Temporal order of bipolar cell genesis in the neural retina
© Morrow et al.; licensee BioMed Central Ltd. 2008
Received: 03 August 2007
Accepted: 23 January 2008
Published: 23 January 2008
Retinal bipolar cells comprise a diverse group of neurons. Cone bipolar cells and rod bipolar cells are so named for their connections with cone and rod photoreceptors, respectively. Morphological criteria have been established that distinguish nine types of cone bipolar cells and one type of rod bipolar cell in mouse and rat. While anatomical and physiological aspects of bipolar types have been actively studied, little is known about the sequence of events that leads to bipolar cell type specification and the potential relationship this process may have with synapse formation in the outer plexiform layer. In this study, we have examined the birth order of rod and cone bipolar cells in the developing mouse and rat in vivo.
Using retroviral lineage analysis with the histochemical marker alkaline phosphatase, the percentage of cone and rod bipolar cells born on postnatal day 0 (P0), P4, and P6 were determined, based upon the well characterized morphology of these cells in the adult rat retina. In this in vivo experiment, we have demonstrated that cone bipolar genesis clearly precedes rod bipolar genesis. In addition, in the postnatal mouse retina, using a combination of tritiated-thymidine birthdating and immunohistochemistry to distinguish bipolar types, we have similarly found that cone bipolar genesis precedes rod bipolar genesis. The tritiated-thymidine birthdating studies also included quantification of the birth of all postnatally generated retinal cell types in the mouse.
Using two independent in vivo methodologies in rat and mouse retina, we have demonstrated that there are distinct waves of genesis of the two major bipolar cell types, with cone bipolar genesis preceding rod bipolar genesis. These waves of bipolar genesis correspond to the order of genesis of the presynaptic photoreceptor cell types.
The retina offers an excellent model system for dissecting the mechanism of neural development in vertebrates [1–3]. The adult retina contains a complement of well-characterized neurons and glia in three cellular layers (outer nuclear, inner nuclear and ganglion cell layers) separated by two distinct plexiform layers (the inner and outer plexiform layers) containing cellular processes and synapses . The inner plexiform layer (IPL) contains bipolar-ganglion cell connections, as well as modulatory amacrine interneuron synapses. The outer plexiform layer (OPL) contains the tripartite ribbon synapses of presynaptic horizontal and photoreceptor cells and the post-synaptic bipolar cells. Given the well characterized cellular morphology and biochemistry of the retina, the developmental processes of neurogenesis, cell fate determination, neuronal and glial differentiation have been actively studied. Bipolar cell type specification and its potential relationship with synaptogenesis have been relatively less well examined [5, 6].
Little is known about the mechanism of bipolar cell specification in the neural retina. In rodents, with respect to bipolar type specification, this is in part due to an incomplete description of the sequence of events during bipolar differentiation. In this study, we describe the kinetics of genesis of bipolar cell types in both the mouse and rat retina in vivo. We have labeled bipolar cells during the terminal mitosis and subsequently studied the relative genesis of cone and rod bipolar cells during the first postnatal week. Both morphology and immunohistochemistry were used to identify types of bipolar cells. We directly demonstrate that there is a clear temporal relationship to bipolar type genesis, with birth of cone bipolar cells distinctly preceding that of rod bipolar cells both in both mouse and rat. This study represents the first example of a description of the kinetics of the genesis of bipolar cell types and contributes to the descriptive framework through which developmental models of cell type specification may be tested.
Postnatal retinal cell genesis as quantified by thymidine birthdating and either immunofluorescent or histological identification of cell fate
2.19 ± 1.45
3.98 ± 2.66
12.81 ± 6.16
16.01 ± 6.31
1.67 ± 1.15
2.73 ± 1.90
7.96 ± 3.23
8.20 ± 2.97
0.52 ± 0.32
1.21 ± 1.01
4.85 ± 3.35
7.82 ± 3.41
69.69 ± 12.65
85.73 ± 4.15
71.22 ± 5.88
62.74 ± 8.63
0.68 ± 0.26
0.58 ± 0.43
2.73 ± 1.08
4.04 ± 1.97
Retinal bipolar cells exhibit significant diversity, categorized by biochemical, morphological and physiological properties. In the present study, we addressed the following question directly: is there an order of genesis of bipolar cell types? The answer to this question was not discoverable by classic tritiated-thymidine birthdating studies due to the requirement for morphologic or immunohistochemical markers in order to distinguish bipolar types [19–23]. Using two independent in vivo methods, we demonstrate that there is a distinct order of genesis of bipolar types. Using combined tritiated-thymidine and immunohistochemistry in mouse, and retroviral transduction in rat, we have found that the genesis of cone bipolar cells definitively precedes the genesis of rod bipolar cells.
