BACKGROUND
th controls normal development on a global scale, as evidenced by its central role in processes as diverse as amphibian metamorphosis and human cretinism  <cit> .
while these roles have been known for some time, the mechanism by which the hormone coordinates such events is still under active investigation.
considerable attention has focused on the hormone's regulation of the trs, which are members of the thyroid/steroid nuclear receptor superfamily that bind dna and activate or repress transcription  <cit> .
trs recruit activators such as the smart complex, or repressors such as the hdacs, and alter the transcription of a variety of genes  <cit> .
equally important to the trs are the deiodinases, which are enzymes that modify the prohormone secreted by the thyroid gland  <cit> .
deiodinases are expressed at different times by different tissues, and are thought to control the amount of active hormone available locally in a tissue  <cit> .
some deiodinases, such as deiodinase  <dig> , have 5'- outer ring  catalytic activity, clipping the 5'-iodine off of the t <dig> prohormone to create t <dig>  t <dig> is the preferred ligand for trs.
other deiodinases, such as deiodinase  <dig> , have 5- inner ring  catalytic activity, removing the 5-iodine from either the t <dig> prohormone or the t <dig> active hormone, and thereby inactivating them.
the inactive products do not bind with high affinity to the hormone binding site of the trs.
deiodinase  <dig>  a deiodinase not traditionally found in the nervous system, can have both 5'd and 5d activities.
as a first step in understanding how th affects tissue development, the different trs and deiodinases in a tissue must be identified.
previous work has shown that th components are expressed at critical times during a particular tissue's development.
for example, in the development of the murine inner ear, th activating enzyme is necessary for proper inner ear development.
its expression peaks within a 2-day window, after which expression sharply declines  <cit> .
similarly, in the rodent heart, tr controls cardiac gene expression during the critical switch from embryonic to neonatal life  <cit> .
another tissue in which th action has been studied is the retina.
the retina has numerous advantages as a model tissue for developmental studies.
it has a well-studied sequence of neural cell production, during which  <dig> different cell types are produced in an overlapping order  <cit> .
early reports identified three thyroid receptors, tra, trb <dig>  and trb <dig>  in the developing chick retina using ish  <cit> .
trb <dig> was found in the photoreceptor layer, tra was observed in the progenitor layer, and trb <dig> showed weak signal during later stages in the inner nuclear layer.
subsequent to this study, other investigators have used a hypothyroid rodent model to show that th is important for retinal morphology  <cit> , and in the amphibian retina, th promotes proper morphology and proliferation  <cit> .
in salmonid fish, th appears to regulate the death and reappearance of uv cones during smoltification, a process similar to amphibian metamorphosis  <cit> .
in addition to these studies, th has been investigated with respect to its role in rodent photoreceptor development.
early in vitro studies implicated th as a modulator of photoreceptor genes  <cit> , and characterization of a trb <dig> knock-out mouse showed that trb <dig> is indeed involved in proper cone photoreceptor differentiation  <cit> .
without trb <dig>  cone photoreceptors expressed the short wavelength opsin  gene earlier than normal, and failed to express medium wavelength opsin .
it is unclear whether this represents a fate change, from one cone cell type to another, or misregulation of the opsin genes.
we were intrigued by the number of processes controlled by th, and looked to the chick retina for further insight into the processes regulated by th.
the chick retina is much larger than the rodent retina, thereby revealing spatiotemporal gradients of development more readily.
we identified the expression patterns of the trs as well as the deiodinases that activate and inactivate the hormone locally.
the results show that th components are expressed dynamically during development, in three sequential waves that spread across the retina in a central to peripheral pattern.
the first wave marks the transition between cells that produce only mitotic daughter cells to cells that can produce neurons.
the second wave marks a transition for some types of immature postmitotic cells to their more mature location and differentiation state.
the third wave is correlated with the production of müller glial cells.
furthermore, using dissociated cell ish , we identified the cell types that express the th components, and the kinetics of expression relative to the cell cycle.
together, these data suggest that th plays multiple roles in chick retinal development, including a role within progenitor cells and photoreceptor cells.
RESULTS
the expression of th components in three distinct time windows
using section ish, three discrete developmental stages were characterized for the expression of various th components .
these timepoints were separated by approximately  <dig> days, as developmental events occur first in the center, and then in the periphery, with the interval increasing as the retina grows larger  <cit> .
the first timepoint during which the expression of the three components was examined was hh stage  <dig>  during this stage, tra was observed throughout the retina, although at low levels .
trb was observed in cells that were located throughout the outer neuroblastic layer  of the retina, with more intense staining observed on the scleral side, near the rpe, where the photoreceptors will eventually reside .
the trb in situ probe used in this study encompassed the common 3' end of both trb <dig> and trb <dig> and therefore did not distinguish between the two isoforms  <cit> .
however, other experiments with probes made from the small n-terminal region specific to trb <dig> showed similar, albeit lighter, staining  <cit> .
neither dio <dig>  the deiodinase that activates th in the chick nervous system nor dio <dig>  the deiodinase that inactivates thyroid hormone, was detected in the retina at hh stage  <dig> .
during the second time period, hh stage  <dig>  the expression of all four th components was observed .
tra was broadly expressed throughout the developing retina .
unlike tra, the expression of trb was restricted to the future photoreceptor layer, in a narrow band of cells abutting the rpe .
at this second developmental stage, the punctate trb staining observed in cells distributed throughout the onbl at hh stage  <dig> had disappeared .
dio <dig> was seen in cells in the center of the onbl, where retinal progenitor cells reside  <cit>  .
finally, dio <dig> expression appeared in the retina, in a layer of cells one or two cell bodies below the rpe .
this cell layer is most closely associated with photoreceptors.
the third timepoint, hh stage  <dig>  showed lighter expression of the th components, relative to the levels during the second time period .
again, tra was detected throughout the retina, in a similar expression pattern as at earlier time periods , although at lower levels.
trb expression was also reduced, but still seen tightly abutting the rpe in a photoreceptor pattern .
dio <dig> expression was observed in the center of the onbl, where the few remaining progenitor cells at these late stages would be located  <cit> .
finally, just as in the first time period, dio <dig> was not detectable by ish in the retina at this later stage .
the first time period correlates with early neurogenesis
to better characterize the dynamic expression patterns of the th components, the expression patterns were further examined at several early hh stages.
during hh stages 20– <dig>  trb expression appeared as an enlarging wave, starting from the center and expanding to the periphery, sometimes overlapping the expression domain of dio <dig> .
at stage  <dig>  dio <dig> expression was observed centrally, overlapping the domain containing a small number of trb-positive cells .
at stage  <dig>  dio <dig> and trb expression were both more robust, having now begun to spread more peripherally from the central portion of the retina .
the expression domain of both genes overlapped, with dio <dig> expression sometimes being observed slightly ahead  of the trb domain .
by stage  <dig>  dio <dig> expression had waned significantly, with only faint expression observed in the periphery .
in contrast, at stage  <dig>  trb expression was robust both centrally and peripherally, and was not yet resolved to the photoreceptor layer, as was observed at later stages.
