BACKGROUND
it is evident that pax <dig> is a multiplex contributor to correct cns development.
this is exemplified by dynamic spatiotemporal expression patterns, occurring from early development and persisting in restricted regions throughout adulthood.
pax <dig> expression initially occurs in the neural tube and mesencephalon from very early stages  <cit>  and is required for polarisation of the dorsoventral axis of the neural tube  <cit>  and specification of the superior colliculus/tectum from the mesencephalic alar plate  <cit> .
in the developing superior colliculus, graded expression of pax <dig> establishes rostrocaudal and dorsoventral polarity.
expression of pax <dig> localises within superior collicular neurons as development proceeds.
this expression is upregulated during retinal innervation and axonal arborisation but reduced in pax <dig>  mutant mice  <cit> , with reduced  retinal innervation  <cit> , confirming that pax7-expressing cells are responsive to retinal input.
demonstrated colocalisation in superior collicular neurons with the mapping marker ephrin-a <dig> validates pax <dig> participation in retinotopic mapping  <cit> .
continued, graded expression into adulthood is thought to maintain a small population of dorsal neurons in the mature colliculus  <cit> , although the functional requirement for this feature remains obscure.
given the aforementioned importance of pax <dig> in mesencephalic and superior collicular development, the lack of gross defects in this region in pax <dig> mutant mice is surprising, and points to rescue by the paralogous pax <dig> gene which has overlapping expression domains  <cit> .
here, within the superior colliculus, we seek to determine the developmental role of pax <dig> in specification of neurons, and assess its spatiotemporal relationship with pax <dig>  we have analysed pax <dig> mutant mice  <cit>  relative to wildtype at key stages of development and results indicate that a subpopulation of neurons is lost during early postnatal stages.
we show that this apparent loss of neurons is not due to aberrant specification or proliferation, or cell-fate switching/transdifferentiation to the astrocytic lineage, but rather appears due to the inability of pax <dig> mutant mice to maintain a subpopulation of dorsal superior collicular neurons.
furthermore, analysis of pax <dig> expression in embryonic wildtype and pax <dig> mutant mice indicates crossregulation between paralogues, and illustrates a functional divergence during superior collicular development.
we propose that within the superior colliculus initial overlapping pax <dig> expression ensures correct neuronal specification, and temporospatial separation of expression patterns leads to solitary expression of pax <dig> during a critical period of neuronal maturation, which abrogates the ability of pax <dig> to compensate, revealing the aberrant phenotype.
RESULTS
pax gene expression patterns
the pax <dig> antibody has previously been demonstrated to be suitable for use in mouse tissue by western blot analysis  <cit> .
the pax <dig> antibody has been tested in vitro and in vivo for specificity in recognition of mouse pax <dig> by western blot analysis  <cit> .
pax <dig> expression in wildtype mice
within the anlage of the tectum, at the earliest embryonic stage examined , pax <dig> expression occurs from the mesencephalic ventricular zone to the most superficial layer.
immunoreactive cells are most dense in the ventricular and subventricular zones, then decrease in the intermediate zone, which is larger rostrally compared to the caudal region at this stage .
pax <dig> expression can also be detected within the subthalamus, pretectum, pons, and in the ventricular zones of the cerebellar primordium, pons and myelencephalon .
this profile concurs with in situ hybridisation results reported previously at e <dig>  <cit> , with the exception of expression detected at the 4th ventricle.
at e <dig> , a large number of immunoreactive cells are detected  in the presumptive superior colliculus whilst expression declines in the caudal tectum, reflecting the emerging distinction of the tectum into the inferior and superior colliculi.
pax7+ cells also recede from the mesencephalic ventricular zone, although remnants of expression can still be detected at the dorsal ventricular surface, with cells also noted at the ventral ventricular surface at certain mediolateral positions.
pax7+ cells can be detected up to the pial surface of the superior colliculus with the exception of the stratum zonale, which is now becoming evident .
expression remains robust within the pons.
pax <dig> expression at e <dig>  is similar to that noted at e <dig>  , however immunoreactivity is no longer detected within the ventricular zone and expression within the subthalamus is waning.
at p <dig>  while a rostrallow to caudalhigh, ventrallow to dorsalhigh gradient is maintained, pax7+ cell numbers are reduced throughout the superior colliculus .
cellular protein levels are similarly graded .
expression is also reduced at the midbrain/hindbrain boundary, pons and subthalamus.
even fewer pax7+ cells are detected throughout the young adult superior colliculus  .
expression persists at the midbrain/hindbrain boundary and rostral to the choroid plexus , with weak expression in the subthalamus .
