BACKGROUND
the development of the central nervous system depends on the actions and interactions of transcription factors and morphogens linked together in complex gene regulatory networks.
these networks serve to finely control processes such as tissue patterning and neuronal subtype specification  <cit> .
the bar homeobox-like  family of transcription factors, barhl <dig> and barhl <dig>  are the mammalian homologues of the drosophila bar homeobox  transcription factors barh <dig> and barh <dig>  <cit> .
barhl <dig> is strongly expressed in the proliferative zones of specific regions in the mammalian forebrain  <cit> .
its interactions with the many other transcription factors expressed in these regions are likely to be critical for normal forebrain development.
the proteins encoded by bar genes and their homologues in other species are characterised by the presence of a homeodomain along with either one or two fil domains—dna-binding regions that are rich in the amino acids phenylalanine , isoleucine , and leucine   <cit> .
transcription factors containing fil domains can act as transcriptional repressors  <cit>  via a mechanism involving their recruitment of the drosophila co-repressor groucho or its homologues in other species .
the drosophila barh genes are known to prevent ectopic neurogenesis in the fly retina by inhibiting the expression of atonal   <cit> , a proneural transcription factor featuring a basic helix–loop–helix  motif.
there is evidence that the actions of the mammalian barhl genes are mediated at least in part by their regulation of atonal-related bhlh transcription factors, such as those of the neurogenin  family  <cit> .
barhl <dig> plays roles in neuronal subtype specification in the vertebrate nervous system.
in the retina, barhl <dig> is required for amacrine cell  subtype specification.
loss of barhl <dig> leads to the specification of increased numbers of cholinergic acs at the expense of glycinergic and gabaergic acs  <cit> , while the premature expression of barhl <dig> in the zebrafish retina induces the differentiation of gabaergic acs at the expense of non-gabaergic acs and photoreceptors  <cit> .
in the mouse spinal cord barhl <dig> serves to specify dl <dig> interneuron subtype, and the loss of barhl <dig> leads to an increase in the number of contralaterally-projecting interneurons, with a reduction in the number that project ipsilaterally  <cit> .
studies in xenopus have shown that the xenopus barh <dig> homologue, xbarhl <dig>  <cit> , promotes the formation of the zona limitans intrathalamica   <cit> , a forebrain organizer region that patterns the diencephalon via the secretion of morphogens including sonic hedgehog   <cit> .
another transcription factor, paired-box  <dig> , has an opposite effect on the zli, limiting its size .
published data on barhl <dig> expression, which is limited, suggests that it might complement that of pax <dig> throughout much of the embryonic mouse forebrain with the possible exception of the thalamic ventricular zone, in which both genes appear to be strongly expressed at some embryonic stages .
we carried out a comprehensive analysis of the forebrain expression of barhl <dig> in embryonic mice at a range of developmental stages, using qualitative and quantitative methods to examine its relationship with the expression of pax <dig>  we examined expression of barhl <dig> in the pax6-null mutant mouse to test for a functional relationship between the expression patterns of barhl <dig> and pax <dig>
methods
experimental animals and ethics statement
all experimental work was carried out in accordance with the uk animals  act  <dig> and uk home office guidelines  <cit> .
all protocols were reviewed and approved by the named veterinary surgeons of the college of medicine and veterinary medicine, the university of edinburgh, prior to the commencement of experimental work.
wild-type mice used were of the mus musculus strain cd-1®  <cit> .
timed matings were set up between cd-1® males and females.
the day on which a vaginal plug could be observed was taken to be embryonic day  <dig>  .
embryos were harvested at e <dig> –e <dig> .
pax6-null mice used were of the mus musculus strain sey
ed  <cit> .
crosses were set up between pax6
+/sey males and pax6
+/sey females to generate litters comprising pax6
+/+, pax6
+/sey, and pax6
sey/sey embryos.
embryos were harvested at e <dig> –e <dig> .
pax6
sey/sey embryos were identified by their lack of eyes.
in situ hybridization
harvested embryos were fixed in a solution of 4% paraformaldehyde  in phosphate buffered saline   at 4 °c overnight before being sucrose-sunk as previously described  <cit>  and fixed in a 1: <dig> mixture of 30% sucrose solution in pbs optimal cutting temperature  medium .
for chromogenic in situ hybridisation embryos were then cryosectioned at a thickness of 10 µm before the protocol was performed as previously described  <cit> .
for fluorescence in situ hybridization embryos were sectioned at a thickness of 16 µm before the protocol was performed as previously described  <cit> .
the rna riboprobe for pax <dig> was that described in pinson et al.
<cit> .
the rna riboprobe for barhl <dig>  <cit>  was a kind gift from asuka suzuki-hirano and tomomi shimogori.
the ngn <dig> probe was that described in gradwohl et al.
<cit> .
the shh probe was that described in echelard et al.
<cit> .
immunohistochemistry
following fluorescence in situ hybridization for barhl <dig>  antigen retrieval was performed by microwaving sections in a 10 mm aqueous solution of sodium citrate.
immunohistochemistry for pax <dig> protein was performed as previously described  <cit> .
the primary antibody used was rabbit poly pax <dig>  at a concentration of 1: <dig> in blocking solution.
the secondary antibody used was goat anti-rabbit conjugated to alexa-fluor 488®  at a concentration of 1: <dig> in blocking solution.
anti-nestin primary antibody was used at 1: <dig> .
imaging
brightfield images were recorded with the leica dmlb microscope and leica application suite software.
fluorescence images were recorded using the nikon a1r-flim confocal microscope and nikon elements software.
the “grab large image free shape” function of elements was used to compile a tiled image from several square images recorded at different regions of the tissue section.
for each of the three channels  a 12-bit greyscale image was recorded at a resolution of 1028 ×  <dig> pixels.
each set of three greyscale images was saved as a stack.
all confocal images were recorded at the image analysis multiphoton and confocal technologies  imaging facility, the university of edinburgh.
quantification of image data
the quantitative analysis was performed on images of coronal sections cut at the plane illustrated in figs.
<dig> and 8a from each of three different embryos harvested at each of four developmental stages from e <dig>  to e <dig>  inclusive .
the 12-bit greyscale images recorded for the pax <dig> immunostaining channel  and barhl <dig> in situ hybridisation channel  were analysed using the fiji software package  <cit> .
for each image, the segmented line tool was used to draw a line through the pax <dig> and barhl2-expressing progenitor populations, parallel with the ventricular surface of the diencephalon from the dorsal midline to the zli .
the intensity plot profile tool was then used to obtain average pixel greyscale values  along the line.
the process was carried out on both left and right sides of the brain and the average values at each position along the line were plotted against distance from the dorsal midline.
for images of embryonic tissue harvested at e <dig> –e <dig> , the line was 40 µm wide along its entire length.
for embryos harvested at e <dig> , the thickness of the neuroepithelium close to the dorsal midline had fallen below 40 µm, and so for this small region a 16 µm-wide line was used and intensity data from the two lines were subsequently combined.
a linear regression trend line was calculated for each plot.
the gradients of the trend lines for each of the three embryos analysed at each developmental stage were used to calculate the mean gradients of pax <dig> and barhl <dig> expression for each stage.