since the center-to-periphery trb wave resembled that seen with the neurogenic genes otx <dig>  <cit> , neurod  <cit> , and cath <dig>  <cit> , serial section ish was used to see if the different expression patterns were correlated .
otx <dig> and neurod were expressed in cells scattered throughout the onbl, with a higher concentration of labeled cells located near the scleral side .
both genes are thought to mark early photoreceptor cells, as well as other cell types  <cit> .
cath <dig>  on the other hand, was expressed in a bilayered pattern, present in cells in the ganglion cell layer as well as in cells that have not been defined, but which overlap the developing photoreceptor layer   <cit> .
the expression pattern of neurod and cath <dig> appeared to extend slightly more toward the peripheral retina than trb and otx <dig>  although all  <dig> genes were similar in the extent of their central-peripheral expression .
these data suggest that trb expression is correlated with a more general wave of neurogenesis spreading across the retina at early stages.
trb expression also was compared to the expression of brn3b, crx, and shh, three genes marking the early differentiation of neurons, which also occurs in a central-to-peripheral pattern.
in mouse, brn3b marks early ganglion cells  <cit>  and crx marks early photoreceptor cells  <cit> .
shh is expressed by ganglion cells in the chick  <cit> , and possibly in other cell types as well.
in zebrafish, ganglion cells and a subset of amacrine cells express shh  <cit> , and in mouse, there is a similar pattern of expression  <cit> , and there is a history of expression of shh in a subset of photoreceptors, likely cone photoreceptors  <cit> .
at stage  <dig>  the location of cells expressing brn3b, crx, and shh expression was similar with respect to the central/peripheral gradient, within the expression domain located more centrally then trb .
the second time period corresponds to a wave of maturation of some cell types
in a second time period, hh stages 29– <dig>  a second wave of th components involving dio <dig> and dio <dig> swept across the retina .
using flat mount ish, dio <dig> expression was observed at stage  <dig> as heavy ventral staining, some dorsal staining, and an absence of expression in a spot and stripe centered on the dorsoventral border .
over the next few stages, dio <dig> expression was confined in narrower stripes toward the periphery .
in a nearly complementary pattern, dio <dig> started at stage  <dig> as a spot and stripe centered on the dorsoventral border .
over the next few stages, dio <dig> expression expanded dorsally and ventrally to occupy the entire retina .
ish performed on serial sections of the peripheral retina at hh stage  <dig> showed that the ebbing dio <dig> and expanding dio <dig> expression patterns were nearly complementary .
during hh stages 20– <dig>  there was a correlation of expression of th components and genes involved in neurogenesis and differentiation .
it was of interest to determine whether the second wave of expression of th components also was correlated with other genes.
the first candidate examined was lim <dig>  which has been shown to mark horizontal cells as they move from the vitreal side to their final scleral position during development  <cit> .
on serial sections, the interface between dio <dig> and dio <dig> occurred in the same place as lim <dig> cell migration .
the dio2/dio <dig> wave also paralleled changes the photoreceptor marker, visinin .
at the dio2/dio <dig> wavefront/transition, visinin expression became more organized, transitioning from a weak diffuse pattern to a more intense band in the maturing photoreceptor layer .
hence, the transition in the expression of dio <dig> and dio <dig> occurred along with the transition of a couple of genes to their mature expression patterns.
the third time period corresponds to the disappearance of progenitor cells
in a later time period, hh stages 35– <dig>  a third wave involving dio <dig> swept across the retina .
during this time period, dio <dig> expression ceased, first from the center  and then in the periphery .
similar to the previous  <dig> waves of expression of th components, this period showed a correlation with the expression of genes involved in retinal development.
on serial sections, dio <dig> was expressed throughout the area occupied by progenitor cells as defined by the expression of notch .
in more central areas, where dio <dig> expression was absent, notch appeared in the layer that seemed to overlap with the expression of a glucose transporter  , in a pattern consistent with müller glia cell expression  <cit> .
birthdating studies have shown that müller glia cells are among the last born of retinal cell types  <cit> , and thus this transition appears with the end of proliferation.
trb is expressed in early photoreceptor cells
previous data concerning trb expression in the murine retina  <cit> , along with the location of the ish signal in the scleral region of the chick retina , suggested that trb is expressed by photoreceptor cells.
however, the scleral region of the retina also harbors cells in m phase, as well as cells that are newly postmitotic, but not photoreceptor cells.
in order to define the cell type expressing trb, dissociated cell ish  was carried out.
probes for cell type-specific markers along with probes for th components were used.
in addition, to investigate whether th components were expressed in mitotic cells, newly postmitotic cells, or postmitotic cells that had not recently exited, and were thus further along in their differentiation, embryos were labeled with -thymidine in ovo.
dish was used to detect expression of one or two genes in cells labeled with -thymidine .
cells were labeled in ovo with -thymidine for  <dig> hour, and tissue was harvested and dissociated at the indicated times.
double dish for the indicated genes and autoradiography for  were carried out on the dissociated cells.
cells were quantified for labeling by a single gene probe, both gene probes, and labeling with .
cells were labeled in ovo with -thymidine for  <dig> hour, and tissue was harvested and dissociated at the indicated times.
double dish for the indicated genes and autoradiography for  were carried out on the dissociated cells.
cells were quantified for labeling by a single gene probe, both gene probes, and labeling with .
* % of cells that express gene  <dig> that also express gene 2
a one hour pulse with -thymidine, followed by an immediate harvest, would be expected to label primarily cells in s phase, along with cells in the early part of g <dig>  <cit> .
dish for trb applied to cells labeled with -thymidine for one hour showed that most of the trb expressing cells were not co-labeled for -thymidine and thus were not in s phase at stages  <dig>   <dig>  or  <dig> .
in addition, the majority of cells in s phase or early g <dig> at these stages did not express trb .
however, since a small fraction of cells,  <dig>  ±  <dig> %, labeled with -thymidine after one hour expressed trb  it is likely that trb turns on in some g <dig> cells.
these data are in keeping with data from the ish on tissue sections, in which the majority of trb+ cells were located in the area of g <dig> cells and photoreceptor cells .
cells were labeled in ovo with -thymidine for  <dig> hour, and tissue was harvested and dissociated at the indicated times.
dish for the indicated genes and autoradiography for  were carried out on the dissociated cells.
examination of -thymidine+ cells at intervals after the initial addition of the label allows a determination of whether a gene is expressed in g <dig>  m, or g1/g <dig>  since injection of -thymidine in ovo results in continuous labeling with -thymidine  <cit> .
dish for trb was performed on cells from stage  <dig> or  <dig> embryos 1– <dig> hours after addition of -thymidine .
these data show a higher percentage of cells that express trb were labeled with -thymidine over time, resulting in  <dig>  ±  <dig> %  of trb+ cells also being labeled with -thymidine by  <dig> hours at stage  <dig>  and  <dig> %  at stage  <dig> by  <dig> hours.