+/+; wildtype +/-; pax7+/-
a values are mean cell numbers ± sem .
# indicates a significant p value from paired t-test assessing variation between axis points, within wildtype and pax7+/- mice.
pax <dig> expression in pax7+/- mutant mice
in pax7+/- embryos, the pax <dig> expression profile is grossly indistinguishable from that of wildtype mice ; cell counts, wt =  <dig>  ±  <dig> ; pax7+/- =  <dig>  ±  <dig>  .
however, at p <dig>  there is a dramatic reduction in the number of pax7+ cells in the most superficial region of pax7+/- mice relative to those of wildtype mice .
this manifests as a distinct area near the pial surface that is almost devoid of pax7+ cells uniformly across the entire rostrocaudal  and mediolateral  axes.
moreover, the number of pax7+ cells is reduced throughout the dorsal half of the superior colliculus.
although the reduction in the total number of pax7+ cells in pax7+/- relative to wildtype mice at p <dig> does not reach statistical significance, this phenomenon results in the loss of graded cellular distribution across the dorsoventral axis .
concomitantly there is some variation in cellular protein levels ventrally creating a lack of graded expression medially .
interestingly, at p <dig>  we did not discern any differences in pax <dig> expression between heterozygous and wildtype mice; cell numbers, wt =  <dig>  ±  <dig> ; pax7+/- =  <dig>  ±  <dig>  .
pax <dig> expression in wildtype and pax <dig> mutant mice
at e <dig> , in wildtype and pax <dig> mutant mice pax <dig> expression can be detected from the pretectum to the tectum in the ventricular and subventricular zones, with rostral expression lower in intensity relative to the mid-caudal region .
immunoreactivity is also evident in the ventricular zones of the cerebellar primordium, pons and myelencephalon, and at the midbrain/hindbrain boundary.
by e <dig>  pax <dig> expression within the superior colliculus of wildtype mice is primarily restricted to the dorsal ventricular zone, and the pons .
in pax7+/- mutant mice at this stage, pax3+ expression expands ventrally within the myelencephalon and pons .
in striking contrast to wildtype  and pax7+/- mice, pax7-/- mice demonstrate large numbers of pax3+ cells above the rostral ventricular zone  .
furthermore, pax3+ cells can be detected at the ventral ventricular surface in pax <dig> mutant mice but not in wildtype mice .
at e <dig> , in wildtype  and heterozygous mice the pretectum and ventricular zone of the superior colliculus remain pax3+, although only a remnant of expression within a small number of weakly stained cells remains within the superior colliculus and in the pons.
by contrast, pax <dig> immunoreactivity cannot be detected within the ventricular zone of pax7-/- mice  .
pax <dig> expression could not be detected at p <dig> in wildtype or mutant mice .
spatiotemporal assessment of neuronal proliferation and differentiation within the superior colliculus
to assess whether the loss of pax7+ cells in the dorsal superior collicular region of pax <dig> mutant mice was due to altered proliferation we explored ki <dig> expression at e <dig> .
we could not detect any variation in expression patterns between wildtype and mutant mice , indicating that cellular proliferation proceeds normally, thus excluding neuronal precocity at this stage .
we next sought to relate observed differences in the pax <dig> and pax <dig> expression profiles to temporal differences in neuronal differentiation.
we have shown previously that pax <dig> co-localises with βiii-tubulin and ephrin-a <dig> in the mouse superior colliculus at p <dig>  <cit> .
we therefore used these and other markers of early neuronal differentiation, α-internexin and doublecortin as well as the mature neuronal marker, neun to analyse neuronal differentiation in wildtype and mutant mice.
the efficacy of βiii-tubulin to discriminate differentiating neurons and tract formation in the mouse embryo has been demonstrated previously  <cit> .
at e <dig>  we observed βiii-tubulin+ cells predominantly located within the intermediate zone at the pial surface of the tectum, identifying the emerging stratum profundum   containing the first differentiating neurons of the superior colliculus  <cit> .
