BACKGROUND
amacrine cells are a distinct class of retinal neuron that participate in the processing and refinement of visual information.
the amacrine cells receive input from other amacrine cells and bipolar cells, release inhibitory neurotransmitters  at synapses that are formed with ganglion cells and other amacrine cells, and participate in retinal image processing.
amacrine cells are a highly diverse class of neuron; there may be as many as  <dig> distinct types  <cit> .
the classical findings of cajál  <cit>  and additional work  <cit>  suggest that there may be as many as  <dig> different types of amacrine cells in the avian retina.
although many studies have identified mechanisms that promote or suppress amacrine cell fate, little is known about the mechanisms involved in the differentiation of specific types of amacrine cells.
the factors that instruct cells from a pool of amacrine-fated neurons to form particular types of amacrine cells remain largely unknown.
in this study we use the cholinergic cells in the embryonic chick retina as a model system to study the mechanisms involved in the differentiation of one particular type of amacrine cell.
cholinergic neurons use acetylcholine  as a transmitter and are found in the retina of all vertebrate classes.
cholinergic amacrine cells have somata located at the proximal margin of the inner nuclear layer  and displaced to the ganglion cell layer  with processes confined to two strata in the inner plexiform layer   <cit> .
type-i and type-ii cholinergic amacrine cells are arrayed in a mosaic pattern with near-mirror symmetry around a horizontal plane through the ipl  <cit> .
however, the patterned spacing of cells within one layer is independent of the spacing of the cells in the other layer  <cit> .
the retinas of birds and reptiles contain a third type of cholinergic amacrine cell, with somata located near the middle of the inl and processes diffusely distributed in sub-laminae  <dig> through  <dig> of the ipl  <cit> .
furthermore, avian type-iii cholinergic amacrine cells can be segregated into  <dig> subtypes: type-iiia cells that contain enkephalin, neurotensin, and somatostatin immunoreactivities, and type-iiib cells that do not  <cit> .
during embryonic development, type-i and type-ii cholinergic amacrine cells express chat  as early as embryonic day  <dig>  , whereas chat-immunoreactivity in type-iii cells is not detected until  <dig> days prior to hatching at about e <dig>  <cit> .
cholinergic amacrine cells arise from a pool of undifferentiated post-mitotic neuronal cells and begin to differentiate in the middle of the presumptive ipl where they, coincidently, accumulate gaba  <cit> .
although a great deal is known about the morphological and temporal development of these amacrine cells, little is known about the genes that they express during differentiation and the factors that influence their development.
however, it has recently been shown that extra-cellular atp and the p2x receptor coordinate, in part, the mosaic patterning and survival of the cholinergic cells in the rodent retina  <cit>  and that visual activity is required for maintenance of the mosaic after eye opening in the mouse  <cit> .
the purpose of this study was to better characterize the development of type-i and type-ii cholinergic amacrine cells in the chick retina and identify factors that influence their development.
we find that the type-i and type-ii cholinergic amacrine cells express p27kip <dig>  pax <dig> and islet <dig> during development.
in addition, we show that these cells are not homotypic during embryonic development; type-ii cells transiently express elevated levels of cellular retinoic acid binding protein  and neurofilament, while type-i cells do not.
furthermore, we show that the relative abundance of type-i and type-ii cholinergic cells is influenced by local microenvironment and signalling through muscarinic acetylcholine receptors.
we propose that autologous signalling between cells acts to drive the differentiation and mosaic patterning of distinct types of retinal amacrine cells.
RESULTS
cholinergic amacrine cells in the embryonic chick retina were detected by using antibodies to choline acetyltransferase .
chat-immunoreactive amacrine cells were first detected in the retina at embryonic day  <dig> , consistent with a previous report  <cit> .
one day later at e <dig>  in central regions of the retina, these cells formed two uniform rows of cells, with type-i cells located at the distal border of the presumptive ipl and type-ii cells located within the proximal half of the presumptive ipl , similar to previous findings  <cit> .
cholinergic amacrine cells express islet <dig> and pax <dig> as they differentiate
in the developing rodent retina, cholinergic amacrine cells are known to express islet <dig>  <cit> .
islet <dig> is a homeotic lim-domain transcription factor that is known to be expressed by bipolar, horizontal, and ganglion cells in the retina  <cit> .
in the developing avian retina at e <dig>  islet1-immunoreactivity was detected in the nuclei of presumptive ganglion and amacrine cells in the gcl and in differentiating cells in the distal retina .
the islet1-positive nuclei in the distal retina presumably were post-mitotic neurons migrating away from the ventricular surface of the retina to their final position within the retina.
at e <dig>  islet <dig> was expressed by chat-immunoreactive cells prior to the formation of the presumptive ipl .
these cells were intermingled with islet1-expressing cells in the gcl, suggesting that the cholinergic cells begin to differentiate among a heterogeneous pool of post-mitotic islet1+/chat- cells.
at e <dig>  islet1-positive nuclei were found in the gcl, putative differentiating bipolar cells in distal layers  of the retina, and two rows of cells between the gcl and bipolar cells .
the islet1-positive nuclei at the vitread border of the inl and within the ipl were those of type-i and type-ii cholinergic amacrine cells, respectively .
at e <dig> and e <dig>  the islet1/chat-positive cells remained as distinctive rows of cells .
by e <dig> the somata of the type-ii cells had migrated into the gcl.
in the retina of postnatal chickens, islet1-immunoreactivity was present in horizontal cells, many bipolar cells, most if not all ganglion cells, and type-i and type-ii cholinergic amacrine cells, but not type-iii cells .
to test whether homeodomain transcription factors in addition to islet <dig> are expressed by developing cholinergic amacrine cells, we double-labeled sections with antibodies to chat and pax <dig>  ap2α, prox <dig> or brn3b.
pax <dig> is paired-class homeodomain transcription factor that is known to be expressed by neural progenitors in the embryonic and postnatal chick retina  <cit> .
pax <dig> is expressed at low levels in progenitors and becomes expressed at high levels in amacrine and ganglion cells as they differentiate  <cit> .
we used a monoclonal antibody to pax <dig> that is known to label horizontal, amacrine, ganglion cells and progenitors in the avian retina  <cit> .
in e <dig> and e <dig> retinas, type-i and type-ii cholinergic amacrine cells expressed relatively low levels of pax6; pax6-immunolabeling was more intense in the nuclei of non-cholinergic neurons in the gcl and inl compared to the labeling intensity of the nuclei of the chat-immunoreactive cells .
the cholinergic amacrine cells did not express ap2α, prox <dig>  or brn3a during embryonic development or in the postnatal retina .
the cell cycle inhibitor p27kip <dig> has been shown to be important for the development of the retina and differentiation of müller glia  <cit> .
in e <dig> retina, immunoreactivity for p27kip <dig> was seen in numerous nuclei scattered throughout the retina, but was absent from the nuclei of proliferating progenitors in distal layers of the retina .
the distribution of p27kip1-immunoreactive nuclei in e <dig> retina was consistent with the known location of differentiating cells.