since the cell cycle length is  <dig> hours at these stages  <cit> , the majority of trb+ cells are within one cell cycle length of labeling.
given the location of trb cells in the tissue section in the area of photoreceptor cells, the lack of s phase labeling for the majority of trb+ cells, the low numbers of trb+ cells in the g <dig> population , most trb+ cells are likely newly postmitotic, and are likely to be photoreceptors.
this was confirmed by double dish using photoreceptor markers .
as mentioned above, neurod and otx <dig> are transcription factors that are expressed by newborn photoreceptors  <cit> , and by other retinal cell types as well.
the expression of otx <dig> and neurod in the cell cycle was examined as described above, and was compared to the labeling of trb .
cells that expressed neurod or otx <dig> showed kinetics of labeling with -thymidine almost identical to the labeling kinetics of trb+ cells .
double dish at stage  <dig> for trb and neurod, or trb and otx <dig>  revealed that  <dig> % of all trb+ cells expressed neurod , and  <dig> % expressed otx <dig> .
the expression of otx <dig> and neurod was examined among the + cells labeled in a one hour exposure to -thymidine .
of the + cells at stage  <dig>   <dig> % were otx2+, and  <dig> % were neurod+, while only  <dig> % were trb+.
thus, among the + cells that expressed otx <dig>  or neurod, only approximately one out of  <dig> also expressed trb .
these data demonstrate that while the kinetics of onset of expression of otx <dig>  neurod, and trb are similar, many more cells, particularly at the later stages examined, expressed neurod and otx <dig> than trb.
co-expression of two other markers of photoreceptors, visinin and crx, along with trb was examined by double dish.
visinin is expressed exclusively by photoreceptors  <cit> , and  <dig> % of all trb+ cells expressed visinin at stage  <dig> .
the percentage of trb+ cells that expressed crx was  <dig> % at stage  <dig>  and  <dig> % at stage  <dig> .
trb and crx co-expression was compared further.
crx can be considered a later photoreceptor marker compared to neurod and otx <dig>  since, at stage  <dig>  < <dig> % of  + cells were crx+; at stage  <dig>  < <dig> % of  + cells were crx+; and, at stage  <dig>  4– <dig> hours after addition of -thymidine, only  <dig> – <dig> % of the + cells were crx+ .
trb and crx expression on section ish displays an inverse relationship: before stage  <dig>  trb was observed in a greater number of cells than crx, whereas after stage  <dig>  the opposite was observed .
this pattern was confirmed using dish.
at stage  <dig>  trb was expressed in approximately 9% of cells, and crx was observed in 7% of all cells, whereas at stage  <dig> trb was mildly reduced to approximately 7%, while crx increased to 14% of all cells.
the transient nature of the co-expression of trb and crx in differentiating photoreceptors was confirmed by double dish with trb and crx.
at stage  <dig>  26% of trb+ cells expressed crx, and 94% of crx+ cells expressed trb .
at stage  <dig>   <dig> % of trb cells expressed crx, whereas  <dig> % of crx cells expressed trb .
at stage  <dig>   <dig>  ±  <dig> % of trb cells expressed crx, and  <dig>  ±  <dig> % of crx cells expressed trb .
this pattern is consistent with an earlier onset of trb than of crx in newly postmitotic photoreceptor cells, followed by a period of co-expression of trb and crx, and then a reduction in expression of trb in photoreceptors, which retain expression of crx.
it also suggests that there is a greater interval between the onset of trb and the onset of crx at the later stages.
the stability of expression of crx among photoreceptors is reflected in the high co-expression of crx and visinin  at stage  <dig> .
dio <dig> is expressed in a subset of photoreceptor cells
dio <dig> expression also was investigated using dish.
dio <dig> was not detected at stage  <dig>  but later was expressed in a small but increasing percentage of all cells: stage  <dig>  approximately  <dig> %, stage  <dig>  approximately  <dig> %, and stage  <dig>  approximately 3% .
double dish showed that at stage  <dig>  almost all dio2+ cells expressed neurod  and otx <dig> , while  <dig> % expressed crx and  <dig> % expressed trb .
at stage  <dig>  85–100% of dio2+ cells expressed cath <dig>  but they likely turn it off as only 17% expressed cath <dig> by stage  <dig>  these data suggest that dio <dig> is present in photoreceptor cells, but in a subset that appears at a later time in development.
with respect to its kinetics in the cell cycle, dio <dig> was expressed later than cath <dig>  trb, neurod, and otx <dig> since dio <dig> expression was almost never observed in s/g <dig> cells .
combined with the section ish results, dio <dig> appeared in a subset of photoreceptors as they transitioned from newly postmitotic cells to later stages of differentiation.
dio <dig> is expressed in mitotic cells
dio <dig>  whose expression appeared to be in progenitor cells on section ish, was also studied using dish and -thymidine incorporation.
at stage  <dig>   <dig> % of dio3+ cells were + after a one hour pulse , and by  <dig> hours of -thymidine labeling, all dio3+ cells were labeled with  .
this is in contrast to the plateau of -thymidine labeling for populations that include postmitotic cells, such as neurod  and trb .
dio <dig> was not present in cells that expressed crx , or dio <dig> , and only very few cells that expressed dio <dig> also expressed trb , at stage  <dig>  however, many cells that expressed dio <dig> also expressed otx <dig>  and neurod  at stage  <dig>  of these dio3+otx2+ or dio3+neurod+ cells,  <dig>  ±  <dig> % and  <dig> ±  <dig> % respectively of them were also + after a  <dig> hour pulse.
these data indicate that cells that co-express dio <dig> and otx <dig>  and/or neurod, are in s phase and/or early g <dig>
discussion
in this report, we show that th components are expressed in three sequential waves that spread across the retina, and that these waves are correlated with expression changes in a number of developmentally important genes.
furthermore, we identify which cell types express which th components, and determine their expression kinetics with respect to the cell cycle.
waves are a common theme in retinal development
in a diverse array of organisms, retinal development occurs in waves.
the best example of this is in drosophila melanogaster, in which the eye differentiates in a wave defined by the morphogenetic furrow  <cit> .
cells ahead of the furrow express genes involved in cell division and the undifferentiated state, whereas cells behind the furrow express cell differentiation markers as well as genes signaling terminal cell divisions.
the phenomenon of genes expressed in differentiation waves also extends to vertebrate eye development.
in zebrafish and xenopus, a program of cell differentiation spreads across the early retina, from the center to the periphery, as exemplified by the expression of shh  <cit> .
similarly, in the chick retina, the expression of neurogenesis markers has been observed to spread in an orderly fashion from the center to the periphery  <cit> .
in drosophila, there is a relationship between a nuclear hormone receptor, the ecdysone receptor, and hedgehog  <cit> .
it appears that the relief of repression from ecdysone receptor occurs just anterior to the morphogenetic furrow, while hedgehog initiates expression within the mf.
as trs are functionally similar to the ecdysone receptor  <cit> , and trb expression in the chick retina is spatially and temporally similar to that of shh, there is a possibility that trb and shh may be coordinated in a manner similar to that in the drosophila retina.