RESULTS
expression of pax <dig> and barhl <dig> in the embryonic forebrain
we first used chromogenic in situ hybridization to examine the expression of pax <dig> and barhl <dig> separately, on adjacent coronal sections through a series of embryonic brains of increasing age.
at e <dig>  , neural tube closure is not yet complete and the two dorsal edges of the neural tube can be observed prior to their fusing to form the roofplate .
at this stage pax <dig> is expressed throughout most of the alar diencephalon and telencephalon  but is absent from their basal plate .
barhl <dig> is also expressed throughout much of the diencephalon, with strongest expression in alar regions overlapping the middle of the diencephalic domain of pax <dig> expression .
barhl <dig> expression is absent from the telencephalon .fig.
<dig>
a–l in situ hybridization data for pax <dig> and barhl <dig> mrna in adjacent sections cut from embryos at e <dig>  and e <dig> .
m, n schematics to illustrate the planes of the sections in a–l and to summarize the results.
scale bar for a–f 200 μm, g–l 500 μm.
tel telencephalon, di diencephalon, bp basal plate
at e <dig> , following the closure of the neural tube and the formation of the roofplate , pax <dig> continues to be strongly expressed throughout alar regions of the forebrain but not the basal plate .
barhl <dig> expression remains absent from the telencephalon .
in the diencephalon, the barhl <dig> expression domain has consolidated into a band of neuroepithelium running from ventral  to dorsal , flanked by regions of diencephalic neuroepithelium expressing little or no barhl <dig> .
the alar component of this band overlaps a central strip of the diencephalic domain of pax <dig> expression.
previous work has shown that major regions of the diencephalon can be distinguished based on their morphology and their patterns of gene expression by e <dig> .
the zli is developing as a narrow shh-expressing domain that spreads across the alar neural tube from ventral to dorsal, separating the prethalamus, which is rostral to the zli, from the thalamus, which is caudal to it  <cit> .
pax <dig> expression levels now show greater regional variation within the alar forebrain .
it is strongly expressed in the pretectum, prethalamus and telencephalon but is absent from the zli .
it is also absent from the mantle cells of the developing eminentia thalami , which link the diencephalon and the telencephalon on each side of the brain.
in the thalamus, pax <dig> expression levels are graded from low near the zli to high at the boundary with the pretectum .
strong pax <dig> expression can also be seen in the retina of the developing eye .
these expression patterns are summarized in fig.
2m.fig.
<dig>
a–l in situ hybridization data for pax <dig> and barhl <dig> mrna in adjacent sections cut from embryos at e <dig>  and e <dig> .
m schematics to illustrate the planes of the sections and to summarize results at e <dig> .
scale bars 500 μm.
pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica, emt eminentia thalami, hyp hypothalamus, pspb pallial–subpallial boundary, tel telencephalon, vtel ventral telencephalon, dtel dorsal telencephalon
unlike pax <dig>  barhl <dig> is expressed within the developing zli itself at e <dig>  .
it is also expressed in the pretectum and throughout the majority of the thalamic ventricular zone, in the region of progenitor cells known as the pth-c  <cit> .
barhl <dig> is not expressed in a narrow strip of progenitor cells immediately caudal to the zli, a region known as the pth-r  <cit>  .
barhl <dig> is also not expressed in the prethalamus , where pax <dig> expression is strong .
in more rostral sections a domain of barhl <dig> expression can be seen within the mantle zone of the developing eminentia thalami , where pax <dig> is not expressed .
barhl <dig> expression remains absent from the eye and the telencephalon .
these expression patterns are summarized in fig.
2m: essentially, significant complementarity between the patterns of expression of these two genes is emerging at e <dig> , with residual overlap in the caudal diencephalon .
by e <dig>  , the complementarity of pax <dig> and barhl <dig> expression has become increasingly obvious throughout much of the forebrain.
whereas many telencephalic progenitor cells express pax <dig>  they do not express barhl <dig>  barhl <dig> is, however, now expressed by differentiating cells in the mantle zone of the ventral telencephalon, in regions where pax <dig> is not expressed .
pax <dig> and barhl <dig> expression patterns in the hypothalamus also show striking complementarity .
the exception is the pretectum and the pth-c domain of the thalamus, where both genes are expressed .
barhl <dig> and pax <dig> both remain low or absent from the pth-r  but only barhl <dig> is strongly expressed within the zli .
the complementarity of pax <dig> and barhl <dig> expression patterns in the telencephalon, eminentia thalami, hypothalamus and prethalamus is well-developed at e <dig> –e <dig>  .
neither gene is expressed specifically in progenitor or postmitotic zones: for example, pax <dig> is expressed in both zones in the prethalamus  but in progenitor zones alone within the dorsal telencephalon and thalamus .
barhl <dig> is expressed in the progenitor zone of the thalamus  but in the postmitotic zone of the eminentia thalami .
overlap between pax <dig> and barhl <dig> expression patterns continues in the pretectum and pth-c: pax <dig> is still expressed in a gradient but its levels are relatively low compared to those in other forebrain regions  whereas levels of barhl <dig> are as high or higher than those in other regions such as the zli  and eminentia thalami .
barhl <dig> expression levels appear to be graded across pth-c by e <dig> , with levels increasing from caudal to rostral sections .fig.
<dig>
a–l in situ hybridization data for pax <dig> and barhl <dig> mrna in adjacent sections cut from embryos at e <dig>  and e <dig> .
m schematic to illustrate the planes of the sections.
scale bar for a–f 500 μm, g–l 250 μm.
pt pretectum, th thalamus, pth prethalamus, hyp hypothalamus, zli zona limitans intrathalamica, emt eminentia thalami, pspb pallial–subpallial boundary, tel telencephalon
co-expression of pax <dig> and barhl <dig> in the diencephalon
the analysis above indicates that diencephalic patterns of expression and co-expression of pax <dig> and barhl <dig> are complex and dynamic.
to confirm and clarify the conclusions drawn from single-colour in situ hybridizations on adjacent coronal sections, we carried out fluorescence double-labelling with both probes on parasagittal sections of the brains of embryos of increasing age .fig.
<dig>
a–o sagittal sections of embryos treated with immunostaining for pax <dig> protein and in situ hybridization for barhl <dig> mrna.
p schematic to illustrate the approximate plane of section.
scale bars for a–i 250 μm, j– <dig> 500 μm.
tel telencephalon, di diencephalon, mes mesencephalon, bp basal plate, ap alar plate, pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica
at e <dig> , this analysis confirmed conclusions from coronal sections .
pax <dig> is expressed throughout alar diencephalon and barhl <dig> is expressed in a narrower region, extending from the floorplate to the roofplate, whose alar domain overlaps a central portion of the diencephalic domain of pax <dig> expression .
at high magnification expression of both pax <dig> and barhl <dig> could be seen in the presumptive zli  and also in the region of prethalamic neuroepithelium directly rostral to it .
barhl <dig> mrna expression is detected primarily in the cytoplasm whereas pax <dig> is located in the nucleus .
in the alar prethalamic neuroepithelium, all cells expressed pax <dig> at this age .
given the fact that barhl <dig> is present in surrounding cytoplasm between the pax6-positive nuclei, we can deduce that many cells in this region co-express pax <dig> and barhl <dig> .fig.