Photoreceptors are well known to be generated in two distinct waves [19–23]. Across many species, cone photoreceptors are generated first. In rats and mice, cone genesis occurs during embryonic retinal development and is largely completed prior to bipolar cell genesis [19, 20, 23]. Rod genesis peaks at birth, and bipolar genesis begins around birth, with a peak during the first postnatal week. Here we show that bipolar cell types also appear to be specified in two distinct, but overlapping, waves. There is a striking correspondence of the waves of bipolar cell specification with the waves of genesis of the presynaptic photoreceptor. Cone photoreceptors are generated early during retinal development and begin to express a subset of markers during embryonic development [24–26], thereby likely reflecting early differentiation, that is, preceding bipolar genesis and differentiation. As cone differentiation precedes bipolar differentiation, these data allow a model whereby the presynaptic cone induces the postsynaptic bipolar to terminally differentiate as a cone bipolar. In a study of bipolar synapse formation in both OPL and IPL, the glutamatergic vesicular transporter VGLUT1 was first detected in cone photoreceptor terminals at P2, several days before initiation of cone ribbon synapses in the OPL at P4-5, and preceding the later appearance of rod spherules in the OPL . Additional studies have confirmed the early, first appearance of cone photoreceptor synaptogenesis in the OPL preceding that of rods [28, 29].
These studies, combined with the bipolar birthdating data presented here, are consistent with the possibility that cones may induce specification and/or differentiation of cone bipolar cells. Indeed this may be an early event in retinal circuit formation in the OPL; however, existing functional data may not fully support this model. At present, there are few mouse models where cone photoreceptors have been disrupted and subsequent bipolar differentiation has been examined in detail. In the example of the retina-restricted Otx2 knock-out, cone and rod photoreceptors fail to develop . In these retina, Chx10-positve cells are noted to be dispersed across the retina. However, analysis of bipolar type and OPL development have not been reported, though expression of at least one bipolar cell marker, PKCα, is still present. As this marker can also be expressed by a type of amacrine cell, it is not clear if this reflects expression in rod bipolar cells. Further, we do not favor the hypothesis that rod photoreceptors induce rod bipolars, as there is an absence of rods in the Nrl mutant retina, yet the level of expression of rod bipolar markers appears to be similar to those found in wild-type retinas . Overall, the data presented here draw attention to the need for further experiments testing the relationships between the development of synapses in the OPL with bipolar cell type specification.
Finally, the data presented here also provide a quantitative analysis of postnatal retinal cell genesis that is distinct from the one classic study on this topic . In the classic study, cell type is determined by position of heavily labeled nuclei in retinal sections, whereas in this study, we employed immunocytochemistry with established cell type-specific markers. Further, the quantification in the study by Young relies on counting all heavily labeled cells in samples of sections from the central and peripheral retina. Here, we achieve whole retina sampling by quantification on dissociated cell preparations from adult retina. As shown in Table 1, while the relative timing of the genesis of major cell types is consistent, Young's study reports a higher percentage of INL cell types at each developmental time in lieu of rod photoreceptors. We believe that this distinction is most likely due to the difference in sampling, that is, finite sampling of sections in the classic method versus a complete retina averaging from the dissociated cell/immunohistochemical method. For example, at the P6 developmental time, the measures are based entirely on cells from the peripheral sections as histogenesis was reported complete in the central retinal sample .
Cone bipolar genesis precedes that of rod bipolar genesis. The order of specification of bipolar cell types therefore mimics the order of genesis of the presynaptic photoreceptors. As several studies have demonstrated that cone differentiation and synaptogenesis precedes that of rods, as well as bipolar differentiation [27–29], these data together are consistent with a model wherein interactions between bipolars and cones in the OPL may play a role in at least leading cone bipolar terminal differentiation. Future studies, specifically those examining bipolar differentiation in mutants that disrupt formation of cone photoreceptors, will be necessary to further dissect the mechanisms used for bipolar cell specification and synapse formation in the OPL. In addition to elucidating basic mechanisms of neural development, these future studies may have importance to understanding the potential for tissue transplantation and regenerative therapy in retinal degenerative disease. In these diseases, transplanted neural progenitors would be required not only to undergo appropriate photoreceptor differentiation, but also to integrate into functional retinal circuitry.