beyond this potential relationship revealed by the spatio-temporal gradient of expression, other developmental regulators appear in central-peripheral waves in the chick retina, including cath <dig>  otx <dig>  neurod, dio <dig>  dio <dig>  ngn <dig> and markers of various differentiated cell types.
the mechanism regulating these patterns is currently unknown.
cell types expressing th components
the expression of trb with respect to cell cycle kinetics and markers of photoreceptor cells show that this receptor is expressed in mitotic cells about to produce a postmitotic cell that will become a photoreceptor .
it appears that such progenitor cells express otx <dig> and neurod at almost exactly the same time as trb.
that the expression is in a g <dig> phase progenitor cell is an intriguing observation in light of retinal lineage data from embryonic mouse  <cit> .
infections of embryonic mice in utero with retroviral vectors were carried out at equivalent developmental stages to chick stages 26– <dig> when cones and rods are being produced in both species  <cit> .
the majority of embryonic mouse retinal clones of > <dig> cell were not  <dig> cell clones, and this included the majority of clones that contained rods and cones.
this observation suggests that when a postmitotic cell is made, it is not from a symmetric division in which both daughter cells become postmitotic, or many  <dig> cell clones would have been observed.
if a similar result were to hold for the chick, the expression of trb in g <dig> cells would not be maintained in both daughters of that division.
presumably, the postmitotic daughter would retain expression, at least transiently, as it initiated its differentiation into a photoreceptor cell.
the other daughter would extinguish the expression of trb as it re-entered s phase, as very few s phase cells express trb.
the expression of otx <dig> and neurod is also likely maintained in the photoreceptor cell, as these genes appear to be expressed in the area of differentiating photoreceptor cells.
they may also continue to be expressed in some of the s phase daughter cells, as there is higher number of s phase cells that express otx <dig> and neurod, particularly at later stages.
it is interesting to note that dio <dig> is expressed in a subset of photoreceptor cells.
the dio <dig> subset can be defined in three ways.
it is highest in the ventral retina, and is missing in a central spot and horizontal stripe.
the only other similar expression pattern is that of rhodopsin, a gene marking rod photoreceptors  <cit> , which does not commence until stage  <dig>   <dig> days after the dio <dig> expression wanes.
a second way to define the subset of dio2+ cells is by the time at which dio <dig> appears in development, appearing later than the expression of otx <dig>  neurod, and trb.
rod photoreceptors also appear to be generated later than cone photoreceptors in most species  <cit> , although this has not been defined for the chick.
finally, the kinetics of expression of dio <dig> in the cell cycle provides a third way to define it.
dio <dig> appears after the earlier expression of otx <dig>  neurod, and trb cells in the subset of cells that express it.
from all of these observations, it is possible that dio <dig> is in rod photoreceptors.
confirmation of this hypothesis will await an early marker of rod photoreceptors in the chick as dio <dig> disappears before expression of definitive rod-specific markers, such as rhodopsin.
model of th signaling in retinal development
the first wave of expression of th components is characterized by mitotic cells transitioning from producing only mitotic daughters to a phase in which they begin to produce neurons.
tra appears to be the receptor present in all cells as they make this transition.
dio <dig> is present in progenitor cells at certain times, whereas trb is expressed in some g <dig> cells and in newly postmitotic cells that begin to differentiate into photoreceptor cells.
based on these observations, it is possible that dio <dig> keeps tra cells in the state where they only generate mitotic daughter cells.
when dio <dig> expression ceases, the tra cells can produce a postmitotic cell, and some of these daughter cells express trb.
whether dio <dig> promotes cell division and blocks differentiation remains an unanswered question.
studies in the xenopus retina during metamorphosis seem to challenge this notion, because in these experiments, dio <dig> blocked cell division and antagonism of dio <dig> promoted cell division  <cit> .
however, these studies measured brdu incorporation over a span of many days after manipulation of dio <dig>  the increased brdu incorporation seen after antagonizing dio <dig> could be due to several mechanisms, including an increase in cells undergoing their last cell division.
this interpretation would agree with dio <dig> promoting the progenitor cell state and a lack of dio <dig> promoting cell cycle exit.
the second wave of expression of th components is characterized by the maturation and migration of certain cell types to their final positions.
before the second wave, trb photoreceptors appear to be in an immature photoreceptor state.
during wave  <dig>  they progress in their differentiation, as shown by expression of crx.
locally activated th might influence trb cells during this period.
the second wave ends with dio <dig> sweeping across the retina, which might halt tra cells from producing postmitotic cells which express trb, and might instead change the types of cells produced, or the balance of mitotic and postmitotic daughters made by tra progenitor cells.
a model consistent with this expression pattern is that trb, in conjunction with th produced by dio <dig>  serves to block photoreceptor differentiation.
in this model, trb appears in the early photoreceptor just as it is being produced, significantly before the cell expresses crx.
eventually, trb and/or dio <dig> levels are reduced and the cell is allowed to continue differentiation and express crx.
in the ventral retina, where retinal dio <dig> is present, the delay may be longer, which might contribute to the development of rod photoreceptors in this area.
this repressive activity of trb in the presence of dio <dig> might be similar to trb activity in other parts of the nervous system, including the inhibitory activity of trb in the presence of t <dig> on transcription of the tshb gene  <cit> .
previous data from mice support a repression and activation role of trb in retinal photoreceptors.
loss of trb led to cones that appeared to differentiate early, as seen by early s-opsin expression.
interestingly, in the same mutant, m-opsin failed to be expressed.
recently, it was shown that mutation of trb <dig> to an allele that could not bind th exhibited both aspects of the phenotype, demonstrating that both repression of s-opsin and activation of m-opsin requires th binding  <cit> .
in addition, th was found at higher levels in the dorsal than the ventral retina  <cit> , and dio <dig> was found to be expressed at higher levels in the dorsal than in the ventral retina  <cit> .
thus, the coordination of th levels through dio <dig> activity, and the activity of the trb <dig>  controls at least one aspect of the dorsoventral gradient of both s and m-opsins.
neurod was also shown to play a role in regulation of s and m-opsins, at least in part via its regulation of trb <dig>  <cit> .
mice deficient in neurod showed the same s and m-opsin phenotypes as the trb <dig> mutant, along with a lack of maintenance of trb <dig> expression.
neurod was shown to bind directly to a regulatory element for trb <dig>  <cit> , but was not itself sufficient to activate trb <dig>  in keeping with the observation that many more cells express neurod than express trb <dig> .
it is interesting to note that in the chick, neurod expression is concomitant with trb <dig> expression in its onset, but at least in the mouse, the onset of expression of trb <dig> occurred in the neurod mutant, but was not maintained.
finally, the third wave of expression of th components is characterized by waning dio <dig> expression and the transition from progenitor cells to müller glial cells.
similar to the models described above, dio <dig> at this stage may keep tra cells in the progenitor state.
when dio <dig> expression ends, the tra cells may be free to exit cell cycle and begin differentiating.
however, at this late stage, cells may no longer be competent to generate photoreceptors.
instead, they may exit cell cycle and become notch+ müller glia cells.
this latter suggestion follows the observations of th signaling in optic nerve cell cultures where th has been demonstrated to promote the differentiation of oligodendrocyte cells  <cit> .
in this well studied system, th can promote the exit of oligodendrocyte precursor cells from the cell cycle and initiate differentiation.
this action appears to be through tra  <cit> .
lack of th can block this process, promoting the expression of cell cycle genes and keeping cells in the proliferative state.