at this stage of development, immature chat-positive cells contained p27kip1-immunoreactive nuclei .
at e <dig>  numerous p27kip1-positive nuclei were observed in the inl, ipl and gcl .
the p27kip1-positive nuclei in the ipl were immunoreactive for chat, and the labeling intensity of these nuclei was greater than that seen in the nuclei of non-cholinergic cells of the inl and gcl .
cholinergic amacrine cells transiently express neurofilament and crabp as they differentiate
neurofilament is known to be expressed by ganglion cells soon after they begin to differentiate, and the ganglion cells in the chick retina are thought to be the only type of neuron that expresses this intermediate filament  <cit> .
surprisingly, we found that the neurites of type-i and type-ii cholinergic amacrine cells were immunoreactive for neurofilament in the retinas from e <dig>  through e <dig> .
increased levels of neurofilament-immunoreactivity were detected in the type-ii cholinergic cells, compared to levels seen in the type-i cells .
at e <dig>  neurofilament-immunoreactivity was present in a "tuft" of processes at the distal edge of the type-ii cholinergic amacrine cells, while little or no immunoreactivity was detected in the type-i cells .
by e <dig>  neurofilament-immunoreactivity was present throughout the neurites of type-ii cells, while sparse immunoreactivity was present in the neurites of type-i cells .
in e <dig> and postnatal retinas, neurofilament-immunoreactivity was not present in the cholinergic amacrine cells .
in the embryonic chick retina, crabp was expressed by numerous differentiating amacrine cells , consistent with a previous report  <cit> .
at e <dig>  crabp-immunoreactive cells were found near the developing gcl  and a few of these cells were also immunoreactive for chat .
at e <dig>  most of the crabp-immunoreactive cells were located in the presumptive ipl and these cells were also immunoreactive for chat  .
low levels of crabp could be detected in a few  presumptive type-i cells in the proximal inl .
this finding indicates that a minority of type-i cells may express low levels of crabp during a narrow window of development.
in e <dig> retina, high levels of crabp-immunoreactivity were detected in type-ii cholinergic cells located in the proximal ipl and weak crabp-immunoreactivity was detected in non-cholinergic cells as they differentiate in the presumptive inl .
the cell bodies of type-i cells contained little or no crabp-immunoreactivity in the e <dig> retina.
differential expression of crabp was most noticeable at e <dig>  after the cells have extended their neurites into the ipl.
type-ii cells expressed high levels of crabp in their cell bodies, as well as neurites, whereas crabp was absent from somata of type-i cells and low levels may have been present in the neurites .
type-ii cholinergic cells continued to express crabp as they differentiated through e <dig> .
at e <dig>   <dig> days before hatching, immunoreactivity for crabp was no longer detectable in the type-ii cholinergic cells .
to better assess whether the dendrites of the cholinergic cells contained crabp, high-magnification confocal images were obtained.
in e <dig> retinas, we observed significant crabp-immunoreactivity in the stratified neurites of the type-ii cells.
by contrast, crabp-immunoreactivity was no longer detectable in the processes of type-ii amacrine cells at p <dig> .
in postnatal chick retina, crabp-immunoreactivity was not observed in type-ii cholinergic cells , but was present in non-cholinergic amacrine cells and bipolar cells, consistent with a previous report  <cit> .
taken together, these findings indicate that during embryonic development the cholinergic cells are not homotypic.
the phenotypic differences between type-i and type-ii cells may occur because of differences imposed by the local microenvironment.
in other words, the local microenvironment provided by the inl likely influences the phenotype of type-i cells, whereas the proximal ipl and/or gcl may influence the phenotype of the type-ii cells; a spatial separation of  <dig> to  <dig> μm within the developing retina.
differentiation of cholinergic amacrine cells in dissociated cell cultures
to test whether the microenvironment influences the phenotype of type-i and type-ii cholinergic amacrine cells, we dissociated retinas from e <dig> chick embryos and maintained these cells in culture for  <dig> days.
dissociated cell cultures will not accurately recapitulate the microenvironment provided in vivo where different cells, and the cues provided by these cells, are highly spatially organized into discrete laminae.
to discriminate between type-i and type-ii cholinergic amacrine cells we double-labeled cells with antibodies to chat and crabp.
cells immunoreactive for chat and crabp  were counted as type-ii cells and cells that were immunoreactive for chat alone were counted as type-i cells .
in vivo, numbers of type-i and type-ii cholinergic amacrine cells in the developing retina are approximately equal .
in dissociated cell cultures of embryonic retina, however, type-i cholinergic cells are approximately twice  as abundant as type-ii cells .
the decrease in the relative abundance of type-ii to type-i cells did not change significantly with differences in original plating density .
prada and colleagues  <cit>  showed that cholinergic cells are postmitotic by about e <dig>  to exclude the possibility of de novo generation of cholinergic cells in culture, we cultured e <dig> retina in the presence of brdu  for  <dig> days.
none  of the chat-positive cells in cultures of e <dig> retina accumulated brdu , indicating that de novo generation of cholinergic amacrine cells does not occur under culture conditions.
taken together, these findings suggest that the environment provided by the intact retina is required to maintain equal numbers of type-i and type-ii cholinergic cells.
since the type-ii cholinergic cells are in close proximity  to developing ganglion cells, whereas the type-i cells are further away , it is possible that signals provided by the differentiating ganglion cells influence the development and phenotype of the type-ii cells.
ganglion cells are known to produce sonic hedgehog  as they differentiate  <cit>  and shh is known to establish morphogenic gradients over short distances  <cit> .
thus, it is possible that shh stimulates type-ii cholinergic amacrine cells to transiently express elevated levels of crabp during embryonic development.
however, the relative abundance of type-ii cells was not significantly affected by the addition of shh or the blockade of shh-signaling with kaad  cyclopamine.
shh-treated samples contained  <dig> %  more type-ii cells, and cultures treated with kaad-cyclopamine contained  <dig> %  fewer type-ii cells compared to control preparations.
statistical analyses indicated that these changes were not significant.
there is some evidence that type-ii cholinergic amacrine cells express muscarinic receptors in the postnatal retina  <cit> .
in addition, antibodies to the m <dig> and m <dig> isoforms of the muscarinic receptor in the embryonic chick retina appear to label type-ii-like cells in the developing ipl  <cit> .
thus, it is possible that the type-ii cholinergic amacrine cells communicate to each other through paracrine muscarinic interactions.
to test this hypothesis we applied a muscarinic antagonist  or agonist  to cultures of e <dig> retinas.
we found that activation and suppression of muscarinic signaling reduced the relative abundance of type-ii cholinergic amacrine cells .
since the relative abundance of type-ii to type-i cholinergic amacrine cells is reduced by in vitro conditions and by influencing muscarinic signaling, we sought to assess whether similar mechanisms influence the phenotype of cholinergic cells in the intact retina.
to test this, we made intraocular injections of  <dig> ng of atropine  or  <dig> ng carbachol  into the vitreous chamber of e <dig> chick embryos.