immunolabelled cells were also observed further ventrally at the rostral tectum, but were reduced in number throughout the mid-caudal region, indicating a rostral to caudal cellular maturation, with the occasional immunoreactive cell seen in more ventral regions.
at e <dig>  the sp is clearly visible in the ventral tectum, and immunolabelled, horizontally-oriented processes are now visible extending throughout the dorsorostral superior colliculus .
these processes extend more caudally at e <dig>  and most likely represent the incoming axons of retinal ganglion cells, the major afferent projection to the superficial superior colliculus  <cit> .
when we compared βiii-tubulin expression in the embryonic superior colliculus of wildtype mice to that of pax <dig> mutant mice we could not discern any differences in expression profiles  .
similar results were obtained at e <dig>  for α-internexin , and doublecortin , early markers of postmitotic  <cit>  and migrating and differentiating  <cit>  neurons, respectively.
results confirm that in mutant mice  at e <dig> , neuronal differentiation occurs normally within the intermediate zone close to the pial surface in a rostralhigh to mid-caudallow manner.
during all embryonic stages investigated, we could not detect any difference in the cellular distribution of neun+ within the superior colliculus of pax <dig> mutant mice relative to that of wildtype mice .
the tectum of wildtype mice at e <dig>  contains a small number of postmitotic, neun+ cells within the intermediate zone, distributed in a rostralhigh to caudallow manner .
at all other embryonic stages examined, neun+ cells are distributed throughout the superior colliculus from ventral to pial surfaces .
by contrast, at p <dig>  the most superficial region of the superior colliculus of pax <dig> mutant mice is almost completely devoid of neun+ cells  indicating dorsal neuronal loss at this important developmental time point.
neun+ cells can be detected throughout the superior colliculus and are in close proximity to the pial surface in wildtype mice  .
interestingly, within the adult mesencephalon  neun, like pax <dig>  shows similar expression in both mutant mice and wildtype mice .
alterations to superior collicular polarity in pax <dig> mutant mice
loss of pax7+ cells and neurons in the dorsal superior colliculus at p <dig> , when retinal ganglion cell axonal innervation and arborisation is occurring  <cit> , would be expected to impact on superior collicular polarity and retinotopic mapping.
we therefore assessed ephrin-a <dig> expression in pax <dig> mutant mice relative to expression in wildtype littermates.
consistent with the altered pax <dig> and neun profile, at p <dig> ephrin-a2+ cells are missing in the most superficial region , in contrast to wildtype expression displaying immunoreactivity close to the pial surface , providing further evidence of neuronal loss and changes to polarity.
neuronal loss is not due to astrocytic cell-fate switching or transdifferentiation
to assess the likelihood of astrocytic cell-fate switching or transdifferentiation, we analysed gfap expression in postnatal stages as the majority of gliogenesis occurs within the first postnatal week  <cit> .
we could not detect co-expression of pax <dig> and gfap at p <dig> or p <dig>  in either wildtype or pax <dig> mutant mice, consistent with previous results determined in the chick tectum  <cit> .
gfap+ processes can be seen extending from cells located at the pial surface, however the dorsal regions of the superior colliculus in pax <dig> mutant mice are similar to wildtype mice and are not populated by gfap+ cells within the region of perturbation at p <dig> or p <dig>  , indicating that cell fate switching or transdifferentiation to the astrocytic lineage is not responsible for the reduced dorsal expression of pax <dig>  neun and ephrin-a <dig> at this stage .
neuronal loss may be attributable to cellular regression
to evaluate whether neuronal loss was due to apoptosis, we investigated activated caspase  <dig> expression from e <dig>  to p <dig>  however we did not detect either co-expression with pax <dig> at any stage, or increased numbers of cells in mutant mice relative to wildtype .
this marker, however, proved difficult to analyse as elevated and widespread immunoreactivity detected at the earlier embryonic stages tested may be due to the emerging role of caspase  <dig> in neuronal differentiation  <cit> .
we therefore utilised hoechst as an indicator of pyknotic nuclei, and assessed co-expression with activated caspase  <dig> as confirmation of the apoptotic status of the cells, but only a few apoptotic cells were detected at any given time point.
the identification of apoptotic cells would, however, require knowledge of the exact timeframe in which the neurons were lost, due to rapid neuronal degeneration noted previously  <cit> .