CONCLUSIONS
in summary, th components are expressed in dynamic waves across the developing chick retina, and involve a number of cell types including progenitor cells and photoreceptors.
through the expression of deiodinases that activate and degrade thyroid hormone, in conjunction with cells expressing trs, a complex interplay between the different th components likely results.
an attractive hypothesis is that the hormone controls the timing of cell cycle exit for both photoreceptors and müller glia cells, as well as the timing of differentiation for photoreceptors.
future functional studies will shed more light on these proposed activities.

 th controls normal development on a global scale, as evidenced by its central role in processes as diverse as amphibian metamorphosis and human cretinism. while these roles have been known for some time, the mechanism by which the hormone coordinates such events is still under active investigation. considerable attention has focused on the hormone's regulation of the trs, which are members of the thyroid/steroid nuclear receptor superfamily that bind dna and activate or repress transcription. trs recruit activators such as the smart complex, or repressors such as the hdacs, and alter the transcription of a variety of genes. equally important to the trs are the deiodinases, which are enzymes that modify the prohormone secreted by the thyroid gland. deiodinases are expressed at different times by different tissues, and are thought to control the amount of active hormone available locally in a tissue. some deiodinases, such as deiodinase, have 5'- outer ring catalytic activity, clipping the 5'-iodine off of the t prohormone to create t t is the preferred ligand for trs. other deiodinases, such as deiodinase, have 5- inner ring catalytic activity, removing the 5-iodine from either the t prohormone or the t active hormone, and thereby inactivating them. the inactive products do not bind with high affinity to the hormone binding site of the trs. deiodinase a deiodinase not traditionally found in the nervous system, can have both 5'd and 5d activities. as a first step in understanding how th affects tissue development, the different trs and deiodinases in a tissue must be identified. previous work has shown that th components are expressed at critical times during a particular tissue's development. for example, in the development of the murine inner ear, th activating enzyme is necessary for proper inner ear development. its expression peaks within a 2-day window, after which expression sharply declines. similarly, in the rodent heart, tr controls cardiac gene expression during the critical switch from embryonic to neonatal life. another tissue in which th action has been studied is the retina. the retina has numerous advantages as a model tissue for developmental studies. it has a well-studied sequence of neural cell production, during which different cell types are produced in an overlapping order. early reports identified three thyroid receptors, tra, trb and trb in the developing chick retina using ish. trb was found in the photoreceptor layer, tra was observed in the progenitor layer, and trb showed weak signal during later stages in the inner nuclear layer. subsequent to this study, other investigators have used a hypothyroid rodent model to show that th is important for retinal morphology, and in the amphibian retina, th promotes proper morphology and proliferation. in salmonid fish, th appears to regulate the death and reappearance of uv cones during smoltification, a process similar to amphibian metamorphosis. in addition to these studies, th has been investigated with respect to its role in rodent photoreceptor development. early in vitro studies implicated th as a modulator of photoreceptor genes, and characterization of a trb knock-out mouse showed that trb is indeed involved in proper cone photoreceptor differentiation. without trb cone photoreceptors expressed the short wavelength opsin gene earlier than normal, and failed to express medium wavelength opsin. it is unclear whether this represents a fate change, from one cone cell type to another, or misregulation of the opsin genes. we were intrigued by the number of processes controlled by th, and looked to the chick retina for further insight into the processes regulated by th. the chick retina is much larger than the rodent retina, thereby revealing spatiotemporal gradients of development more readily. we identified the expression patterns of the trs as well as the deiodinases that activate and inactivate the hormone locally. the results show that th components are expressed dynamically during development, in three sequential waves that spread across the retina in a central to peripheral pattern. the first wave marks the transition between cells that produce only mitotic daughter cells to cells that can produce neurons. the second wave marks a transition for some types of immature postmitotic cells to their more mature location and differentiation state. the third wave is correlated with the production of müller glial cells. furthermore, using dissociated cell ish, we identified the cell types that express the th components, and the kinetics of expression relative to the cell cycle. together, these data suggest that th plays multiple roles in chick retinal development, including a role within progenitor cells and photoreceptor cells.  the expression of th components in three distinct time windows using section ish, three discrete developmental stages were characterized for the expression of various th components. these timepoints were separated by approximately days, as developmental events occur first in the center, and then in the periphery, with the interval increasing as the retina grows larger. the first timepoint during which the expression of the three components was examined was hh stage during this stage, tra was observed throughout the retina, although at low levels. trb was observed in cells that were located throughout the outer neuroblastic layer of the retina, with more intense staining observed on the scleral side, near the rpe, where the photoreceptors will eventually reside. the trb in situ probe used in this study encompassed the common 3' end of both trb and trb and therefore did not distinguish between the two isoforms. however, other experiments with probes made from the small n-terminal region specific to trb showed similar, albeit lighter, staining. neither dio the deiodinase that activates th in the chick nervous system nor dio the deiodinase that inactivates thyroid hormone, was detected in the retina at hh stage. during the second time period, hh stage the expression of all four th components was observed. tra was broadly expressed throughout the developing retina. unlike tra, the expression of trb was restricted to the future photoreceptor layer, in a narrow band of cells abutting the rpe. at this second developmental stage, the punctate trb staining observed in cells distributed throughout the onbl at hh stage had disappeared. dio was seen in cells in the center of the onbl, where retinal progenitor cells reside. finally, dio expression appeared in the retina, in a layer of cells one or two cell bodies below the rpe. this cell layer is most closely associated with photoreceptors. the third timepoint, hh stage showed lighter expression of the th components, relative to the levels during the second time period. again, tra was detected throughout the retina, in a similar expression pattern as at earlier time periods, although at lower levels. trb expression was also reduced, but still seen tightly abutting the rpe in a photoreceptor pattern. dio expression was observed in the center of the onbl, where the few remaining progenitor cells at these late stages would be located. finally, just as in the first time period, dio was not detectable by ish in the retina at this later stage. the first time period correlates with early neurogenesis to better characterize the dynamic expression patterns of the th components, the expression patterns were further examined at several early hh stages. during hh stages 20– trb expression appeared as an enlarging wave, starting from the center and expanding to the periphery, sometimes overlapping the expression domain of dio. at stage dio expression was observed centrally, overlapping the domain containing a small number of trb-positive cells. at stage dio and trb expression were both more robust, having now begun to spread more peripherally from the central portion of the retina. the expression domain of both genes overlapped, with dio expression sometimes being observed slightly ahead of the trb domain. by stage dio expression had waned significantly, with only faint expression observed in the periphery. in contrast, at stage trb expression was robust both centrally and peripherally, and was not yet resolved to the photoreceptor layer, as was observed