the e <dig> eye was estimated to have  <dig> mm <dig> of volume, which should have resulted in initial maximum vitreal concentrations of carbachol and atropine of about  <dig> μm.
atropine and carbachol have been used from  <dig> nm to  <dig> mm to elicit effects on retinal cells and chick muscarinic acetylcholine receptors   <cit> .
similar to the findings of the in vitro studies, we found that the number of cells that express chat and crabp  was decreased by muscarinic ligands.
treatment with carbachol or atropine reduced the number of chat-positive cells that are immunoreactive for crabp in the presumptive ipl .
there was a reduction in the relative abundance of chat/crabp-positive cells by about 23% with atropine treatment  or by about 38% with carbachol treatment  .
the abundance of type-ii-like cells in the ipl was not significantly different between treatments with atropine or carbachol.
we consistently found that the type-i cells in the inl did not express detectable levels of crabp in retinas that were treated with muscarinic ligands.
these findings suggest that gains or losses in muscarinic signaling decrease the relative abundance of crabp-expressing cells among the type-ii cholinergic amacrine cells in vivo.
in addition to a decrease in the percentage of type-ii cholinergic amacrine cells, we found that the density of chat-positive cells was decreased by treatment with both a muscarinic achr antagonist and agonist .
the number of chat+ cells per  <dig>  μm <dig> in the inl  and in the gcl  was significantly decreased by intraocular injections of atropine .
similarly, treatment with the muscarinic agonist carbachol decreased the density of cholinergic amacrine cells in the inl and gcl .
decreases in the density of cholinergic cells were not caused by increased apoptosis; we failed to find increased numbers of cleaved caspase3-positive cells in retinas treated with atropine or carbachol .
additionally, the muscarinic ligands did not have any obvious effect on non-cholinergic neurons; ganglion cells labeled for brn3a and horizontal, bipolar, amacrine and ganglion cells labeled for calretinin displayed normal distributions when treated with atropine or carbachol .
in retinas treated with atropine or carbachol, we found that the regularly patterned mosaic of cholinergic amacrine cells was disrupted .
by visual inspection, the type-i and type-ii cells in retinas treated with atropine or carbachol appeared irregularly spaced compared to saline-treated retinas .
to quantify whether the mosaic patterning of the cholinergic cells was disrupted by the muscarinic ligands, we performed a nearest neighbor analysis.
nearest neighbor regularity indices with values above  <dig> are considered to be regular and values below  <dig> represent a random distribution.
in saline-treated retinas the regularity index of the cholinergic amacrine cells was high, with a value of  <dig>  ±  <dig>  .
the regularity index of the cholinergic cells was significantly decreased in retinas treated with atropine  or carbachol  compared to the regularity index of the cholinergic cells in saline-treated retinas .
these findings suggest that perturbation of muscarinic signaling disrupted the mosaic patterning of the cholinergic amacrine cells.
discussion
the genes that are expressed by differentiating cholinergic amacrine cells are distinctly different from those that are expressed by other types of amacrine cells.
unlike other types of amacrine cells, the cholinergic cells express islet <dig> with the onset of differentiation and into the mature retina, consistent with reports in the embryonic rat retina  <cit> .
as the cholinergic amacrine cells differentiate they express proteins common to other types of amacrine cells such as pax <dig>  crabp and p27kip <dig>  however, the relative levels of immunolabeling for these proteins within the cholinergic cells are either decreased  or increased  compared to levels seen in other types of differentiating amacrine cells.
it is likely that islet <dig> and pax <dig> play important roles in the development and maintenance of phenotype of the type-i and type-ii cholinergic amacrine cells.
the significance of elevated expression of p27kip <dig> and crabp in the cholinergic cells remains unknown.
the type-i and type-ii cholinergic amacrine cells are equally abundant, morphologically identical, and are arranged with near-mirror symmetry across a horizontal plane through the ipl  <cit> .
in addition to using acetylcholine, type-i and type-ii cholinergic amacrine cells are known to utilize gaba as a neurotransmitter  <cit>  and begin to accumulate gaba soon after they begin to differentiate  <cit> .
furthermore, type-i and type-ii cholinergic amacrine cells in the chick retina have been shown to originate from a common pool of migrating, post-mitotic undifferentiated neurons  <cit> .
these findings have lead to the assumption that these cells are phenotypically identical and differ only by the position of their somata.
however, we provide evidence that these two cell types differ by more than the location of their cell bodies.
we found that during differentiation, type-ii cells transiently expressed high levels of crabp and neurofilament, whereas the type-i cells do not.
further, in the postnatal chick retina, type-ii cells may express machr <dig>  whereas type-i cells do not  <cit> , and these cells have different sensitivities to excitotoxins, suggesting that different types of glutamate receptors are expressed by these cells, and/or that they have different susceptibilities to large fluctuations in ion concentrations  <cit> .
embryonic expression of machr <dig>  machr <dig>  and machr <dig> appear in non-overlapping laminae of the developing ipl at e <dig>  <cit> , suggesting that these receptors may be expressed by different types of amacrine cells.
the machr <dig> may not exist in the chick and  <cit>  the retinal expression of machr <dig>  <cit>  remains uncertain, but has been detected in the sclera of guinea pigs  <cit>  and human  <cit> .
differential expression of receptor isoforms is a possible mechanism to explain the differing effects of muscarinic ligands on the type-i and type-ii cholinergic cells.
the cholinergic cells may be among the first types of amacrine cell to differentiate, with the onset of chat and islet <dig> expression occurring while these cells are interspersed among the developing ganglion cells at e <dig>  by comparison, amacrine cells that utilize different neurotransmitters such as dopamine, glucagon, enkephalin or vasoactive intestinal peptide differentiate 6– <dig> days after the cholinergic cells  <cit> .
as development proceeds, the islet1/chat-positive cells migrate away from the ganglion cells into the presumptive ipl where they begin to differentiate.
when the type-i and type-ii cholinergic cells become segregated into different laminae, the type-ii cells begin to express elevated levels of crabp and neurofilament.
the close proximity of type-ii cells to developing ganglion cells may expose these cells to environmental cues that influence the differentiation of ganglion cells.
accordingly, this microenvironment may promote the expression of genes common to both ganglion and type-ii cells .
secreted signals produced by ganglion cells that influence the type-ii cholinergic cells could include shh  <cit>  and bmp2/ <dig>  <cit> .
however, we failed to find evidence that shh influences the differentiation of type-ii cholinergic cells.
we found that the 1: <dig> ratio of type-i to type-ii cholinergic cells from the intact retina was disrupted when the cells were dissociated and grown in culture; type-i cells were twice as abundant as the type-ii cells in vitro.
in our in vitro paradigm, it is likely that crabp expression is down-regulated by the type-ii cells making them indistinguishable from the type-i cells.
the disruption of the in vivo microenvironment did not favor the differentiation of type-ii cholinergic cells – the co-expression of chat and crabp.