<dig>
a sagittal section of e <dig>  embryo immunostained for pax <dig> protein and in situ hybridization for barhl <dig> mrna.
scale bar 200 μm.
b detail of area outlined in a. scale bar 25 μm.
c, d dapi staining and triple-staining for dapi, pax <dig> and barhl <dig> within the prethalamus in the area outlined in b. scale bar 10 μm
at e <dig>  the zli emerges as a thin barhl2-positive, pax6-negative domain between the barhl2-positive thalamus and the prethalamus, which is now barhl2-negative .
this confirms conclusions summarized in fig.
2m.
the separation of the barhl2-positive zli from the barhl2-positive pth-c by the pth-r, a narrow strip of tissue expressing low or no pax <dig> and barhl <dig>  becomes clearer by e <dig>  .
the barhl2-positive zli continues to be obvious at e <dig> – <dig>  .
we next considered the relationship between the expression of pax <dig> and barhl <dig> in the pretectum and thalamus where, unusually, both are expressed in the same region for a prolonged period.
we first considered whether pax <dig> and barhl <dig> are expressed by the same cells in the pretectum and thalamus by double-labelling for pax <dig> protein and barhl <dig> mrna in the same coronal sections at e <dig>  .
in these experiments we did not delineate the exact position of the boundary between the pretectum and thalamus, preferring to analyse the two together since the gradients of pax <dig> and barhl <dig> expression were continuous across the two regions .
as shown in fig.
6a, c, g, cells express higher levels of pax <dig> the closer they are to the pretectum.
close to the pretectum, almost all cells express detectable levels of pax <dig> .
these cells also stain for barhl <dig> in the cytoplasm around their pax6-positive nuclei .
this contrasts with other forebrain regions, such as the eminentia thalami, where pax6-expressing and barhl2-expressing cells are clearly segregated .fig.
<dig>
a coronal section of e <dig>  embryo treated with immunostaining for pax <dig> protein and in situ hybridization for barhl <dig> mrna.
b–e, f–i higher magnification of tissue outlined in a. j–m detail of the eminentia thalami.
scale bars for a 100 μm, b–m 25 μm.
pt pretectum, th thalamus, pth prethalamus
to study the relationship between the gradient of pax <dig> and expression levels of barhl <dig> across the thalamus, we examined coronal sections double-labelled with immunohistochemistry for pax <dig> and fluorescence in situ hybridization for barhl <dig> .
the plane at which these sections were cut  offers a clear view of the gradient of pax <dig> expression  and, therefore, the opportunity to correlate this gradient with variations in barhl <dig> expression.
in the examples shown in fig.
<dig>  there is evidence of countergradients of barhl <dig>  with levels increasing towards the zli, at e <dig> –e <dig> .
by e <dig> , pax <dig> levels in the thalamus are very low and there is no longer any obvious gradient of barhl <dig>  which is relatively strongly expressed in the ventricular zone of the thalamus .fig.
<dig>
a–l coronal sections of embryos treated with immunostaining for pax <dig> protein and in situ hybridization for barhl <dig> mrna.
m schematic to illustrate the approximate plane of section.
scale bars 500 μm.
pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica, emt eminentia thalami, tel telencephalon
to gain more objective data on these countergradients and their variation between embryos of the same and different ages, we quantified the levels of pax <dig> and barhl <dig> expression in three embryos at each of four ages, e <dig> , e <dig> , e <dig>  and e <dig>  .fig.
<dig>
a schematic to illustrate the area of the ventricular zone across which the intensity plots were calculated.
scale bar 100 μm.
b intensity plots for  <dig> embryos, three at each of four ages from e <dig>  to e <dig> , with linear regression trend lines.
c table of the gradients of the trend lines for each developmental stage, and the mean gradient for each set of three embryos.
d bar chart of the mean values of the gradients recorded for each group of three embryos, with standard errors.
pt pretectum, zli zona limitans intrathalamica, dpc days post conception
at all the ages studied—even at e <dig> , when pax <dig> levels are overall low throughout the thalamus —the intensity of staining for pax <dig> showed a consistently negative correlation with distance from pretectum to zli .
this is shown by the green lines and the mean gradients for each individual embryo  and by relatively low variance around the means at each age .
at e <dig> , all three embryos show a countergradient of barhl <dig> , as shown by the magenta lines and the mean gradients for each e <dig>  embryo in fig.
8b, c and by the low variance around the mean at e <dig>  .
at earlier ages, clear gradients of barhl <dig> expression were not always detected, although where strong gradients were detected they ran counter to those of pax <dig> .
at the latest age examined, e <dig> , when pax <dig> levels are generally very low , no countergradients were observed .
these data suggest that an inverse relationship between thalamic gradients of pax <dig> and barhl <dig> becomes established over the 2 days between e <dig>  and e <dig> , with variability in the timing of its emergence between individuals.
the gradient of pax <dig> appears to be established robustly before that of barhl <dig>
expression of barhl <dig> in the pax6-null forebrain
the findings above suggested that pax <dig> might repress the forebrain expression of barhl <dig>  in order to investigate this possibility further we performed in situ hybridization for barhl <dig> mrna on cryosections from pax6
sey/sey forebrains .fig.
<dig>
a–f in situ hybridization for barhl <dig> mrna on comparable sections from pax6
+/+ and pax6
sey/sey embryos at e <dig> .
g–l in situ hybridization for barhl <dig> mrna on comparable sections from pax6
+/+ and pax6
sey/sey embryos at e <dig> .
pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica, emt eminentia thalami, tel telencephalon, hyp hypothalamus.
scale bars 500 μm
as has been described before, the morphology of the pax6
sey/sey mutant forebrain differs from that of the wild-type.
the zli is expanded , much of the neuroepithelium is reduced in thickness  <cit>  and the third ventricle expands laterally  <cit>  as a result of two diencephalic structures, the paraventricular nucleus and the caudal zona incerta, failing to develop correctly  <cit> .
despite these differences in morphology, structures such as the thalamus and prethalamus can be distinguished in both the wild type and mutant forebrain  <cit> , making it possible to compare the expression of barhl <dig> in these regions.
nestin staining for radial glial cells in the ventricular zone  <cit>  was similar in pax6
+/+ and pax6
sey/sey embryos and indicated that there was no major change in the depth of the ventricular zone in mutants .fig.