mesencephalic boundary formation
to explore the effects of perturbed pax <dig> expression on formation of superior collicular boundaries, ie between the diencephalon and mesencephalon and between the superior colliculus  and tegmentum , we assessed pax <dig> expression at the rostral and ventral boundaries of the superior colliculus  <cit> .
at e <dig> , there are a few weakly-stained pax6+ cells superior to the interpeduncular fossa.
at e <dig>  in wildtype mice, pax6+ cells can be detected dorsally in the pretectum  sometimes spreading to the rostral margin of the ventricular zone.
pax <dig> immunoreactivity could not be detected within the pretectum at e <dig>  in either wildtype or mutant mice.
we could not detect any abnormality in the expression domain of pax <dig> in pax <dig> mutant mice at these midgestational stages .
to investigate alterations to mesencephalic/metencephalic boundary formation, we assessed the expression of the marker en- <dig>  <cit>  from e <dig>  to p <dig> .
en-1+ cells can be detected at the midbrain/hindbrain boundary, from the interpeduncular fossa to the rhombencephalic isthmus  and expression wanes as development proceeds until p <dig> , when en- <dig> expression in the midbrain is usually restricted to a cluster of cells superior to the interpeduncular fossa.
throughout all stages examined, we could not detect any difference between wildtype and mutant mice .
discussion
in order to understand the role of pax <dig> in superior collicular development we investigated the superior colliculus of pax <dig> mutant mice relative to wildtype at a variety of developmental stages.
whilst both pax <dig> and pax <dig> are known to define the early tectum subsequent to mesencephalic determination, and the ability to form ectopic tectum subsequent to misexpression within the diencephalon and ventral mesencephalon testifies to their critical nature in specification of tectal identity  <cit> , individual roles during further development have remained elusive.
here we show that loss of pax <dig> dramatically alters pax <dig> expression domains and disturbs the neuronal profile in the dorsal layers of the developing superior colliculus.
pax <dig> and pax <dig> have separate roles during neuronal differentiation in the superior colliculus
since pax <dig> and pax <dig> have similar expression domains in the developing superior colliculus at mid-gestation  <cit> , we undertook a thorough, comparative analysis of their expression patterns in wildtype and mutant mice.
it was anticipated that this analysis would highlight any changes caused by loss of pax <dig> and shed light on their individual roles during superior collicular development.
results indicate that as the superior colliculus emerges and the dorsal laminae are formed by cellular migration and differentiation, pax <dig> expression becomes progressively more refined towards the upper, developing strata whereas pax <dig> expression becomes increasingly restricted towards the proliferative region .
at e <dig> , pax7- and pax3-expressing cells overlap in the ventricular and subventricular zones, but not in the emerging, differentiating intermediate zone.
at e <dig>  tectal pax <dig> expression within the ventricular zone decreases, and is no longer evident at e <dig> , with expression persisting within the upper developing primary layers .
however, pax <dig> expression becomes restricted to the ventricular zone, with a few scattered cells within the intermediate region at e <dig>  decreasing at e <dig> .
postnatally, expression of pax <dig> is detected throughout the superior colliculus, numerically declining with maturation yet persisting at all developmental stages examined, whilst pax <dig> expression is extinguished.
the divergent pax3/pax <dig> expression profiles, as demonstrated in this paper, suggest a dichotomy of function with respect to neuronal differentiation.
the pax <dig> expression profile, without co-expression of pax <dig>  appears spatially associated with neuronal differentiation and maturation of the superior colliculus, whilst pax <dig> expression appears to be negatively associated with these characteristics.
a role for pax <dig> in maintaining the undifferentiated phenotype has been demonstrated previously, both in vitro and in vivo for neuronal cells  <cit> , for schwann cells  <cit>  and for melanoblasts  <cit> .
conversely, transfection of pax <dig> into p <dig> mouse embryonal cells in vitro directs these cells along a neuronal pathway  <cit> .
this dichotomy may, therefore, represent an important functional divergence between paralogues which may have implications for future stem cell therapies designed to treat midbrain disorders.
to investigate this further, we performed a comparative assessment of βiii-tubulin, α-internexin, doublecortin and neun expression at e <dig> , just prior to the normal birthdate of the superficially located neurons.
