the basis of their location. From E14, the expressions of these ADAMs by bipolar cells decrease except for ADAM10 (Figs 3, 4). Whether the ADAMs are involved in the proliferation and differentiation of bipolar cells should be further studied.

Horizontal cells are interneurons located in the outer part of the INL of retina. They help integrate and regulate the input from multiple photoreceptor cells and are responsible for allowing eyes to adjust to bright and dim light conditions (Masland 2001). Our results show that ADAM9 and ADAM10 mRNAs are expressed in the horizontal cells (Figs 1, 3).

Expression of ADAMs in ONL

Rod photoreceptor cells are responsible for sensing motion, contrast and brightness, while cone photoreceptor cells are essential for color vision and resolution of the fine detail (Kolb et al. 2001). During the development, the nuclei of rod and cone cells localize in the ONL and their dendrites form the OPL (Olson 1972; Prada et al. 1991). In the present study, ADAM9, ADAM10, ADAM17 and ADAM23 are expressed in the ONL at E9 (Figs 1C, 2C) and the expression of them are decreased from the ONL at later stages (Figs 3, 4).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Dr S. Nakagawa and Dr M. Takeichi for their kind gifts of the antibodies, and Dr E. Mix for critical reading of this manuscript. This work was supported by a grant from the German Research Foundation (DFG; LU1455/1-1) and a fund from the National Natural Science Foundation of China (31000475).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References