<dig> immunohistochemistry for nestin on sections of the e <dig>  pax6
+/+ and pax6
sey/sey diencephalon.
th thalamus, pth prethalamus.
scale bars 500 µm in a, c and 250 µm in b, d
in the e <dig> –e <dig>  pax6
+/+ diencephalon the domain of barhl <dig> within the pth-c was restricted to the ventricular zone .
in the pax6
sey/sey mutant the thalamic barhl <dig> domain spanned all or most of the mediolateral width of the thalamic neuroepithelium, most likely due to the absence of normal mantle zone development .
barhl <dig> was expressed within the expanded zli of pax6
sey/sey mutants.
the most striking difference between barhl <dig> expression in pax6
+/+ and pax6
sey/sey diencephalon was its elevated expression in the thalamus and pretectum of mutants at e <dig> , but not earlier.
the pth-r was clearer in the pax6
+/+ diencephalon, where it was barhl2-negative, than in the pax6
sey/sey diencephalon, where it showed weaker barhl <dig> expression than surrounding pth-c and zli .
the pattern of barhl <dig> expression in other forebrain regions appeared similar in both genotypes at e <dig> –e <dig>  .
the prethalamus, dorsal telencephalon and much of the ventral telencephalon  remained negative for barhl <dig> in the mutants.
we conclude that the loss of pax <dig> causes increased barhl <dig> specifically in the pretectum and thalamus, where their co-expression is normally prolonged.
comparison of barhl <dig> and ngn <dig> expression in the embryonic forebrain
barhl <dig> has been suggested as an inhibitor of the expression of bhlh transcription factors.
we compared its diencephalic expression to that of the bhlh transcription factor neurogenin <dig>  using double fluorescence in situ hybridization with riboprobes for barhl <dig> and ngn <dig> on sections cut at the level of the central diencephalon from embryos at e <dig> , e <dig>  and e <dig>  .
in the telencephalon and the eminentia thalami, expression of barhl <dig> complemented that of ngn <dig> .
ngn <dig> was expressed in the dorsal telencephalon and the proliferative zone of the eminentia thalami.
barhl <dig>  on the other hand, was not expressed in dorsal telencephalon and was expressed in the differentiating but not the proliferating zone of the eminentia thalami.fig.
<dig>
a–l in situ hybridization for barhl <dig> and ngn <dig> mrna on coronal sections from pax6
+/+ embryos at e <dig> –e <dig> .
scale bars for a–i 500 μm, j–l 50 μm.
m–o in situ hybridization for barhl <dig> and shh mrna, high magnification image showing detail of the zli.
scale bar 50 μm.
pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica, emt eminentia thalami, tel telencephalon, emt eminentia thalami
in the diencephalon, however, the expression patterns of barhl <dig> and ngn <dig> were similar.
neither barhl <dig> nor ngn <dig> was expressed in the prethalamus and the pth-r, while both were expressed in the zli and pth-c .
within the zli, barhl <dig> expression corresponded with that of ngn <dig>  which is known to be expressed mainly in the more caudal region of the zli  <cit> .
to confirm the location of barhl <dig> expression in the zli, double fluorescence in situ hybridization was performed using riboprobes for barhl <dig> and shh, the definitive marker of the entire zli  <cit> .
the domain of barhl <dig> was slightly narrower than that of shh within the zli and was centred caudally, as anticipated .
in the thalamus and pretectum, barhl <dig> expression was confined to the proliferative zone whereas ngn <dig> expression extended through the proliferative zone and into the overlying differentiating mantle zone.
discussion
evidence for interactions between barhl <dig> and pax6
the expression of barhl <dig> complements that of pax <dig> in the telencephalon and eminentia thalami from early stages of its development, but the expression domains of barhl <dig> and pax <dig> overlap in diencephalic regions.
early in development, there is co-expression of the two genes from prethalamus to pretectum, but barhl <dig> expression is rapidly turned off in prethalamus, where pax <dig> levels remain high.
co-expression persists in the thalamus and pretectum, where pax <dig> and barhl <dig> develop countergradients.
our data suggest that a robust gradient of pax <dig> expression across this region predates the establishment of a robust countergradient of barhl <dig> expression.
soon after consistent countergradients are established, pax <dig> levels become undetectable across most of this region, while barhl <dig> levels remain high.
these results suggested that pax <dig> expression might have a repressive effect on barhl <dig> expression.
this might be direct or indirect.
a screen designed to predict pax <dig> target genes identified  <dig> potential pax <dig> binding sites around the transcription start site of barhl <dig>  suggesting that pax <dig> protein may be capable of exerting a direct effect on the expression of barhl <dig> mrna  <cit> .
we found that the absence of pax <dig> did not induce barhl <dig> expression in telencephalic regions that normally express pax <dig> but not barhl <dig>  nor did the absence of pax <dig> prevent the loss of barhl <dig> expression from the prethalamus.
absence of pax <dig> did, however, increase barhl <dig> expression in the thalamus and pretectum.
this interesting finding suggests a functional interaction between pax <dig> and barhl <dig> only in the region of the forebrain in which co-expression of the two genes persists much longer than in any other region.
whereas factors other than pax <dig> appear to control the overall expression patterns of barhl <dig>  pax <dig> is likely to play an important part in modulating the expression levels of barhl <dig> in just this one specific part of the forebrain.
our findings indicated that absence of pax <dig> led to barhl <dig> upregulation in thalamus and pretectum after e <dig> , suggesting that pax <dig> exerts its repressive effect on barhl <dig> only once the production of neurons is getting underway.
the importance of this might be to limit the anti-proneural activity of barhl <dig>  reducing its ability to interfere with the proneural activity of factors such as ngn <dig>  it is possible that proneural inhibition by unfettered upregulation of barhl <dig> expression in the absence of pax <dig> is a cause of the reduced size of the differentiating  zone in pax6-null mutants.
previous studies have provided limited evidence for the possibility that barhl <dig> and pax <dig> might be mutually repressive.
one study reported an expansion of the pax <dig> expression domain in response to a morpholino knockdown of xbarhl <dig> in the xenopus embryo  <cit> .
further work is needed to test whether loss of barhl <dig> in mice affects the expression of pax <dig>
the relationship between barhl <dig> and pax <dig> expression in the pretectum and thalamus may differ from that observed in other forebrain regions for many reasons.
the regions of neuroepithelium fated to become the thalamus and prethalamus are established prior to zli formation and differ significantly in molecular character from the forebrain regions rostral to the zli  <cit> .
the thalamic anlage expresses both irx <dig> and pax <dig>  which together confer competence to develop into the thalamus in response to the shh signal  <cit> .
the prethalamic anlage strongly expresses pax <dig> but not irx <dig>  <cit>  and also expresses six <dig>  which confers competence on the neuroepithelium to develop into more rostral forebrain structures  <cit> .
both regions of neuroepithelium are competent to respond to secreted signals from the zli  <cit> , but respond in different ways, and signalling from the zli therefore exerts changes in gene expression in an asymmetric fashion  <cit> .
these differences in molecular character may also influence the degree to which pax <dig> and barhl <dig> can be co-expressed.
barhl <dig> and pax <dig> in zli development
the zli is a thin shh-expressing strip of tissue that separates the thalamus and prethalamus.
morpholino knockdown of barhl <dig> expression in xenopus results in a failure of the zli to develop  <cit> , while the loss of functional pax <dig> in mice causes the zli to undergo an expansion along the rostrocaudal axis of the diencephalon .
together these findings suggest that barhl <dig> is required for zli initiation, while pax <dig> may play a later role in the shaping of the zli.
it is possible that pax6’s inhibition of barhl <dig> might contribute to its role in limiting the region of diencephalon that is competent to develop into the zli.
in the developing drosophila retina hh acts upstream of barh <dig> and barh <dig> and is required to initiate their expression  <cit> .
in the vertebrate forebrain the need for barhl <dig> in the induction of the zli suggests that barhl <dig> may act upstream of shh in this process and may be required to induce shh expression.
alternatively, barhl <dig> may be required to render a central region of diencephalic neuroepithelium competent to develop into zli.
while barhl <dig> is expressed in the anlage of the zli, and later within the mature zli itself, it is not expressed throughout the whole of the mature zli.
its expression is confined to the more caudal, ngn2-expressing region.
it is not clear what function barhl <dig> serves in the mature zli, if any, and why it is not expressed throughout the entire zli.