Materials and methods
Timed pregnant rats (Sprague-Dawley) and mice (C57/B6) were acquired from either Taconic Farms, Germantown, NY, USA or Charles River, Wilmington, MA, USA and handled in accordance with IACUC guidelines and Harvard Center for Animal Resources and Comparative Medicine.
pELY is a retroviral vector modified from the pLIA vector . The 5' portion of the retroviral gag gene, with its translation initiation codon mutated, is present within pLIA, creating a 5' untranslated region that is the same as the gag gene mRNA. In pELY, the gag gene was completely deleted, creating a shortened 5' untranslated region, which may increase the expression level. pELY was also modified to carry multiple cloning sites (Cla I/Bst BI/Sna BI/Xho I/Eco RI) to facilitate the cloning process.
Bacterial expression plasmids containing GST fusion of mouse Chx10 amino-terminal and carboxy-terminal fragments were constructed as following: The Eco RI/Xho I fragments of amino-terminal and carboxy-terminal regions of mouse Chx10 were PCR amplified using GAGAATTCCGGGAGATGACGGGGAAAGC and TCGAGTCACTTGCTCTGGTTTAAAGCCG primer pairs and GAGAATTCCTGGAGGCAGCAGCTGAGTC and TTCTCGAGGCCCTAAGCCATGTCCTC primer pairs and subcloned into the Eco RI/Xho I site of the pGEX4T1 vector to generate pGEXmChx10N and pGEXmChx10C, respectively.
Retroviral production and clonal analysis
High titer retroviral stocks were produced in the ecotropic producer cell line (Phoenix-E) and viral titer was determined on NIH-3T3 cells. In vivo infection in retina and clonal analysis were done as described previously .
Generation of Chx10 antiserum
GST fusion constructs containing the mouse Chx10 amino-terminal domain and carboxy-terminal domain, pGEXmChx10N and pGEXmChx10C, were expressed in Escherichia coli. The fusion proteins were affinity purified using glutathione beads and injected into rabbits as a mixture to raise Chx10 antiserum.
Immunostaining of retinal cryosections or dissociated cells were performed as described previously . The primary antibodies for staining sections were used at the following dilution: Chx10 antiserum, 1:2,000; PKCα, 1:100 (Oncogene Science, Cambridge, MA, USA); glutamine synthetase, 1:500 (Chemicon, Billerica, MA, USA); Rho4D2, 1: 100 .
Mouse pups were injected intraperitoneally with a single dose of [3H]-thymidine according to their body weight (10 μCi per gram of body weight). In general, 15 μCi were injected to each P0 pup, 20 μCi to each P2 pup, 25–30 μCi to each P4 pup, and 40 μCi to each P6 pup. Retinas were dissected at P16 and dissociated with papain as described previously . Antibody staining described above and autoradiography were carried out as described previously .
Neural retinae were dissected free of other ocular tissues and incubated for 10 minutes at room temperature in Hank's Buffered Salt Solution (HBSS) lacking Ca2+/Mg2+ (Life Technologies, Gaithersburg, MD, USA) to which trypsin (Worthington, Freehold, NJ, USA) was added to a final concentration of 1 mg/ml. After trypsinization, soybean trypsin inhibitor (Sigma, St Louis, MO, USA) was added to a final concentration of 2 mg/ml. The cells were then pelleted by centrifugation (1,200 rpm, 5 minutes), resuspended, and gently triturated to a single cell suspension in HBSS containing 100 μg/ml DNase I (Sigma). Cells were then plated on poly-D-lysine-coated (Sigma), eight-well glass slides (Cel-Line Associates, Newfield, NJ, USA) before fixation.
inner plexiform layer
outer plexiform layer
protein kinase C.
We thank Bob Molday for Rho4D2 antisera. This work was supported by NIH EY09676. AC and CLC were supported by HHMI. EMM holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund and is also grateful for support from the Rappaport Research Scholarship in Neuroscience at MGH and the Charles H. Hood Foundation Child Health Research Award.
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