at later stages. since the center-to-periphery trb wave resembled that seen with the neurogenic genes otx, neurod, and cath, serial section ish was used to see if the different expression patterns were correlated. otx and neurod were expressed in cells scattered throughout the onbl, with a higher concentration of labeled cells located near the scleral side. both genes are thought to mark early photoreceptor cells, as well as other cell types. cath on the other hand, was expressed in a bilayered pattern, present in cells in the ganglion cell layer as well as in cells that have not been defined, but which overlap the developing photoreceptor layer. the expression pattern of neurod and cath appeared to extend slightly more toward the peripheral retina than trb and otx although all genes were similar in the extent of their central-peripheral expression. these data suggest that trb expression is correlated with a more general wave of neurogenesis spreading across the retina at early stages. trb expression also was compared to the expression of brn3b, crx, and shh, three genes marking the early differentiation of neurons, which also occurs in a central-to-peripheral pattern. in mouse, brn3b marks early ganglion cells and crx marks early photoreceptor cells. shh is expressed by ganglion cells in the chick, and possibly in other cell types as well. in zebrafish, ganglion cells and a subset of amacrine cells express shh, and in mouse, there is a similar pattern of expression, and there is a history of expression of shh in a subset of photoreceptors, likely cone photoreceptors. at stage the location of cells expressing brn3b, crx, and shh expression was similar with respect to the central/peripheral gradient, within the expression domain located more centrally then trb. the second time period corresponds to a wave of maturation of some cell types in a second time period, hh stages 29– a second wave of th components involving dio and dio swept across the retina. using flat mount ish, dio expression was observed at stage as heavy ventral staining, some dorsal staining, and an absence of expression in a spot and stripe centered on the dorsoventral border. over the next few stages, dio expression was confined in narrower stripes toward the periphery. in a nearly complementary pattern, dio started at stage as a spot and stripe centered on the dorsoventral border. over the next few stages, dio expression expanded dorsally and ventrally to occupy the entire retina. ish performed on serial sections of the peripheral retina at hh stage showed that the ebbing dio and expanding dio expression patterns were nearly complementary. during hh stages 20– there was a correlation of expression of th components and genes involved in neurogenesis and differentiation. it was of interest to determine whether the second wave of expression of th components also was correlated with other genes. the first candidate examined was lim which has been shown to mark horizontal cells as they move from the vitreal side to their final scleral position during development. on serial sections, the interface between dio and dio occurred in the same place as lim cell migration. the dio2/dio wave also paralleled changes the photoreceptor marker, visinin. at the dio2/dio wavefront/transition, visinin expression became more organized, transitioning from a weak diffuse pattern to a more intense band in the maturing photoreceptor layer. hence, the transition in the expression of dio and dio occurred along with the transition of a couple of genes to their mature expression patterns. the third time period corresponds to the disappearance of progenitor cells in a later time period, hh stages 35– a third wave involving dio swept across the retina. during this time period, dio expression ceased, first from the center and then in the periphery. similar to the previous waves of expression of th components, this period showed a correlation with the expression of genes involved in retinal development. on serial sections, dio was expressed throughout the area occupied by progenitor cells as defined by the expression of notch. in more central areas, where dio expression was absent, notch appeared in the layer that seemed to overlap with the expression of a glucose transporter, in a pattern consistent with müller glia cell expression. birthdating studies have shown that müller glia cells are among the last born of retinal cell types, and thus this transition appears with the end of proliferation. trb is expressed in early photoreceptor cells previous data concerning trb expression in the murine retina, along with the location of the ish signal in the scleral region of the chick retina, suggested that trb is expressed by photoreceptor cells. however, the scleral region of the retina also harbors cells in m phase, as well as cells that are newly postmitotic, but not photoreceptor cells. in order to define the cell type expressing trb, dissociated cell ish was carried out. probes for cell type-specific markers along with probes for th components were used. in addition, to investigate whether th components were expressed in mitotic cells, newly postmitotic cells, or postmitotic cells that had not recently exited, and were thus further along in their differentiation, embryos were labeled with -thymidine in ovo. dish was used to detect expression of one or two genes in cells labeled with -thymidine. cells were labeled in ovo with -thymidine for hour, and tissue was harvested and dissociated at the indicated times. double dish for the indicated genes and autoradiography for were carried out on the dissociated cells. cells were quantified for labeling by a single gene probe, both gene probes, and labeling with. cells were labeled in ovo with -thymidine for hour, and tissue was harvested and dissociated at the indicated times. double dish for the indicated genes and autoradiography for were carried out on the dissociated cells. cells were quantified for labeling by a single gene probe, both gene probes, and labeling with. * % of cells that express gene that also express gene 2 a one hour pulse with -thymidine, followed by an immediate harvest, would be expected to label primarily cells in s phase, along with cells in the early part of g. dish for trb applied to cells labeled with -thymidine for one hour showed that most of the trb expressing cells were not co-labeled for -thymidine and thus were not in s phase at stages or. in addition, the majority of cells in s phase or early g at these stages did not express trb. however, since a small fraction of cells, ± %, labeled with -thymidine after one hour expressed trb it is likely that trb turns on in some g cells. these data are in keeping with data from the ish on tissue sections, in which the majority of trb+ cells were located in the area of g cells and photoreceptor cells. cells were labeled in ovo with -thymidine for hour, and tissue was harvested and dissociated at the indicated times. dish for the indicated genes and autoradiography for were carried out on the dissociated cells. examination of -thymidine+ cells at intervals after the initial addition of the label allows a determination of whether a gene is expressed in g m, or g1/g since injection of -thymidine in ovo results in continuous labeling with -thymidine. dish for trb was performed on cells from stage or embryos 1– hours after addition of -thymidine. these data show a higher percentage of cells that express trb were labeled with -thymidine over time, resulting in ± % of trb+ cells also being labeled with -thymidine by hours at stage and % at stage by hours. since the cell cycle length is hours at these stages, the majority of trb+ cells are within one cell cycle length of labeling. given the location of trb cells in the tissue section in the area of photoreceptor cells, the lack of s phase labeling for the majority of trb+ cells, the low numbers of trb+ cells in the g population, most trb+ cells are likely newly postmitotic, and are likely to be photoreceptors. this was confirmed by double dish using photoreceptor markers. as mentioned above, neurod and otx are transcription factors that are expressed by newborn photoreceptors, and by other retinal cell types as well. the expression of otx and neurod in the cell cycle was examined as described above, and was compared to the labeling of trb. cells that expressed neurod or otx showed kinetics of labeling with -thymidine almost identical to the labeling kinetics of trb+ cells. double dish at stage for trb and neurod, or trb and otx revealed that % of all trb+ cells expressed neurod, and % expressed otx. the expression of otx and neurod was examined among the + cells labeled in a one hour exposure to -thymidine. of the + cells at stage % were otx2+, and % were neurod+, while only % were trb+. thus, among the + cells that expressed otx or neurod, only approximately one out of also expressed trb. these data demonstrate