CONCLUSIONS
the expression of barhl <dig> in the thalamus and pretectum is related to, and regulated by, the expression of pax <dig>  pax <dig> is known to be required for normal diencephalic development.
the findings presented here suggest that some of its actions might be mediated by its maintenance of a repressive influence over barhl <dig> expression in the thalamus and pretectum.
abbreviations
bhlhbasic helix–loop–helix
acamacrine cell
pbsphosphate-buffered saline
octoptimal cutting temperature
teltelencephalon
didiencephalon
mesmesencephalon
rhrhombencephalon
scspinal cord
ptpretectum
ththalamus
pthprethalamus
zli
zona limitans intrathalamica
emt
eminentia thalami
hyphypothalamus
vtelventral telencephalon
dteldorsal telencephalon
pspbpallial–subpallial boundary
bpbasal plate
apalar plate
dpcdays post conception
authors’ contributions
evp designed and carried out all experiments, recorded all image data and performed the quantitative analysis.
djp and jom advised on the design of experiments.
djp directed the study.
evp and djp drew the illustrations and compiled the figures.
all authors read and approved the final manuscript.

 the development of the central nervous system depends on the actions and interactions of transcription factors and morphogens linked together in complex gene regulatory networks. these networks serve to finely control processes such as tissue patterning and neuronal subtype specification. the bar homeobox-like family of transcription factors, barhl and barhl are the mammalian homologues of the drosophila bar homeobox transcription factors barh and barh. barhl is strongly expressed in the proliferative zones of specific regions in the mammalian forebrain. its interactions with the many other transcription factors expressed in these regions are likely to be critical for normal forebrain development. the proteins encoded by bar genes and their homologues in other species are characterised by the presence of a homeodomain along with either one or two fil domains—dna-binding regions that are rich in the amino acids phenylalanine, isoleucine, and leucine. transcription factors containing fil domains can act as transcriptional repressors via a mechanism involving their recruitment of the drosophila co-repressor groucho or its homologues in other species. the drosophila barh genes are known to prevent ectopic neurogenesis in the fly retina by inhibiting the expression of atonal, a proneural transcription factor featuring a basic helix–loop–helix motif. there is evidence that the actions of the mammalian barhl genes are mediated at least in part by their regulation of atonal-related bhlh transcription factors, such as those of the neurogenin family. barhl plays roles in neuronal subtype specification in the vertebrate nervous system. in the retina, barhl is required for amacrine cell subtype specification. loss of barhl leads to the specification of increased numbers of cholinergic acs at the expense of glycinergic and gabaergic acs, while the premature expression of barhl in the zebrafish retina induces the differentiation of gabaergic acs at the expense of non-gabaergic acs and photoreceptors. in the mouse spinal cord barhl serves to specify dl interneuron subtype, and the loss of barhl leads to an increase in the number of contralaterally-projecting interneurons, with a reduction in the number that project ipsilaterally. studies in xenopus have shown that the xenopus barh homologue, xbarhl, promotes the formation of the zona limitans intrathalamica, a forebrain organizer region that patterns the diencephalon via the secretion of morphogens including sonic hedgehog. another transcription factor, paired-box, has an opposite effect on the zli, limiting its size. published data on barhl expression, which is limited, suggests that it might complement that of pax throughout much of the embryonic mouse forebrain with the possible exception of the thalamic ventricular zone, in which both genes appear to be strongly expressed at some embryonic stages. we carried out a comprehensive analysis of the forebrain expression of barhl in embryonic mice at a range of developmental stages, using qualitative and quantitative methods to examine its relationship with the expression of pax we examined expression of barhl in the pax6-null mutant mouse to test for a functional relationship between the expression patterns of barhl and pax methods experimental animals and ethics statement all experimental work was carried out in accordance with the uk animals act and uk home office guidelines. all protocols were reviewed and approved by the named veterinary surgeons of the college of medicine and veterinary medicine, the university of edinburgh, prior to the commencement of experimental work. wild-type mice used were of the mus musculus strain cd-1®. timed matings were set up between cd-1® males and females. the day on which a vaginal plug could be observed was taken to be embryonic day. embryos were harvested at e –e. pax6-null mice used were of the mus musculus strain sey ed. crosses were set up between pax6 +/sey males and pax6 +/sey females to generate litters comprising pax6 +/+, pax6 +/sey, and pax6 sey/sey embryos. embryos were harvested at e –e. pax6 sey/sey embryos were identified by their lack of eyes. in situ hybridization harvested embryos were fixed in a solution of 4% paraformaldehyde in phosphate buffered saline at 4 °c overnight before being sucrose-sunk as previously described and fixed in a 1: mixture of 30% sucrose solution in pbs optimal cutting temperature medium. for chromogenic in situ hybridisation embryos were then cryosectioned at a thickness of 10 µm before the protocol was performed as previously described. for fluorescence in situ hybridization embryos were sectioned at a thickness of 16 µm before the protocol was performed as previously described. the rna riboprobe for pax was that described in pinson et al.. the rna riboprobe for barhl was a kind gift from asuka suzuki-hirano and tomomi shimogori. the ngn probe was that described in gradwohl et al.. the shh probe was that described in echelard et al.. immunohistochemistry following fluorescence in situ hybridization for barhl antigen retrieval was performed by microwaving sections in a 10 mm aqueous solution of sodium citrate. immunohistochemistry for pax protein was performed as previously described. the primary antibody used was rabbit poly pax at a concentration of 1: in blocking solution. the secondary antibody used was goat anti-rabbit conjugated to alexa-fluor 488® at a concentration of 1: in blocking solution. anti-nestin primary antibody was used at 1:. imaging brightfield images were recorded with the leica dmlb microscope and leica application suite software. fluorescence images were recorded using the nikon a1r-flim confocal microscope and nikon elements software. the “grab large image free shape” function of elements was used to compile a tiled image from several square images recorded at different regions of the tissue section. for each of the three channels a 12-bit greyscale image was recorded at a resolution of 1028 ×  pixels. each set of three greyscale images was saved as a stack. all confocal images were recorded at the image analysis multiphoton and confocal technologies imaging facility, the university of edinburgh. quantification of image data the quantitative analysis was performed on images of coronal sections cut at the plane illustrated in figs.  and 8a from each of three different embryos harvested at each of four developmental stages from e to e inclusive. the 12-bit greyscale images recorded for the pax immunostaining channel and barhl in situ hybridisation channel were analysed using the fiji software package. for each image, the segmented line tool was used to draw a line through the pax and barhl2-expressing progenitor populations, parallel with the ventricular surface of the diencephalon from the dorsal midline to the zli. the intensity plot profile tool was then used to obtain average pixel greyscale values along the line. the process was carried out on both left and right sides of the brain and the average values at each position along the line were plotted against distance from the dorsal midline. for images of embryonic tissue harvested at e –e, the line was 40 µm wide along its entire length. for embryos harvested at e, the thickness of the neuroepithelium close to the dorsal midline had fallen below 40 µm, and so for this small region a 16 µm-wide line was used and intensity data from the two lines were subsequently combined. a linear regression trend line was calculated for each plot. the gradients of the trend lines for each of the three embryos analysed at each developmental stage were used to calculate the mean gradients of pax and barhl expression for each stage.  