these findings suggest that the factors promoting the type-ii phenotype are diluted under culture conditions and that the phenotype of type-ii cells relies, in part, on the microenvironment provided by the intact retina.
alternatively, the survival of type-ii cholinergic amacrine cells may be selectively compromised by acute dissociation and culture conditions .
it is unlikely that changes in the relative abundance of type-i to type-ii cholinergic cells resulted from cell fate decisions made during the terminal mitosis.
we did not find any cholinergic cells in our in vitro and in vivo paradigms that were labeled for brdu, indicating that none of the cholinergic cells were generated after the cultures were established or after compounds were delivered to the developing eye.
similar to the results of our in vitro studies, we found that the in vivo development of the cholinergic amacrine cells was influenced by muscarinic ligands.
in saline-treated retinas, the vast majority of cholinergic cells in the gcl express crabp.
however, treatment with muscarinic ligands resulted in increased numbers of type-ii cells that failed to express crabp.
these findings suggest that appropriate levels of muscarinic signaling are required for the maintenance of crabp expression in the type-ii cells; perturbation of optimal levels of muscarinic signaling between cholinergic cells by agonist or antagonist disrupted the phenotype of these cells.
it remains uncertain whether the effects of the muscarinic ligands were mediated by direct or indirect actions on the type-ii cells.
the patterning of the cholinergic amacrine cells is likely coordinated by several distinct signaling pathways in addition to the muscarinic pathway.
for example, extra-cellular atp and the p2x receptor influence the patterning and survival of the cholinergic cells in the rodent retina  <cit> .
in addition, zhang and colleagues  <cit>  have shown that maintenance of array of type-ii amacrine cells in the mouse gcl is dependent upon visual activity; after  <dig> days of rearing pups in the dark, chat-positive cells are undetectable in the gcl, but remain present in the inl.
inhibiting machrs with atropine or activating machrs with carbachol disrupted the regular spacing between the cholinergic amacrine cells.
we propose that the proper patterning of the cholinergic cells requires optimal levels of muscarinic signaling similar to the shh concentration-dependent differentiation of spinal cord interneurons  <cit> .
in principle, release and diffusion of ach from a cholinergic cell should result in a concentration gradient that decreases with increasing distance from the source.
regular spacing between cells can occur if like-cells are repelled from each other; and for the cholinergic amacrine cells this may involve migrating laterally away from neighboring, concentrated sources of ach.
our data are consistent with the hypothesis that the regular spacing of the cholinergic cells requires an optimal level of muscarinic signaling, and if these cells encounter gains or losses in signaling the mosaic patterning is disrupted.
it remains uncertain what effect perturbations in muscarinic signaling have on the spontaneous calcium waves, spread through the cholinergic amacrine cells, that are necessary for the establishment of early retinal circuitry in the ipl  <cit> .
the mosaic pattern of the cholinergic cells may, in part, be required to establish normal waves of activity.
however, this activity was shown to be regulated by nicotinic achr in the embryonic retina  <cit> .
it is possible that the application of muscarinic compounds generally perturbed retinal development and thereby disrupted the patterning of the cholinergic cells.
however, we found that muscarinic compounds did not affect non-cholinergic cells expressing calretinin, islet <dig> or brn3a-positive ganglion cells, and we failed to observe an increase in immunolabeling for cleaved-caspase  <dig>  taken together, these findings indicate muscarinic ligands do not overtly affect non-cholinergic inner retinal neurons.
the type-i and type-ii cells are not likely to influence each other through cholinergic signaling across the developing ipl.
for example, the mosaic pattern of type-ii cholinergic amacrine cells in the gcl has no relation to the pattern of type-i cells in the inl  <cit> .
additionally, acetylcholine esterase  is first detected in the chick retina between e <dig> and e <dig> in the middle of the ipl when the differentiating cholinergic cells are segregating between the gcl and inl  <cit> .
ache that is distributed into discrete laminae in the ipl should prevent any vertical diffusion of ach through the retina.
for example, ach released from the type-ii cells is not likely to act on type-i cells because ache in the ipl is stratified between the dendrites of these cells and should degrade the ach that diffuses distally through the ipl.
however, given the dense intermingling of the dendrites of the cholinergic cells within narrow strata of the ipl, it seems likely that ach released from the dendrites of one cholinergic cell could act on a neighboring cell with overlapping, co-stratified dendritic arbors.
it is possible that decreases in the density of cholinergic cells resulted from increases in cell death.
however, we failed to detect increased numbers of cleaved-caspase  <dig> positive cells in retinas treated with carbachol or atropine.
we cannot exclude the possibility that the death of cholinergic cells occurred in a caspase 3-independent manner resulting in reduced numbers of cholinergic cells and perhaps disruption of the mosaic patterning.
another possibility is that decreases in the abundance of chat/crabp-positive cells in the ipl resulted from the down-regulation of chat and crabp.
consistent with this hypothesis, chat may be transiently expressed by some cells in the gcl similar to the developing turtle retina, indicating that chat expression may be reversible in some types of retinal cells  <cit> .
the large diversity of amacrine cell types that are distributed in mosaic patterns with regular spacing implies that autologous signals are required to coordinate the patterning of unique cell types.
the notion of homotypic interactions underlying mosaic patterning has been proposed for the cholinergic amacrine cells in the retina  <cit> .
furthermore, rossi and colleagues  <cit>  demonstrated that the establishment of regular spacing between horizontal cells in the rodent retina occurs independent of pre-synaptic input from photoreceptors, suggesting that patterning is mediated via homotypic interactions among the horizontal cells.
it is possible that homotypic interactions are responsible for the regular spacing of the different types of horizontal, amacrine and ganglion cells.
consistent with this hypothesis, cones, horizontal cells, ganglion cells, and cholinergic amacrine cells migrate laterally away from their radial column of clonally derived cells, whereas bipolar cells and müller glia do not  <cit> .
to establish a highly ordered mosaic pattern, cells within the retina must have the ability to migrate laterally away from columns of clonally derived cells unless the order of the array is determined by the progenitors.
a likely mechanism underlying the spatial patterning between like-cells is the use of peptide or amino acid-derived transmitters to provide homotypic signals between cells.
for example, dopamine, glucagon, neuropeptide y, substance p, vasoactive intestinal polypeptide or enkephalin, neurotransmitters that are used by distinct types of amacrine cells, are used as autologous signals to coordinate cell type-specific patterning.
consistent with this hypothesis, our data indicate that the disruption of muscarinic ach signaling perturbs the numbers, spacing and phenotype of cholinergic amacrine cells in the developing retina.
CONCLUSIONS
we conclude that, during development, type-ii cholinergic amacrine cells transiently express crabp and neurofilament, while type-i cells do not.
we propose that the transient expression of crabp and neurofilament is regulated by local microenvironment.
additionally, we find that interfering with muscarinic signaling decreases the relative number of type-ii cells and mosaic patterning of the cholinergic amacrine cells.
we propose that the phenotypic differences between developing type-i and type-ii cholinergic amacrine cells are elicited, in part, by paracrine cholinergic signaling; the local microenvironment of the in tact retina allows precise muscarinic signaling to establish proper cellular phenotypes and organization of the type-ii cells.
it remains uncertain whether muscarinic receptors are differentially expressed by the type-i and type-ii cells.