at this timepoint, the intermediate zone appears as an expanded region rostrally  diminishing to a thin region at the pial surface mid-caudally, indicating a rostral to caudal maturity, and is characterised by the expression of pax <dig>  βiii-tubulin , α-internexin, doublecortin and neun, and morphologically by increased internuclear distances.
by contrast, pax <dig> expression cannot be detected within the intermediate zone.
we could not discern any evidence of precocious neuronogenesis at this time, a function previously demonstrated for pax <dig> within the eye of sey mutant mice  <cit> .
however, a more thorough investigation at closer temporal increments from e <dig> would be required to thoroughly preclude this possibility, as precocious or more rapid neuronogenesis may occur between the time frames investigated in this study.
furthermore, the perturbation to pax <dig> expression within the ventricular zone at the later embryonic stages investigated, suggests that the early processes of specification and/or migration may be accelerated somewhat with insufficient pax <dig> levels.
the unaltered proliferation/specification of tectal cells does not preclude a role for pax <dig> in these processes per se, but may indicate compensation via pax <dig>
the observed perturbations in pax <dig> expression in pax <dig> mutant mice may indicate that pax <dig> acts to limit the expression domain of pax <dig>  this is supported by the rostral, ventral and caudal expansion of pax3+ cells in the presence of reduced or absent pax <dig> expression.
these regions of expanded expression occur where pax <dig> expression would normally be encountered, and suggests that pax <dig> acts to dorsalise  pax <dig> expression.
a cell autonomous relationship between pax <dig> and pax <dig> has been determined whereby misexpression of one paralogue represses expression of the other, and a balancing mechanism may exist to produce correct total expression levels  <cit> .
the exact mechanism behind this relationship is ambiguous, however it may be significant that the expression of pax <dig> precedes that of pax <dig> within the headfold and primitive fold ectoderm in the developing chick  <cit> .
moreover, it is clear that this relationship is also dosage-sensitive, as perturbations occur in the haploinsufficient state.
role for pax <dig> in timing of neuronal specification
expression analysis and cellular quantification have demonstrated that pax <dig> haploinsufficiency does not alter the initial proliferation steps in pax7+ cells during mid embryonic stages.
the only notable disparity to pax <dig> expression occurs at p <dig>  where a subpopulation of pax7+ cells, situated in the dorsal half of the superior colliculus, is no longer detectable, presenting with an obvious margin lacking pax7+ cells superficially.
sagittal and coronal sections have identified that this perturbation occurs across the dorsal surface of the entire rostrocaudal axis and most of the mediolateral axis.
to understand the significance of this anomaly we reviewed previous studies, which indicate that the majority of superior collicular neurons are born throughout the period e <dig> to e <dig>  <cit>  and distinct temporal and spatial migration patterns culminate in dictation of the correct cytoarchitecture of the developing superior colliculus .
cells generated at e <dig> initially migrate to populate the superior colliculus, and are subsequently divided dorsoventrally by cells generated at e <dig>  <cit> .
the cells that eventually reside within the most superficial region of the superior colliculus undergo their final postmitotic division at e <dig> while in the ventricular/subventricular zones, and migrate to subdivide their predecessors in the intermediate zone at e <dig>  thereafter, a subpopulation assume their final position superficially at e <dig>  <cit>  which coincides with retinal ganglion cell innervation of both superficial and intermediate zones  <cit> .
the stratum superficiale thus contains cells generated at both e <dig> and e <dig>  with the latter cells being more superficially placed.
in contrast, the ventral regions of the superior colliculus, associated with auditory and motor responses, undergo earlier development than the superficial regions and are thus populated by earlier-born neurons  <cit> .
this temporal specification of cells is similarly observed in the chick tectum, where two populations of cells are generated, each with distinct laminar fates, with later born neurons expressing pax <dig> fated to reside in the more dorsal laminae  <cit> .
it is therefore evident that the timely migration of cells from the ventricular zone contributes to the correct placement of cells within the superficial laminae, and this temporal-based specification of neurons ensures that placement of cells within laminae coincides with their maturation and initiation of circuitry.