expression of pax and barhl in the embryonic forebrain we first used chromogenic in situ hybridization to examine the expression of pax and barhl separately, on adjacent coronal sections through a series of embryonic brains of increasing age. at e, neural tube closure is not yet complete and the two dorsal edges of the neural tube can be observed prior to their fusing to form the roofplate. at this stage pax is expressed throughout most of the alar diencephalon and telencephalon but is absent from their basal plate. barhl is also expressed throughout much of the diencephalon, with strongest expression in alar regions overlapping the middle of the diencephalic domain of pax expression. barhl expression is absent from the telencephalon.fig.  a–l in situ hybridization data for pax and barhl mrna in adjacent sections cut from embryos at e and e. m, n schematics to illustrate the planes of the sections in a–l and to summarize the results. scale bar for a–f 200 μm, g–l 500 μm. tel telencephalon, di diencephalon, bp basal plate at e, following the closure of the neural tube and the formation of the roofplate, pax continues to be strongly expressed throughout alar regions of the forebrain but not the basal plate. barhl expression remains absent from the telencephalon. in the diencephalon, the barhl expression domain has consolidated into a band of neuroepithelium running from ventral to dorsal, flanked by regions of diencephalic neuroepithelium expressing little or no barhl. the alar component of this band overlaps a central strip of the diencephalic domain of pax expression. previous work has shown that major regions of the diencephalon can be distinguished based on their morphology and their patterns of gene expression by e. the zli is developing as a narrow shh-expressing domain that spreads across the alar neural tube from ventral to dorsal, separating the prethalamus, which is rostral to the zli, from the thalamus, which is caudal to it. pax expression levels now show greater regional variation within the alar forebrain. it is strongly expressed in the pretectum, prethalamus and telencephalon but is absent from the zli. it is also absent from the mantle cells of the developing eminentia thalami, which link the diencephalon and the telencephalon on each side of the brain. in the thalamus, pax expression levels are graded from low near the zli to high at the boundary with the pretectum. strong pax expression can also be seen in the retina of the developing eye. these expression patterns are summarized in fig. 2m.fig.  a–l in situ hybridization data for pax and barhl mrna in adjacent sections cut from embryos at e and e. m schematics to illustrate the planes of the sections and to summarize results at e. scale bars 500 μm. pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica, emt eminentia thalami, hyp hypothalamus, pspb pallial–subpallial boundary, tel telencephalon, vtel ventral telencephalon, dtel dorsal telencephalon unlike pax barhl is expressed within the developing zli itself at e. it is also expressed in the pretectum and throughout the majority of the thalamic ventricular zone, in the region of progenitor cells known as the pth-c. barhl is not expressed in a narrow strip of progenitor cells immediately caudal to the zli, a region known as the pth-r. barhl is also not expressed in the prethalamus, where pax expression is strong. in more rostral sections a domain of barhl expression can be seen within the mantle zone of the developing eminentia thalami, where pax is not expressed. barhl expression remains absent from the eye and the telencephalon. these expression patterns are summarized in fig. 2m: essentially, significant complementarity between the patterns of expression of these two genes is emerging at e, with residual overlap in the caudal diencephalon. by e, the complementarity of pax and barhl expression has become increasingly obvious throughout much of the forebrain. whereas many telencephalic progenitor cells express pax they do not express barhl barhl is, however, now expressed by differentiating cells in the mantle zone of the ventral telencephalon, in regions where pax is not expressed. pax and barhl expression patterns in the hypothalamus also show striking complementarity. the exception is the pretectum and the pth-c domain of the thalamus, where both genes are expressed. barhl and pax both remain low or absent from the pth-r but only barhl is strongly expressed within the zli. the complementarity of pax and barhl expression patterns in the telencephalon, eminentia thalami, hypothalamus and prethalamus is well-developed at e –e. neither gene is expressed specifically in progenitor or postmitotic zones: for example, pax is expressed in both zones in the prethalamus but in progenitor zones alone within the dorsal telencephalon and thalamus. barhl is expressed in the progenitor zone of the thalamus but in the postmitotic zone of the eminentia thalami. overlap between pax and barhl expression patterns continues in the pretectum and pth-c: pax is still expressed in a gradient but its levels are relatively low compared to those in other forebrain regions whereas levels of barhl are as high or higher than those in other regions such as the zli and eminentia thalami. barhl expression levels appear to be graded across pth-c by e, with levels increasing from caudal to rostral sections.fig.  a–l in situ hybridization data for pax and barhl mrna in adjacent sections cut from embryos at e and e. m schematic to illustrate the planes of the sections. scale bar for a–f 500 μm, g–l 250 μm. pt pretectum, th thalamus, pth prethalamus, hyp hypothalamus, zli zona limitans intrathalamica, emt eminentia thalami, pspb pallial–subpallial boundary, tel telencephalon co-expression of pax and barhl in the diencephalon the analysis above indicates that diencephalic patterns of expression and co-expression of pax and barhl are complex and dynamic. to confirm and clarify the conclusions drawn from single-colour in situ hybridizations on adjacent coronal sections, we carried out fluorescence double-labelling with both probes on parasagittal sections of the brains of embryos of increasing age.fig.  a–o sagittal sections of embryos treated with immunostaining for pax protein and in situ hybridization for barhl mrna. p schematic to illustrate the approximate plane of section. scale bars for a–i 250 μm, j– 500 μm. tel telencephalon, di diencephalon, mes mesencephalon, bp basal plate, ap alar plate, pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica at e, this analysis confirmed conclusions from coronal sections. pax is expressed throughout alar diencephalon and barhl is expressed in a narrower region, extending from the floorplate to the roofplate, whose alar domain overlaps a central portion of the diencephalic domain of pax expression. at high magnification expression of both pax and barhl could be seen in the presumptive zli and also in the region of prethalamic neuroepithelium directly rostral to it. barhl mrna expression is detected primarily in the cytoplasm whereas pax is located in the nucleus. in the alar prethalamic neuroepithelium, all cells expressed pax at this age. given the fact that barhl is present in surrounding cytoplasm between the pax6-positive nuclei, we can deduce that many cells in this region co-express pax and barhl.fig.  a sagittal section of e embryo immunostained for pax protein and in situ hybridization for barhl mrna. scale bar 200 μm. b detail of area outlined in a. scale bar 25 μm. c, d dapi staining and triple-staining for dapi, pax and barhl within the prethalamus in the area outlined in b. scale bar 10 μm at e the zli emerges as a thin barhl2-positive, pax6-negative domain between the barhl2-positive thalamus and the prethalamus, which is now barhl2-negative. this confirms conclusions summarized in fig. 2m. the separation of the barhl2-positive zli from the barhl2-positive pth-c by the pth-r, a narrow strip of tissue expressing low or no pax and barhl becomes clearer by e. the barhl2-positive zli continues to be obvious at e –. we next considered the relationship between the expression of pax and barhl in the pretectum and thalamus where, unusually, both are expressed in the same region for a prolonged period. we first considered whether pax and barhl are expressed by the same cells in the pretectum and thalamus by double-labelling for pax protein and barhl mrna in the same coronal sections at e. in these experiments we did not delineate the exact position of the boundary between the pretectum and thalamus, preferring to analyse the two together since the gradients of pax and barhl expression were continuous across the two regions. as shown in fig. 6a, c, g, cells express higher levels of pax the closer they are to the