 amacrine cells are a distinct class of retinal neuron that participate in the processing and refinement of visual information. the amacrine cells receive input from other amacrine cells and bipolar cells, release inhibitory neurotransmitters at synapses that are formed with ganglion cells and other amacrine cells, and participate in retinal image processing. amacrine cells are a highly diverse class of neuron; there may be as many as distinct types. the classical findings of cajál and additional work suggest that there may be as many as different types of amacrine cells in the avian retina. although many studies have identified mechanisms that promote or suppress amacrine cell fate, little is known about the mechanisms involved in the differentiation of specific types of amacrine cells. the factors that instruct cells from a pool of amacrine-fated neurons to form particular types of amacrine cells remain largely unknown. in this study we use the cholinergic cells in the embryonic chick retina as a model system to study the mechanisms involved in the differentiation of one particular type of amacrine cell. cholinergic neurons use acetylcholine as a transmitter and are found in the retina of all vertebrate classes. cholinergic amacrine cells have somata located at the proximal margin of the inner nuclear layer and displaced to the ganglion cell layer with processes confined to two strata in the inner plexiform layer. type-i and type-ii cholinergic amacrine cells are arrayed in a mosaic pattern with near-mirror symmetry around a horizontal plane through the ipl. however, the patterned spacing of cells within one layer is independent of the spacing of the cells in the other layer. the retinas of birds and reptiles contain a third type of cholinergic amacrine cell, with somata located near the middle of the inl and processes diffusely distributed in sub-laminae through of the ipl. furthermore, avian type-iii cholinergic amacrine cells can be segregated into subtypes: type-iiia cells that contain enkephalin, neurotensin, and somatostatin immunoreactivities, and type-iiib cells that do not. during embryonic development, type-i and type-ii cholinergic amacrine cells express chat as early as embryonic day, whereas chat-immunoreactivity in type-iii cells is not detected until days prior to hatching at about e. cholinergic amacrine cells arise from a pool of undifferentiated post-mitotic neuronal cells and begin to differentiate in the middle of the presumptive ipl where they, coincidently, accumulate gaba. although a great deal is known about the morphological and temporal development of these amacrine cells, little is known about the genes that they express during differentiation and the factors that influence their development. however, it has recently been shown that extra-cellular atp and the p2x receptor coordinate, in part, the mosaic patterning and survival of the cholinergic cells in the rodent retina and that visual activity is required for maintenance of the mosaic after eye opening in the mouse. the purpose of this study was to better characterize the development of type-i and type-ii cholinergic amacrine cells in the chick retina and identify factors that influence their development. we find that the type-i and type-ii cholinergic amacrine cells express p27kip pax and islet during development. in addition, we show that these cells are not homotypic during embryonic development; type-ii cells transiently express elevated levels of cellular retinoic acid binding protein and neurofilament, while type-i cells do not. furthermore, we show that the relative abundance of type-i and type-ii cholinergic cells is influenced by local microenvironment and signalling through muscarinic acetylcholine receptors. we propose that autologous signalling between cells acts to drive the differentiation and mosaic patterning of distinct types of retinal amacrine cells.  cholinergic amacrine cells in the embryonic chick retina were detected by using antibodies to choline acetyltransferase. chat-immunoreactive amacrine cells were first detected in the retina at embryonic day, consistent with a previous report. one day later at e in central regions of the retina, these cells formed two uniform rows of cells, with type-i cells located at the distal border of the presumptive ipl and type-ii cells located within the proximal half of the presumptive ipl, similar to previous findings. cholinergic amacrine cells express islet and pax as they differentiate in the developing rodent retina, cholinergic amacrine cells are known to express islet. islet is a homeotic lim-domain transcription factor that is known to be expressed by bipolar, horizontal, and ganglion cells in the retina. in the developing avian retina at e islet1-immunoreactivity was detected in the nuclei of presumptive ganglion and amacrine cells in the gcl and in differentiating cells in the distal retina. the islet1-positive nuclei in the distal retina presumably were post-mitotic neurons migrating away from the ventricular surface of the retina to their final position within the retina. at e islet was expressed by chat-immunoreactive cells prior to the formation of the presumptive ipl. these cells were intermingled with islet1-expressing cells in the gcl, suggesting that the cholinergic cells begin to differentiate among a heterogeneous pool of post-mitotic islet1+/chat- cells. at e islet1-positive nuclei were found in the gcl, putative differentiating bipolar cells in distal layers of the retina, and two rows of cells between the gcl and bipolar cells. the islet1-positive nuclei at the vitread border of the inl and within the ipl were those of type-i and type-ii cholinergic amacrine cells, respectively. at e and e the islet1/chat-positive cells remained as distinctive rows of cells. by e the somata of the type-ii cells had migrated into the gcl. in the retina of postnatal chickens, islet1-immunoreactivity was present in horizontal cells, many bipolar cells, most if not all ganglion cells, and type-i and type-ii cholinergic amacrine cells, but not type-iii cells. to test whether homeodomain transcription factors in addition to islet are expressed by developing cholinergic amacrine cells, we double-labeled sections with antibodies to chat and pax ap2α, prox or brn3b. pax is paired-class homeodomain transcription factor that is known to be expressed by neural progenitors in the embryonic and postnatal chick retina. pax is expressed at low levels in progenitors and becomes expressed at high levels in amacrine and ganglion cells as they differentiate. we used a monoclonal antibody to pax that is known to label horizontal, amacrine, ganglion cells and progenitors in the avian retina. in e and e retinas, type-i and type-ii cholinergic amacrine cells expressed relatively low levels of pax6; pax6-immunolabeling was more intense in the nuclei of non-cholinergic neurons in the gcl and inl compared to the labeling intensity of the nuclei of the chat-immunoreactive cells. the cholinergic amacrine cells did not express ap2α, prox or brn3a during embryonic development or in the postnatal retina. the cell cycle inhibitor p27kip has been shown to be important for the development of the retina and differentiation of müller glia. in e retina, immunoreactivity for p27kip was seen in numerous nuclei scattered throughout the retina, but was absent from the nuclei of proliferating progenitors in distal layers of the retina. the distribution of p27kip1-immunoreactive nuclei in e retina was consistent with the known location of differentiating cells. at this stage of development, immature chat-positive cells contained p27kip1-immunoreactive nuclei. at e numerous p27kip1-positive nuclei were observed in the inl, ipl and gcl. the p27kip1-positive nuclei in the ipl were immunoreactive for chat, and the labeling intensity of these nuclei was greater than that seen in the nuclei of non-cholinergic cells of the inl and gcl. cholinergic amacrine cells transiently express neurofilament and crabp as they differentiate neurofilament is known to be expressed by ganglion cells soon after they begin to differentiate, and the ganglion cells in the chick retina are thought to be the only type of neuron that expresses this intermediate filament. surprisingly, we found that the neurites of type-i and type-ii cholinergic amacrine cells were immunoreactive for neurofilament in the retinas from e through e. increased levels of neurofilament-immunoreactivity were detected in the type-ii