that while the kinetics of onset of expression of otx neurod, and trb are similar, many more cells, particularly at the later stages examined, expressed neurod and otx than trb. co-expression of two other markers of photoreceptors, visinin and crx, along with trb was examined by double dish. visinin is expressed exclusively by photoreceptors, and % of all trb+ cells expressed visinin at stage. the percentage of trb+ cells that expressed crx was % at stage and % at stage. trb and crx co-expression was compared further. crx can be considered a later photoreceptor marker compared to neurod and otx since, at stage < % of + cells were crx+; at stage < % of + cells were crx+; and, at stage 4– hours after addition of -thymidine, only – % of the + cells were crx+. trb and crx expression on section ish displays an inverse relationship: before stage trb was observed in a greater number of cells than crx, whereas after stage the opposite was observed. this pattern was confirmed using dish. at stage trb was expressed in approximately 9% of cells, and crx was observed in 7% of all cells, whereas at stage trb was mildly reduced to approximately 7%, while crx increased to 14% of all cells. the transient nature of the co-expression of trb and crx in differentiating photoreceptors was confirmed by double dish with trb and crx. at stage 26% of trb+ cells expressed crx, and 94% of crx+ cells expressed trb. at stage % of trb cells expressed crx, whereas % of crx cells expressed trb. at stage ± % of trb cells expressed crx, and ± % of crx cells expressed trb. this pattern is consistent with an earlier onset of trb than of crx in newly postmitotic photoreceptor cells, followed by a period of co-expression of trb and crx, and then a reduction in expression of trb in photoreceptors, which retain expression of crx. it also suggests that there is a greater interval between the onset of trb and the onset of crx at the later stages. the stability of expression of crx among photoreceptors is reflected in the high co-expression of crx and visinin at stage. dio is expressed in a subset of photoreceptor cells dio expression also was investigated using dish. dio was not detected at stage but later was expressed in a small but increasing percentage of all cells: stage approximately %, stage approximately %, and stage approximately 3%. double dish showed that at stage almost all dio2+ cells expressed neurod and otx, while % expressed crx and % expressed trb. at stage 85–100% of dio2+ cells expressed cath but they likely turn it off as only 17% expressed cath by stage these data suggest that dio is present in photoreceptor cells, but in a subset that appears at a later time in development. with respect to its kinetics in the cell cycle, dio was expressed later than cath trb, neurod, and otx since dio expression was almost never observed in s/g cells. combined with the section ish results, dio appeared in a subset of photoreceptors as they transitioned from newly postmitotic cells to later stages of differentiation. dio is expressed in mitotic cells dio whose expression appeared to be in progenitor cells on section ish, was also studied using dish and -thymidine incorporation. at stage % of dio3+ cells were + after a one hour pulse, and by hours of -thymidine labeling, all dio3+ cells were labeled with. this is in contrast to the plateau of -thymidine labeling for populations that include postmitotic cells, such as neurod and trb. dio was not present in cells that expressed crx, or dio, and only very few cells that expressed dio also expressed trb, at stage however, many cells that expressed dio also expressed otx and neurod at stage of these dio3+otx2+ or dio3+neurod+ cells, ± % and ± % respectively of them were also + after a hour pulse. these data indicate that cells that co-express dio and otx and/or neurod, are in s phase and/or early g discussion in this report, we show that th components are expressed in three sequential waves that spread across the retina, and that these waves are correlated with expression changes in a number of developmentally important genes. furthermore, we identify which cell types express which th components, and determine their expression kinetics with respect to the cell cycle. waves are a common theme in retinal development in a diverse array of organisms, retinal development occurs in waves. the best example of this is in drosophila melanogaster, in which the eye differentiates in a wave defined by the morphogenetic furrow. cells ahead of the furrow express genes involved in cell division and the undifferentiated state, whereas cells behind the furrow express cell differentiation markers as well as genes signaling terminal cell divisions. the phenomenon of genes expressed in differentiation waves also extends to vertebrate eye development. in zebrafish and xenopus, a program of cell differentiation spreads across the early retina, from the center to the periphery, as exemplified by the expression of shh. similarly, in the chick retina, the expression of neurogenesis markers has been observed to spread in an orderly fashion from the center to the periphery. in drosophila, there is a relationship between a nuclear hormone receptor, the ecdysone receptor, and hedgehog. it appears that the relief of repression from ecdysone receptor occurs just anterior to the morphogenetic furrow, while hedgehog initiates expression within the mf. as trs are functionally similar to the ecdysone receptor, and trb expression in the chick retina is spatially and temporally similar to that of shh, there is a possibility that trb and shh may be coordinated in a manner similar to that in the drosophila retina. beyond this potential relationship revealed by the spatio-temporal gradient of expression, other developmental regulators appear in central-peripheral waves in the chick retina, including cath otx neurod, dio dio ngn and markers of various differentiated cell types. the mechanism regulating these patterns is currently unknown. cell types expressing th components the expression of trb with respect to cell cycle kinetics and markers of photoreceptor cells show that this receptor is expressed in mitotic cells about to produce a postmitotic cell that will become a photoreceptor. it appears that such progenitor cells express otx and neurod at almost exactly the same time as trb. that the expression is in a g phase progenitor cell is an intriguing observation in light of retinal lineage data from embryonic mouse. infections of embryonic mice in utero with retroviral vectors were carried out at equivalent developmental stages to chick stages 26– when cones and rods are being produced in both species. the majority of embryonic mouse retinal clones of > cell were not cell clones, and this included the majority of clones that contained rods and cones. this observation suggests that when a postmitotic cell is made, it is not from a symmetric division in which both daughter cells become postmitotic, or many cell clones would have been observed. if a similar result were to hold for the chick, the expression of trb in g cells would not be maintained in both daughters of that division. presumably, the postmitotic daughter would retain expression, at least transiently, as it initiated its differentiation into a photoreceptor cell. the other daughter would extinguish the expression of trb as it re-entered s phase, as very few s phase cells express trb. the expression of otx and neurod is also likely maintained in the photoreceptor cell, as these genes appear to be expressed in the area of differentiating photoreceptor cells. they may also continue to be expressed in some of the s phase daughter cells, as there is higher number of s phase cells that express otx and neurod, particularly at later stages. it is interesting to note that dio is expressed in a subset of photoreceptor cells. the dio subset can be defined in three ways. it is highest in the ventral retina, and is missing in a central spot and horizontal stripe. the only other similar expression pattern is that of rhodopsin, a gene marking rod photoreceptors, which does not commence until stage days after the dio expression wanes. a second way to define the subset of dio2+ cells is by the time at which dio appears in development, appearing later than the expression of otx neurod, and trb. rod photoreceptors also appear to be generated later than cone photoreceptors in most species, although this has not been defined for the chick. finally, the kinetics of expression of dio in the cell cycle provides a third way to define it. dio appears after the earlier expression of otx neurod, and trb cells in the subset of cells that express it. from all of these observations, it is possible that dio is in rod photoreceptors. confirmation of this hypothesis will await an early marker of rod photoreceptors in the chick as dio disappears before expression of definitive rod-specific markers, such as