pretectum. close to the pretectum, almost all cells express detectable levels of pax. these cells also stain for barhl in the cytoplasm around their pax6-positive nuclei. this contrasts with other forebrain regions, such as the eminentia thalami, where pax6-expressing and barhl2-expressing cells are clearly segregated.fig.  a coronal section of e embryo treated with immunostaining for pax protein and in situ hybridization for barhl mrna. b–e, f–i higher magnification of tissue outlined in a. j–m detail of the eminentia thalami. scale bars for a 100 μm, b–m 25 μm. pt pretectum, th thalamus, pth prethalamus to study the relationship between the gradient of pax and expression levels of barhl across the thalamus, we examined coronal sections double-labelled with immunohistochemistry for pax and fluorescence in situ hybridization for barhl. the plane at which these sections were cut offers a clear view of the gradient of pax expression and, therefore, the opportunity to correlate this gradient with variations in barhl expression. in the examples shown in fig.  there is evidence of countergradients of barhl with levels increasing towards the zli, at e –e. by e, pax levels in the thalamus are very low and there is no longer any obvious gradient of barhl which is relatively strongly expressed in the ventricular zone of the thalamus.fig.  a–l coronal sections of embryos treated with immunostaining for pax protein and in situ hybridization for barhl mrna. m schematic to illustrate the approximate plane of section. scale bars 500 μm. pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica, emt eminentia thalami, tel telencephalon to gain more objective data on these countergradients and their variation between embryos of the same and different ages, we quantified the levels of pax and barhl expression in three embryos at each of four ages, e, e, e and e.fig.  a schematic to illustrate the area of the ventricular zone across which the intensity plots were calculated. scale bar 100 μm. b intensity plots for embryos, three at each of four ages from e to e, with linear regression trend lines. c table of the gradients of the trend lines for each developmental stage, and the mean gradient for each set of three embryos. d bar chart of the mean values of the gradients recorded for each group of three embryos, with standard errors. pt pretectum, zli zona limitans intrathalamica, dpc days post conception at all the ages studied—even at e, when pax levels are overall low throughout the thalamus —the intensity of staining for pax showed a consistently negative correlation with distance from pretectum to zli. this is shown by the green lines and the mean gradients for each individual embryo and by relatively low variance around the means at each age. at e, all three embryos show a countergradient of barhl, as shown by the magenta lines and the mean gradients for each e embryo in fig. 8b, c and by the low variance around the mean at e. at earlier ages, clear gradients of barhl expression were not always detected, although where strong gradients were detected they ran counter to those of pax. at the latest age examined, e, when pax levels are generally very low, no countergradients were observed. these data suggest that an inverse relationship between thalamic gradients of pax and barhl becomes established over the 2 days between e and e, with variability in the timing of its emergence between individuals. the gradient of pax appears to be established robustly before that of barhl expression of barhl in the pax6-null forebrain the findings above suggested that pax might repress the forebrain expression of barhl in order to investigate this possibility further we performed in situ hybridization for barhl mrna on cryosections from pax6 sey/sey forebrains.fig.  a–f in situ hybridization for barhl mrna on comparable sections from pax6 +/+ and pax6 sey/sey embryos at e. g–l in situ hybridization for barhl mrna on comparable sections from pax6 +/+ and pax6 sey/sey embryos at e. pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica, emt eminentia thalami, tel telencephalon, hyp hypothalamus. scale bars 500 μm as has been described before, the morphology of the pax6 sey/sey mutant forebrain differs from that of the wild-type. the zli is expanded, much of the neuroepithelium is reduced in thickness and the third ventricle expands laterally as a result of two diencephalic structures, the paraventricular nucleus and the caudal zona incerta, failing to develop correctly. despite these differences in morphology, structures such as the thalamus and prethalamus can be distinguished in both the wild type and mutant forebrain, making it possible to compare the expression of barhl in these regions. nestin staining for radial glial cells in the ventricular zone was similar in pax6 +/+ and pax6 sey/sey embryos and indicated that there was no major change in the depth of the ventricular zone in mutants.fig.  immunohistochemistry for nestin on sections of the e pax6 +/+ and pax6 sey/sey diencephalon. th thalamus, pth prethalamus. scale bars 500 µm in a, c and 250 µm in b, d in the e –e pax6 +/+ diencephalon the domain of barhl within the pth-c was restricted to the ventricular zone. in the pax6 sey/sey mutant the thalamic barhl domain spanned all or most of the mediolateral width of the thalamic neuroepithelium, most likely due to the absence of normal mantle zone development. barhl was expressed within the expanded zli of pax6 sey/sey mutants. the most striking difference between barhl expression in pax6 +/+ and pax6 sey/sey diencephalon was its elevated expression in the thalamus and pretectum of mutants at e, but not earlier. the pth-r was clearer in the pax6 +/+ diencephalon, where it was barhl2-negative, than in the pax6 sey/sey diencephalon, where it showed weaker barhl expression than surrounding pth-c and zli. the pattern of barhl expression in other forebrain regions appeared similar in both genotypes at e –e. the prethalamus, dorsal telencephalon and much of the ventral telencephalon remained negative for barhl in the mutants. we conclude that the loss of pax causes increased barhl specifically in the pretectum and thalamus, where their co-expression is normally prolonged. comparison of barhl and ngn expression in the embryonic forebrain barhl has been suggested as an inhibitor of the expression of bhlh transcription factors. we compared its diencephalic expression to that of the bhlh transcription factor neurogenin using double fluorescence in situ hybridization with riboprobes for barhl and ngn on sections cut at the level of the central diencephalon from embryos at e, e and e. in the telencephalon and the eminentia thalami, expression of barhl complemented that of ngn. ngn was expressed in the dorsal telencephalon and the proliferative zone of the eminentia thalami. barhl on the other hand, was not expressed in dorsal telencephalon and was expressed in the differentiating but not the proliferating zone of the eminentia thalami.fig.  a–l in situ hybridization for barhl and ngn mrna on coronal sections from pax6 +/+ embryos at e –e. scale bars for a–i 500 μm, j–l 50 μm. m–o in situ hybridization for barhl and shh mrna, high magnification image showing detail of the zli. scale bar 50 μm. pt pretectum, th thalamus, pth prethalamus, zli zona limitans intrathalamica, emt eminentia thalami, tel telencephalon, emt eminentia thalami in the diencephalon, however, the expression patterns of barhl and ngn were similar. neither barhl nor ngn was expressed in the prethalamus and the pth-r, while both were expressed in the zli and pth-c. within the zli, barhl expression corresponded with that of ngn which is known to be expressed mainly in the more caudal region of the zli. to confirm the location of barhl expression in the zli, double fluorescence in situ hybridization was performed using riboprobes for barhl and shh, the definitive marker of the entire zli. the domain of barhl was slightly narrower than that of shh within the zli and was centred caudally, as anticipated. in the thalamus and pretectum, barhl expression was confined to the proliferative zone whereas ngn expression extended through the proliferative zone and into the overlying differentiating mantle zone. discussion evidence for interactions between barhl and pax6 the expression of barhl complements that of pax in the telencephalon and eminentia thalami from early stages of its development, but the expression domains of barhl and pax overlap in diencephalic regions. early in development, there is co-expression of the two genes from prethalamus to pretectum, but barhl expression is rapidly turned off in prethalamus, where pax levels remain high. co-expression persists in the thalamus and pretectum, where pax and barhl develop countergradients. our