cholinergic cells, compared to levels seen in the type-i cells. at e neurofilament-immunoreactivity was present in a "tuft" of processes at the distal edge of the type-ii cholinergic amacrine cells, while little or no immunoreactivity was detected in the type-i cells. by e neurofilament-immunoreactivity was present throughout the neurites of type-ii cells, while sparse immunoreactivity was present in the neurites of type-i cells. in e and postnatal retinas, neurofilament-immunoreactivity was not present in the cholinergic amacrine cells. in the embryonic chick retina, crabp was expressed by numerous differentiating amacrine cells, consistent with a previous report. at e crabp-immunoreactive cells were found near the developing gcl and a few of these cells were also immunoreactive for chat. at e most of the crabp-immunoreactive cells were located in the presumptive ipl and these cells were also immunoreactive for chat. low levels of crabp could be detected in a few presumptive type-i cells in the proximal inl. this finding indicates that a minority of type-i cells may express low levels of crabp during a narrow window of development. in e retina, high levels of crabp-immunoreactivity were detected in type-ii cholinergic cells located in the proximal ipl and weak crabp-immunoreactivity was detected in non-cholinergic cells as they differentiate in the presumptive inl. the cell bodies of type-i cells contained little or no crabp-immunoreactivity in the e retina. differential expression of crabp was most noticeable at e after the cells have extended their neurites into the ipl. type-ii cells expressed high levels of crabp in their cell bodies, as well as neurites, whereas crabp was absent from somata of type-i cells and low levels may have been present in the neurites. type-ii cholinergic cells continued to express crabp as they differentiated through e. at e days before hatching, immunoreactivity for crabp was no longer detectable in the type-ii cholinergic cells. to better assess whether the dendrites of the cholinergic cells contained crabp, high-magnification confocal images were obtained. in e retinas, we observed significant crabp-immunoreactivity in the stratified neurites of the type-ii cells. by contrast, crabp-immunoreactivity was no longer detectable in the processes of type-ii amacrine cells at p. in postnatal chick retina, crabp-immunoreactivity was not observed in type-ii cholinergic cells, but was present in non-cholinergic amacrine cells and bipolar cells, consistent with a previous report. taken together, these findings indicate that during embryonic development the cholinergic cells are not homotypic. the phenotypic differences between type-i and type-ii cells may occur because of differences imposed by the local microenvironment. in other words, the local microenvironment provided by the inl likely influences the phenotype of type-i cells, whereas the proximal ipl and/or gcl may influence the phenotype of the type-ii cells; a spatial separation of to μm within the developing retina. differentiation of cholinergic amacrine cells in dissociated cell cultures to test whether the microenvironment influences the phenotype of type-i and type-ii cholinergic amacrine cells, we dissociated retinas from e chick embryos and maintained these cells in culture for days. dissociated cell cultures will not accurately recapitulate the microenvironment provided in vivo where different cells, and the cues provided by these cells, are highly spatially organized into discrete laminae. to discriminate between type-i and type-ii cholinergic amacrine cells we double-labeled cells with antibodies to chat and crabp. cells immunoreactive for chat and crabp were counted as type-ii cells and cells that were immunoreactive for chat alone were counted as type-i cells. in vivo, numbers of type-i and type-ii cholinergic amacrine cells in the developing retina are approximately equal. in dissociated cell cultures of embryonic retina, however, type-i cholinergic cells are approximately twice as abundant as type-ii cells. the decrease in the relative abundance of type-ii to type-i cells did not change significantly with differences in original plating density. prada and colleagues showed that cholinergic cells are postmitotic by about e to exclude the possibility of de novo generation of cholinergic cells in culture, we cultured e retina in the presence of brdu for days. none of the chat-positive cells in cultures of e retina accumulated brdu, indicating that de novo generation of cholinergic amacrine cells does not occur under culture conditions. taken together, these findings suggest that the environment provided by the intact retina is required to maintain equal numbers of type-i and type-ii cholinergic cells. since the type-ii cholinergic cells are in close proximity to developing ganglion cells, whereas the type-i cells are further away, it is possible that signals provided by the differentiating ganglion cells influence the development and phenotype of the type-ii cells. ganglion cells are known to produce sonic hedgehog as they differentiate and shh is known to establish morphogenic gradients over short distances. thus, it is possible that shh stimulates type-ii cholinergic amacrine cells to transiently express elevated levels of crabp during embryonic development. however, the relative abundance of type-ii cells was not significantly affected by the addition of shh or the blockade of shh-signaling with kaad cyclopamine. shh-treated samples contained % more type-ii cells, and cultures treated with kaad-cyclopamine contained % fewer type-ii cells compared to control preparations. statistical analyses indicated that these changes were not significant. there is some evidence that type-ii cholinergic amacrine cells express muscarinic receptors in the postnatal retina. in addition, antibodies to the m and m isoforms of the muscarinic receptor in the embryonic chick retina appear to label type-ii-like cells in the developing ipl. thus, it is possible that the type-ii cholinergic amacrine cells communicate to each other through paracrine muscarinic interactions. to test this hypothesis we applied a muscarinic antagonist or agonist to cultures of e retinas. we found that activation and suppression of muscarinic signaling reduced the relative abundance of type-ii cholinergic amacrine cells. since the relative abundance of type-ii to type-i cholinergic amacrine cells is reduced by in vitro conditions and by influencing muscarinic signaling, we sought to assess whether similar mechanisms influence the phenotype of cholinergic cells in the intact retina. to test this, we made intraocular injections of ng of atropine or ng carbachol into the vitreous chamber of e chick embryos. the e eye was estimated to have mm of volume, which should have resulted in initial maximum vitreal concentrations of carbachol and atropine of about μm. atropine and carbachol have been used from nm to mm to elicit effects on retinal cells and chick muscarinic acetylcholine receptors. similar to the findings of the in vitro studies, we found that the number of cells that express chat and crabp was decreased by muscarinic ligands. treatment with carbachol or atropine reduced the number of chat-positive cells that are immunoreactive for crabp in the presumptive ipl. there was a reduction in the relative abundance of chat/crabp-positive cells by about 23% with atropine treatment or by about 38% with carbachol treatment. the abundance of type-ii-like cells in the ipl was not significantly different between treatments with atropine or carbachol. we consistently found that the type-i cells in the inl did not express detectable levels of crabp in retinas that were treated with muscarinic ligands. these findings suggest that gains or losses in muscarinic signaling decrease the relative abundance of crabp-expressing cells among the type-ii cholinergic amacrine cells in vivo. in addition to a decrease in the percentage of type-ii cholinergic amacrine cells, we found that the density of chat-positive cells was decreased by treatment with both a muscarinic achr antagonist and agonist. the number of chat+ cells per μm in the inl and in the gcl was significantly decreased by intraocular injections of atropine. similarly, treatment with the muscarinic agonist carbachol decreased the density of cholinergic amacrine cells in the inl and gcl. decreases in the density of cholinergic cells were not caused by increased apoptosis; we failed to find increased