rhodopsin. model of th signaling in retinal development the first wave of expression of th components is characterized by mitotic cells transitioning from producing only mitotic daughters to a phase in which they begin to produce neurons. tra appears to be the receptor present in all cells as they make this transition. dio is present in progenitor cells at certain times, whereas trb is expressed in some g cells and in newly postmitotic cells that begin to differentiate into photoreceptor cells. based on these observations, it is possible that dio keeps tra cells in the state where they only generate mitotic daughter cells. when dio expression ceases, the tra cells can produce a postmitotic cell, and some of these daughter cells express trb. whether dio promotes cell division and blocks differentiation remains an unanswered question. studies in the xenopus retina during metamorphosis seem to challenge this notion, because in these experiments, dio blocked cell division and antagonism of dio promoted cell division. however, these studies measured brdu incorporation over a span of many days after manipulation of dio the increased brdu incorporation seen after antagonizing dio could be due to several mechanisms, including an increase in cells undergoing their last cell division. this interpretation would agree with dio promoting the progenitor cell state and a lack of dio promoting cell cycle exit. the second wave of expression of th components is characterized by the maturation and migration of certain cell types to their final positions. before the second wave, trb photoreceptors appear to be in an immature photoreceptor state. during wave they progress in their differentiation, as shown by expression of crx. locally activated th might influence trb cells during this period. the second wave ends with dio sweeping across the retina, which might halt tra cells from producing postmitotic cells which express trb, and might instead change the types of cells produced, or the balance of mitotic and postmitotic daughters made by tra progenitor cells. a model consistent with this expression pattern is that trb, in conjunction with th produced by dio serves to block photoreceptor differentiation. in this model, trb appears in the early photoreceptor just as it is being produced, significantly before the cell expresses crx. eventually, trb and/or dio levels are reduced and the cell is allowed to continue differentiation and express crx. in the ventral retina, where retinal dio is present, the delay may be longer, which might contribute to the development of rod photoreceptors in this area. this repressive activity of trb in the presence of dio might be similar to trb activity in other parts of the nervous system, including the inhibitory activity of trb in the presence of t on transcription of the tshb gene. previous data from mice support a repression and activation role of trb in retinal photoreceptors. loss of trb led to cones that appeared to differentiate early, as seen by early s-opsin expression. interestingly, in the same mutant, m-opsin failed to be expressed. recently, it was shown that mutation of trb to an allele that could not bind th exhibited both aspects of the phenotype, demonstrating that both repression of s-opsin and activation of m-opsin requires th binding. in addition, th was found at higher levels in the dorsal than the ventral retina, and dio was found to be expressed at higher levels in the dorsal than in the ventral retina. thus, the coordination of th levels through dio activity, and the activity of the trb controls at least one aspect of the dorsoventral gradient of both s and m-opsins. neurod was also shown to play a role in regulation of s and m-opsins, at least in part via its regulation of trb. mice deficient in neurod showed the same s and m-opsin phenotypes as the trb mutant, along with a lack of maintenance of trb expression. neurod was shown to bind directly to a regulatory element for trb, but was not itself sufficient to activate trb in keeping with the observation that many more cells express neurod than express trb. it is interesting to note that in the chick, neurod expression is concomitant with trb expression in its onset, but at least in the mouse, the onset of expression of trb occurred in the neurod mutant, but was not maintained. finally, the third wave of expression of th components is characterized by waning dio expression and the transition from progenitor cells to müller glial cells. similar to the models described above, dio at this stage may keep tra cells in the progenitor state. when dio expression ends, the tra cells may be free to exit cell cycle and begin differentiating. however, at this late stage, cells may no longer be competent to generate photoreceptors. instead, they may exit cell cycle and become notch+ müller glia cells. this latter suggestion follows the observations of th signaling in optic nerve cell cultures where th has been demonstrated to promote the differentiation of oligodendrocyte cells. in this well studied system, th can promote the exit of oligodendrocyte precursor cells from the cell cycle and initiate differentiation. this action appears to be through tra. lack of th can block this process, promoting the expression of cell cycle genes and keeping cells in the proliferative state.  in summary, th components are expressed in dynamic waves across the developing chick retina, and involve a number of cell types including progenitor cells and photoreceptors. through the expression of deiodinases that activate and degrade thyroid hormone, in conjunction with cells expressing trs, a complex interplay between the different th components likely results. an attractive hypothesis is that the hormone controls the timing of cell cycle exit for both photoreceptors and müller glia cells, as well as the timing of differentiation for photoreceptors. future functional studies will shed more light on these proposed activities. 
trb expression initiated during g in progenitor cells, concomitant with neurod and otx which are expressed in early photoreceptor cells. the first wave occurred as progenitor cells began to make photoreceptors, the second as some cell types adopted a more mature location and differentiation state, and the third as müller glia were generated. these dynamic patterns suggest that th is playing several roles in retinal development, both within the cycling progenitor cells and possibly with respect to the timing of differentiation of photoreceptor cells. trb was expressed in photoreceptor cells for several days and then was reduced in expression level, as the expression of crx, a later photoreceptor gene, became more evident. dio appeared transiently in a ventral to dorsal gradient, likely in a subset of photoreceptor cells. the cell types expressing the components, as well as the kinetics of expression within the cell cycle, were defined. dio was expressed throughout the cell cycle in progenitor cells. multiple th components were expressed in dynamic patterns in cycling progenitor cells and photoreceptors cells across the developing chick retina. in the current work, the expression of these th components was examined in the chick retina over time.
 thyroid hormone is an important developmental regulator in many tissues, including the retina. th is activated locally via deiodinase, and it is destroyed by deiodinase. the th receptors, tra and trb, mediate th activity through hormone and dna binding, and interactions with transcription regulators.  in the current work, the expression of these th components was examined in the chick retina over time. three waves of expression were characterized and found to be correlated with critical developmental events. the first wave occurred as progenitor cells began to make photoreceptors, the second as some cell types adopted a more mature location and differentiation state, and the third as müller glia were generated. the cell types expressing the components, as well as the kinetics of expression within the cell cycle, were defined. trb expression initiated during g in progenitor cells, concomitant with neurod and otx which are expressed in early photoreceptor cells. trb was expressed in photoreceptor cells for several days and then was reduced in expression level, as the expression of crx, a later photoreceptor gene, became more evident. dio was expressed throughout the cell cycle in progenitor cells. tra was in most, if not all, retinal cells. dio appeared transiently in a ventral to dorsal gradient, likely in a subset of photoreceptor cells.  multiple th components were expressed in dynamic patterns in cycling progenitor cells and photoreceptors cells across the developing chick retina. these dynamic patterns suggest that th is playing several roles in retinal development, both within the cycling progenitor cells and possibly with respect to the timing of differentiation of photoreceptor cells. 