data suggest that a robust gradient of pax expression across this region predates the establishment of a robust countergradient of barhl expression. soon after consistent countergradients are established, pax levels become undetectable across most of this region, while barhl levels remain high. these results suggested that pax expression might have a repressive effect on barhl expression. this might be direct or indirect. a screen designed to predict pax target genes identified potential pax binding sites around the transcription start site of barhl suggesting that pax protein may be capable of exerting a direct effect on the expression of barhl mrna. we found that the absence of pax did not induce barhl expression in telencephalic regions that normally express pax but not barhl nor did the absence of pax prevent the loss of barhl expression from the prethalamus. absence of pax did, however, increase barhl expression in the thalamus and pretectum. this interesting finding suggests a functional interaction between pax and barhl only in the region of the forebrain in which co-expression of the two genes persists much longer than in any other region. whereas factors other than pax appear to control the overall expression patterns of barhl pax is likely to play an important part in modulating the expression levels of barhl in just this one specific part of the forebrain. our findings indicated that absence of pax led to barhl upregulation in thalamus and pretectum after e, suggesting that pax exerts its repressive effect on barhl only once the production of neurons is getting underway. the importance of this might be to limit the anti-proneural activity of barhl reducing its ability to interfere with the proneural activity of factors such as ngn it is possible that proneural inhibition by unfettered upregulation of barhl expression in the absence of pax is a cause of the reduced size of the differentiating zone in pax6-null mutants. previous studies have provided limited evidence for the possibility that barhl and pax might be mutually repressive. one study reported an expansion of the pax expression domain in response to a morpholino knockdown of xbarhl in the xenopus embryo. further work is needed to test whether loss of barhl in mice affects the expression of pax the relationship between barhl and pax expression in the pretectum and thalamus may differ from that observed in other forebrain regions for many reasons. the regions of neuroepithelium fated to become the thalamus and prethalamus are established prior to zli formation and differ significantly in molecular character from the forebrain regions rostral to the zli. the thalamic anlage expresses both irx and pax which together confer competence to develop into the thalamus in response to the shh signal. the prethalamic anlage strongly expresses pax but not irx and also expresses six which confers competence on the neuroepithelium to develop into more rostral forebrain structures. both regions of neuroepithelium are competent to respond to secreted signals from the zli, but respond in different ways, and signalling from the zli therefore exerts changes in gene expression in an asymmetric fashion. these differences in molecular character may also influence the degree to which pax and barhl can be co-expressed. barhl and pax in zli development the zli is a thin shh-expressing strip of tissue that separates the thalamus and prethalamus. morpholino knockdown of barhl expression in xenopus results in a failure of the zli to develop, while the loss of functional pax in mice causes the zli to undergo an expansion along the rostrocaudal axis of the diencephalon. together these findings suggest that barhl is required for zli initiation, while pax may play a later role in the shaping of the zli. it is possible that pax6’s inhibition of barhl might contribute to its role in limiting the region of diencephalon that is competent to develop into the zli. in the developing drosophila retina hh acts upstream of barh and barh and is required to initiate their expression. in the vertebrate forebrain the need for barhl in the induction of the zli suggests that barhl may act upstream of shh in this process and may be required to induce shh expression. alternatively, barhl may be required to render a central region of diencephalic neuroepithelium competent to develop into zli. while barhl is expressed in the anlage of the zli, and later within the mature zli itself, it is not expressed throughout the whole of the mature zli. its expression is confined to the more caudal, ngn2-expressing region. it is not clear what function barhl serves in the mature zli, if any, and why it is not expressed throughout the entire zli.  the expression of barhl in the thalamus and pretectum is related to, and regulated by, the expression of pax pax is known to be required for normal diencephalic development. the findings presented here suggest that some of its actions might be mediated by its maintenance of a repressive influence over barhl expression in the thalamus and pretectum. abbreviations bhlhbasic helix–loop–helix acamacrine cell pbsphosphate-buffered saline octoptimal cutting temperature teltelencephalon didiencephalon mesmesencephalon rhrhombencephalon scspinal cord ptpretectum ththalamus pthprethalamus zli zona limitans intrathalamica emt eminentia thalami hyphypothalamus vtelventral telencephalon dteldorsal telencephalon pspbpallial–subpallial boundary bpbasal plate apalar plate dpcdays post conception authors’ contributions evp designed and carried out all experiments, recorded all image data and performed the quantitative analysis. djp and jom advised on the design of experiments. djp directed the study. evp and djp drew the illustrations and compiled the figures. all authors read and approved the final manuscript. 
existing evidence suggests that its expression in the embryonic forebrain of the mouse is regional and may complement that of another transcription factor that is important for forebrain development, pax the aim of this study is to provide a more detailed description of the barhl expression pattern in the embryonic forebrain than is currently available, to relate its expression domains to those of pax and to examine the effects of pax loss on barhl expression. the transcription factor barhl is an antiproneural transcription factor with roles in neuronal differentiation. its expression initially overlaps that of pax in a central region of the alar diencephalon but over the following days their domains of expression become complementary in most forebrain regions. we found that barhl expression becomes upregulated in specifically the thalamus and pretectum in pax6-null mice. the region-specific expression pattern of barhl makes it likely to be an important player in the development of region-specific differences in embryonic mouse forebrain.
 the transcription factor barhl is an antiproneural transcription factor with roles in neuronal differentiation. the functions of its homologue in drosophila development are better understood than its functions in mammalian brain development. existing evidence suggests that its expression in the embryonic forebrain of the mouse is regional and may complement that of another transcription factor that is important for forebrain development, pax the aim of this study is to provide a more detailed description of the barhl expression pattern in the embryonic forebrain than is currently available, to relate its expression domains to those of pax and to examine the effects of pax loss on barhl expression.  we found that barhl is expressed in the developing diencephalon from the time of anterior neural tube closure. its expression initially overlaps that of pax in a central region of the alar diencephalon but over the following days their domains of expression become complementary in most forebrain regions. the exceptions are the thalamus and pretectum, where countergradients of pax and barhl expression are established by embryonic day, before overall pax levels in these regions decline greatly while barhl levels remain relatively high. we found that barhl expression becomes upregulated in specifically the thalamus and pretectum in pax6-null mice.  the region-specific expression pattern of barhl makes it likely to be an important player in the development of region-specific differences in embryonic mouse forebrain. repression of its expression in the thalamus and pretectum by pax may be crucial for allowing proneural factors to promote normal neuronal differentiation in this region. keywords mousedevelopmentthalamusforebraingene expressionzona limitans intrathalamicapax6barhl2http://dx.doi.org/ /501100000265medical research councilj003662no12291price david j. issue-copyright-statement© the author 2016 