numbers of cleaved caspase3-positive cells in retinas treated with atropine or carbachol. additionally, the muscarinic ligands did not have any obvious effect on non-cholinergic neurons; ganglion cells labeled for brn3a and horizontal, bipolar, amacrine and ganglion cells labeled for calretinin displayed normal distributions when treated with atropine or carbachol. in retinas treated with atropine or carbachol, we found that the regularly patterned mosaic of cholinergic amacrine cells was disrupted. by visual inspection, the type-i and type-ii cells in retinas treated with atropine or carbachol appeared irregularly spaced compared to saline-treated retinas. to quantify whether the mosaic patterning of the cholinergic cells was disrupted by the muscarinic ligands, we performed a nearest neighbor analysis. nearest neighbor regularity indices with values above are considered to be regular and values below represent a random distribution. in saline-treated retinas the regularity index of the cholinergic amacrine cells was high, with a value of ±. the regularity index of the cholinergic cells was significantly decreased in retinas treated with atropine or carbachol compared to the regularity index of the cholinergic cells in saline-treated retinas. these findings suggest that perturbation of muscarinic signaling disrupted the mosaic patterning of the cholinergic amacrine cells. discussion the genes that are expressed by differentiating cholinergic amacrine cells are distinctly different from those that are expressed by other types of amacrine cells. unlike other types of amacrine cells, the cholinergic cells express islet with the onset of differentiation and into the mature retina, consistent with reports in the embryonic rat retina. as the cholinergic amacrine cells differentiate they express proteins common to other types of amacrine cells such as pax crabp and p27kip however, the relative levels of immunolabeling for these proteins within the cholinergic cells are either decreased or increased compared to levels seen in other types of differentiating amacrine cells. it is likely that islet and pax play important roles in the development and maintenance of phenotype of the type-i and type-ii cholinergic amacrine cells. the significance of elevated expression of p27kip and crabp in the cholinergic cells remains unknown. the type-i and type-ii cholinergic amacrine cells are equally abundant, morphologically identical, and are arranged with near-mirror symmetry across a horizontal plane through the ipl. in addition to using acetylcholine, type-i and type-ii cholinergic amacrine cells are known to utilize gaba as a neurotransmitter and begin to accumulate gaba soon after they begin to differentiate. furthermore, type-i and type-ii cholinergic amacrine cells in the chick retina have been shown to originate from a common pool of migrating, post-mitotic undifferentiated neurons. these findings have lead to the assumption that these cells are phenotypically identical and differ only by the position of their somata. however, we provide evidence that these two cell types differ by more than the location of their cell bodies. we found that during differentiation, type-ii cells transiently expressed high levels of crabp and neurofilament, whereas the type-i cells do not. further, in the postnatal chick retina, type-ii cells may express machr whereas type-i cells do not, and these cells have different sensitivities to excitotoxins, suggesting that different types of glutamate receptors are expressed by these cells, and/or that they have different susceptibilities to large fluctuations in ion concentrations. embryonic expression of machr machr and machr appear in non-overlapping laminae of the developing ipl at e, suggesting that these receptors may be expressed by different types of amacrine cells. the machr may not exist in the chick and the retinal expression of machr remains uncertain, but has been detected in the sclera of guinea pigs and human. differential expression of receptor isoforms is a possible mechanism to explain the differing effects of muscarinic ligands on the type-i and type-ii cholinergic cells. the cholinergic cells may be among the first types of amacrine cell to differentiate, with the onset of chat and islet expression occurring while these cells are interspersed among the developing ganglion cells at e by comparison, amacrine cells that utilize different neurotransmitters such as dopamine, glucagon, enkephalin or vasoactive intestinal peptide differentiate 6– days after the cholinergic cells. as development proceeds, the islet1/chat-positive cells migrate away from the ganglion cells into the presumptive ipl where they begin to differentiate. when the type-i and type-ii cholinergic cells become segregated into different laminae, the type-ii cells begin to express elevated levels of crabp and neurofilament. the close proximity of type-ii cells to developing ganglion cells may expose these cells to environmental cues that influence the differentiation of ganglion cells. accordingly, this microenvironment may promote the expression of genes common to both ganglion and type-ii cells. secreted signals produced by ganglion cells that influence the type-ii cholinergic cells could include shh and bmp2/. however, we failed to find evidence that shh influences the differentiation of type-ii cholinergic cells. we found that the 1: ratio of type-i to type-ii cholinergic cells from the intact retina was disrupted when the cells were dissociated and grown in culture; type-i cells were twice as abundant as the type-ii cells in vitro. in our in vitro paradigm, it is likely that crabp expression is down-regulated by the type-ii cells making them indistinguishable from the type-i cells. the disruption of the in vivo microenvironment did not favor the differentiation of type-ii cholinergic cells – the co-expression of chat and crabp. these findings suggest that the factors promoting the type-ii phenotype are diluted under culture conditions and that the phenotype of type-ii cells relies, in part, on the microenvironment provided by the intact retina. alternatively, the survival of type-ii cholinergic amacrine cells may be selectively compromised by acute dissociation and culture conditions. it is unlikely that changes in the relative abundance of type-i to type-ii cholinergic cells resulted from cell fate decisions made during the terminal mitosis. we did not find any cholinergic cells in our in vitro and in vivo paradigms that were labeled for brdu, indicating that none of the cholinergic cells were generated after the cultures were established or after compounds were delivered to the developing eye. similar to the results of our in vitro studies, we found that the in vivo development of the cholinergic amacrine cells was influenced by muscarinic ligands. in saline-treated retinas, the vast majority of cholinergic cells in the gcl express crabp. however, treatment with muscarinic ligands resulted in increased numbers of type-ii cells that failed to express crabp. these findings suggest that appropriate levels of muscarinic signaling are required for the maintenance of crabp expression in the type-ii cells; perturbation of optimal levels of muscarinic signaling between cholinergic cells by agonist or antagonist disrupted the phenotype of these cells. it remains uncertain whether the effects of the muscarinic ligands were mediated by direct or indirect actions on the type-ii cells. the patterning of the cholinergic amacrine cells is likely coordinated by several distinct signaling pathways in addition to the muscarinic pathway. for example, extra-cellular atp and the p2x receptor influence the patterning and survival of the cholinergic cells in the rodent retina. in addition, zhang and colleagues have shown that maintenance of array of type-ii amacrine cells in the mouse gcl is dependent upon visual activity; after days of rearing pups in the dark, chat-positive cells are undetectable in the gcl, but remain present in the inl. inhibiting machrs with atropine or activating machrs with carbachol disrupted the regular spacing between the cholinergic amacrine cells. we propose that the proper patterning of the cholinergic cells requires optimal levels of muscarinic signaling similar to the shh concentration-dependent differentiation of spinal cord interneurons. in principle, release and diffusion of ach from a cholinergic cell should result in a concentration gradient that decreases with increasing