overall, our results tend to suggest that pax <dig> may partially contribute to the correct timing of neuronogenesis by regulating migration of pax3+ cells from the ventricular zone into the mid-collicular region in a timely and orderly fashion, a characteristic that is lost in pax7-/- mice between e <dig>  and e <dig> , at the time when wildtype cells destined to reside in the more superficial strata are migrating and assuming their final destination.
a putative anti-apoptotic role for pax <dig> in postnatal superior collicular development
as earlier quantification did not detect any numerical changes in the pax <dig> cellular profile in pax7+/- mice, it became apparent that these cells had either altered their differentiation profile or had regressed within the first postnatal week.
we assessed gfap expression postnatally and as there was no change in the cellular distribution of gfap+ cells in pax <dig> mutant mice we excluded cell fate switching or transdifferentiation to the astrocytic lineage as a causative factor for the reduced number of pax7+ cells dorsally.
we were, however, unable to detect increased apoptosis within the superior colliculus using activated caspase  <dig>  cresyl violet or hoechst staining.
a more detailed examination commencing from birth would be required to determine the exact time interval when perturbation occurs in order to conclusively demonstrate apoptotic mechanisms.
apoptotic labelling methods such as tunel or caspase  <dig>  concomitant with pax <dig> immunolabelling, would be required to address this issue, however rapid neuronal degeneration coupled with a progressive rostrocaudal maturation to the tissue which is likely to result in a punctuated apoptotic phenomenon, will likely impede the quantitative nature of the analysis required to differentiate between normal  and increased  apoptosis.
however, the loss of neurons evidenced in pax <dig> mutant mice indicates a loss of cells, rather than simply extinction of pax <dig> expression.
relating phenotype to the specification of cells during superior collicular development  <cit>  suggests that the second population of neurons, generated at e <dig>  may be less capable of long-term survival than their earlier-produced counterparts, and as such are sensitive to gene dosage .
whilst the differential placement of late-born neurons identified in previous studies  <cit>  coincides spatially with the perturbed neuronal profile of pax <dig> mutant mice further experimentation is required to distinguish a link between the generation of subpopulations of cells and their relationship with pax <dig> and postnatal cytoarchitecture.
furthermore, differential sensitivity to neuron death has been suggested for different functional populations of neurons within the superior colliculus  <cit> .
anti-apoptotic roles for pax genes are well-documented to-date  <cit>  and, importantly, the ciliary neurotrophic factor receptor has recently been demonstrated as a downstream target of pax <dig> within the mouse embryo  <cit> .
apparent neuronal recovery at p <dig>
we have demonstrated altered pax7+ and neuronal profiles in pax <dig> mutant mice at p <dig>  however at p <dig>  we can no longer detect these variations between pax <dig> mutant and wildtype mice.
whilst it is feasible that the similar number of pax7+ cells in pax7+/- mice relative to wildtype at p <dig>  occurs because pax7+ cells in the adult do not normally include the subpopulation of cells which are missing at p <dig> in pax <dig> mutant mice, the apparent recovery of the neuronal profile by p <dig>  has proven enigmatic.
we have drawn from established studies of cns and superior collicular development to address this issue.
it is clear that early postnatal stages of superior collicular development are tumultuous.
the colliculus becomes invaded by retinal afferents just prior to birth and this continues within the first postnatal week, with formation of collaterals giving rise to axonal arborisation and commencement of synaptogenesis  <cit> .
during this process, a dramatic transformation occurs in which almost half of all fibre bundles are removed from the upper half of the stratum superficiale   <cit> .
this process coincides with naturally occurring neuron death in the superior colliculus  <cit> , indicating neuronal formation in excess of adult requirements.
excessive neuron formation is a recurrent theme in cns development  <cit> .
therefore it is likely that excessive neuron formation masks the loss of the more susceptible, pax7-deficient neurons, with sufficient cells remaining to satisfy functional requirements.
subsequently, displacement of cells due to fibre invasion and the ensuring maturation of the tissue could possibly account for the apparent phenotypic equivalence of neun+ cells noted at p <dig> .
it should be noted that functional studies have not been performed on pax <dig> mutant mice to-date to assess visual acuity.
further studies
a possible cause for the loss of superficial neurons in early postnatal stages could be altered circuitry.
neurons of the superficial superior colliculus communicate with the deeper collicular regions through superficial neuronal axons  <cit> , or via dendrites from neurons of the deep layers  <cit>  which in turn contain a variety of efferent projections from regions such as the cortex, retina, zona incerta   <cit>  and indirectly from the subthalamus via the substantia nigra pars reticulata or enteropeduncular nucleus  <cit> .