distance from the source. regular spacing between cells can occur if like-cells are repelled from each other; and for the cholinergic amacrine cells this may involve migrating laterally away from neighboring, concentrated sources of ach. our data are consistent with the hypothesis that the regular spacing of the cholinergic cells requires an optimal level of muscarinic signaling, and if these cells encounter gains or losses in signaling the mosaic patterning is disrupted. it remains uncertain what effect perturbations in muscarinic signaling have on the spontaneous calcium waves, spread through the cholinergic amacrine cells, that are necessary for the establishment of early retinal circuitry in the ipl. the mosaic pattern of the cholinergic cells may, in part, be required to establish normal waves of activity. however, this activity was shown to be regulated by nicotinic achr in the embryonic retina. it is possible that the application of muscarinic compounds generally perturbed retinal development and thereby disrupted the patterning of the cholinergic cells. however, we found that muscarinic compounds did not affect non-cholinergic cells expressing calretinin, islet or brn3a-positive ganglion cells, and we failed to observe an increase in immunolabeling for cleaved-caspase taken together, these findings indicate muscarinic ligands do not overtly affect non-cholinergic inner retinal neurons. the type-i and type-ii cells are not likely to influence each other through cholinergic signaling across the developing ipl. for example, the mosaic pattern of type-ii cholinergic amacrine cells in the gcl has no relation to the pattern of type-i cells in the inl. additionally, acetylcholine esterase is first detected in the chick retina between e and e in the middle of the ipl when the differentiating cholinergic cells are segregating between the gcl and inl. ache that is distributed into discrete laminae in the ipl should prevent any vertical diffusion of ach through the retina. for example, ach released from the type-ii cells is not likely to act on type-i cells because ache in the ipl is stratified between the dendrites of these cells and should degrade the ach that diffuses distally through the ipl. however, given the dense intermingling of the dendrites of the cholinergic cells within narrow strata of the ipl, it seems likely that ach released from the dendrites of one cholinergic cell could act on a neighboring cell with overlapping, co-stratified dendritic arbors. it is possible that decreases in the density of cholinergic cells resulted from increases in cell death. however, we failed to detect increased numbers of cleaved-caspase positive cells in retinas treated with carbachol or atropine. we cannot exclude the possibility that the death of cholinergic cells occurred in a caspase 3-independent manner resulting in reduced numbers of cholinergic cells and perhaps disruption of the mosaic patterning. another possibility is that decreases in the abundance of chat/crabp-positive cells in the ipl resulted from the down-regulation of chat and crabp. consistent with this hypothesis, chat may be transiently expressed by some cells in the gcl similar to the developing turtle retina, indicating that chat expression may be reversible in some types of retinal cells. the large diversity of amacrine cell types that are distributed in mosaic patterns with regular spacing implies that autologous signals are required to coordinate the patterning of unique cell types. the notion of homotypic interactions underlying mosaic patterning has been proposed for the cholinergic amacrine cells in the retina. furthermore, rossi and colleagues demonstrated that the establishment of regular spacing between horizontal cells in the rodent retina occurs independent of pre-synaptic input from photoreceptors, suggesting that patterning is mediated via homotypic interactions among the horizontal cells. it is possible that homotypic interactions are responsible for the regular spacing of the different types of horizontal, amacrine and ganglion cells. consistent with this hypothesis, cones, horizontal cells, ganglion cells, and cholinergic amacrine cells migrate laterally away from their radial column of clonally derived cells, whereas bipolar cells and müller glia do not. to establish a highly ordered mosaic pattern, cells within the retina must have the ability to migrate laterally away from columns of clonally derived cells unless the order of the array is determined by the progenitors. a likely mechanism underlying the spatial patterning between like-cells is the use of peptide or amino acid-derived transmitters to provide homotypic signals between cells. for example, dopamine, glucagon, neuropeptide y, substance p, vasoactive intestinal polypeptide or enkephalin, neurotransmitters that are used by distinct types of amacrine cells, are used as autologous signals to coordinate cell type-specific patterning. consistent with this hypothesis, our data indicate that the disruption of muscarinic ach signaling perturbs the numbers, spacing and phenotype of cholinergic amacrine cells in the developing retina.  we conclude that, during development, type-ii cholinergic amacrine cells transiently express crabp and neurofilament, while type-i cells do not. we propose that the transient expression of crabp and neurofilament is regulated by local microenvironment. additionally, we find that interfering with muscarinic signaling decreases the relative number of type-ii cells and mosaic patterning of the cholinergic amacrine cells. we propose that the phenotypic differences between developing type-i and type-ii cholinergic amacrine cells are elicited, in part, by paracrine cholinergic signaling; the local microenvironment of the in tact retina allows precise muscarinic signaling to establish proper cellular phenotypes and organization of the type-ii cells. it remains uncertain whether muscarinic receptors are differentially expressed by the type-i and type-ii cells. 
we found that as chat-immunoreactive cells differentiate they expressed the homeodomain transcription factors pax and islet and the cell-cycle inhibitor p27kip as differentiation proceeds, type-ii cholinergic cells, displaced to the ganglion cell layer, transiently expressed high levels of cellular retinoic acid binding protein and neurofilament, while type-i cells in the inner nuclear layer did not. although there is a 1: ratio of type-i to type-ii cells in vivo, in dissociated cell cultures the type-i cells out-numbered the type-ii cells by 2: the relative abundance of type-i to type-ii cells was not influenced by sonic hedgehog, but was affected by compounds that act at muscarinic acetylcholine receptors. many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase. in addition, the abundance and mosaic patterning of type-ii cholinergic amacrine cells is disrupted by interfering with muscarinic signaling. we conclude that: during development type-i and type-ii cholinergic amacrine cells are not homotypic, the phenotypic differences between these subtypes of cells is controlled by the local microenvironment, and appropriate levels of muscarinic signaling between the cholinergic amacrine cells are required for proper mosaic patterning.
 many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase.  we found that as chat-immunoreactive cells differentiate they expressed the homeodomain transcription factors pax and islet and the cell-cycle inhibitor p27kip as differentiation proceeds, type-ii cholinergic cells, displaced to the ganglion cell layer, transiently expressed high levels of cellular retinoic acid binding protein and neurofilament, while type-i cells in the inner nuclear layer did not. although there is a 1: ratio of type-i to type-ii cells in vivo, in dissociated cell cultures the type-i cells out-numbered the type-ii cells by 2: the relative abundance of type-i to type-ii cells was not influenced by sonic hedgehog, but was affected by compounds that act at muscarinic acetylcholine receptors. in addition, the abundance and mosaic patterning of type-ii cholinergic amacrine cells is disrupted by interfering with muscarinic signaling.  we conclude that: during development type-i and type-ii cholinergic amacrine cells are not homotypic, the phenotypic differences between these subtypes of cells is controlled by the local microenvironment, and appropriate levels of muscarinic signaling between the cholinergic amacrine cells are required for proper mosaic patterning. 