taking this into consideration, an interesting finding from this research is the persistent pax7+/pax3- expression profile of the subthalamus throughout all timepoints examined.
providing pax <dig> is not expressed in this region at an earlier developmental stage than examined in this study, the subthalamus may represent a novel opportunity to further dissect the functional repertoire of pax <dig> from that of pax <dig>  additional work would therefore be required to characterize the subthalamus of pax <dig> mutant mice to address this issue, as this important brain region is a target of deep brain stimulation to treat disorders such as parkinson's disease and epilepsy  <cit> .
further studies on the role of pax <dig> in superior collicular development would benefit from investigation into the axonal projection and circuity of pax7+ cells, which may be elaborated by the generation of pax7/tau-labelled x pax <dig> mutant mice.
cell lineage tracing experiments in pax <dig> mutant mice would be required to trace the progression of cells generated at e <dig> and e <dig> to conclusively resolve the questions related to the early postnatal perturbation and the apparent neuronal recovery in the adult.
moreover, functional studies assessing the ability of pax <dig> mutant mice to evoke a co-ordinated response to stimuli will be required to assess the integrity of the mature superior colliculus.
CONCLUSIONS
in summary pax <dig>  while not required for neuronogenesis or neuronal differentiation at early stages of superior collicular development, is absolutely requisite in a dosage-dependent manner for long term maintenance of a subpopulation of dorsal mesencephalic neurons.
this characteristic may well impart prophylactic properties to stem cells utilised in future replacement therapies to enhance treatment of neurodegenerative diseases of the midbrain.
furthermore, comparative expression analyses indicate a functionally divergent role for pax <dig> and pax <dig> during neuronal differentiation within the superior colliculus.

 it is evident that pax is a multiplex contributor to correct cns development. this is exemplified by dynamic spatiotemporal expression patterns, occurring from early development and persisting in restricted regions throughout adulthood. pax expression initially occurs in the neural tube and mesencephalon from very early stages and is required for polarisation of the dorsoventral axis of the neural tube and specification of the superior colliculus/tectum from the mesencephalic alar plate. in the developing superior colliculus, graded expression of pax establishes rostrocaudal and dorsoventral polarity. expression of pax localises within superior collicular neurons as development proceeds. this expression is upregulated during retinal innervation and axonal arborisation but reduced in pax mutant mice, with reduced retinal innervation, confirming that pax7-expressing cells are responsive to retinal input. demonstrated colocalisation in superior collicular neurons with the mapping marker ephrin-a validates pax participation in retinotopic mapping. continued, graded expression into adulthood is thought to maintain a small population of dorsal neurons in the mature colliculus, although the functional requirement for this feature remains obscure. given the aforementioned importance of pax in mesencephalic and superior collicular development, the lack of gross defects in this region in pax mutant mice is surprising, and points to rescue by the paralogous pax gene which has overlapping expression domains. here, within the superior colliculus, we seek to determine the developmental role of pax in specification of neurons, and assess its spatiotemporal relationship with pax we have analysed pax mutant mice relative to wildtype at key stages of development and results indicate that a subpopulation of neurons is lost during early postnatal stages. we show that this apparent loss of neurons is not due to aberrant specification or proliferation, or cell-fate switching/transdifferentiation to the astrocytic lineage, but rather appears due to the inability of pax mutant mice to maintain a subpopulation of dorsal superior collicular neurons. furthermore, analysis of pax expression in embryonic wildtype and pax mutant mice indicates crossregulation between paralogues, and illustrates a functional divergence during superior collicular development. we propose that within the superior colliculus initial overlapping pax expression ensures correct neuronal specification, and temporospatial separation of expression patterns leads to solitary expression of pax during a critical period of neuronal maturation, which abrogates the ability of pax to compensate, revealing the aberrant phenotype.  pax gene expression patterns the pax antibody has previously been demonstrated to be suitable for use in mouse tissue by western blot analysis. the pax antibody has been tested in vitro and in vivo for specificity in recognition of mouse pax by western blot analysis. pax expression in wildtype mice within the anlage of the tectum, at the earliest embryonic stage examined, pax expression occurs from the mesencephalic ventricular zone to the most superficial layer. immunoreactive cells are most dense in the ventricular and subventricular zones, then decrease in the intermediate zone, which is larger rostrally compared to the caudal region at this stage. pax expression can also be detected within the subthalamus, pretectum, pons, and in the ventricular zones of the cerebellar primordium, pons and myelencephalon. this profile concurs with in situ hybridisation results reported previously at e, with the exception of expression detected at the 4th ventricle. at e, a large number of immunoreactive cells are detected in the presumptive superior colliculus whilst expression declines in the caudal tectum, reflecting the emerging distinction of the tectum into the inferior and superior colliculi. pax7+ cells also recede from the mesencephalic ventricular zone, although remnants of expression can still be detected at the dorsal ventricular surface, with cells also noted at the ventral ventricular surface at certain mediolateral positions. pax7+ cells can be detected up to the pial surface of the superior colliculus with the exception of the stratum zonale, which is now becoming evident. expression remains robust within the pons. pax expression at e is similar to that noted at e, however immunoreactivity is no longer detected within the ventricular zone and expression within the subthalamus is waning. at p while a rostrallow to caudalhigh, ventrallow to dorsalhigh gradient is maintained, pax7+ cell numbers are reduced throughout the superior colliculus. cellular protein levels are similarly graded. expression is also reduced at the midbrain/hindbrain boundary, pons and subthalamus. even fewer pax7+ cells are detected throughout the young adult superior colliculus. expression persists at the midbrain/hindbrain boundary and rostral to the choroid plexus, with weak expression in the subthalamus. +/+; wildtype +/-; pax7+/- a values are mean cell numbers ± sem. # indicates a significant p value from paired t-test assessing variation between axis points, within wildtype and pax7+/- mice. pax expression in pax7+/- mutant mice in pax7+/- embryos, the pax expression profile is grossly indistinguishable from that of wildtype mice; cell counts, wt = ±; pax7+/- = ±. however, at p there is a dramatic reduction in the number of pax7+ cells in the most superficial region of pax7+/- mice relative to those of wildtype mice. this manifests as a distinct area near the pial surface that is almost devoid of pax7+ cells uniformly across the entire rostrocaudal and mediolateral axes. moreover, the number of pax7+ cells is reduced throughout the dorsal half of the superior colliculus. although the reduction in the total number of pax7+ cells in pax7+/- relative to wildtype mice at p does not reach statistical significance, this phenomenon results in the loss of graded cellular distribution across the dorsoventral axis. concomitantly there is some variation in cellular protein levels ventrally creating a lack of graded expression medially. interestingly, at p we did not discern any differences in pax expression between heterozygous and wildtype mice; cell numbers, wt = ±; pax7+/- = ±. pax expression in wildtype and pax mutant mice at e, in wildtype and pax mutant mice pax expression can be detected from the pretectum to the tectum in the ventricular and subventricular zones, with rostral expression lower in intensity relative to the mid-caudal region. immunoreactivity is also evident in the ventricular zones of the cerebellar primordium, pons and myelencephalon, and at the midbrain/hindbrain boundary. by e pax expression within the superior colliculus of wildtype mice is primarily restricted to the dorsal ventricular zone, and the pons. in pax7+/- mutant mice at this stage, pax3+ expression expands ventrally within the myelencephalon and pons. in striking contrast to wildtype and pax7+/- mice, pax7-/- mice demonstrate large numbers of pax3+ cells above the rostral ventricular zone. furthermore, pax3+ cells can be detected at the ventral ventricular surface in pax mutant mice but not in wildtype mice. at e, in wildtype and heterozygous mice the pretectum and ventricular zone of the superior colliculus remain pax3+, although only a remnant of expression within a small number of weakly stained cells remains within the superior colliculus and in the pons. by contrast, pax immunoreactivity cannot be detected within the ventricular zone of pax7-/- mice. pax expression could not be detected at p in wildtype or mutant mice. spatiotemporal assessment of neuronal proliferation and differentiation within the superior colliculus to assess whether the loss of pax7+ cells in the dorsal superior collicular region of pax mutant mice was due to altered proliferation we explored ki expression at e. we could not detect any variation in expression patterns between wildtype and mutant mice, indicating that cellular proliferation proceeds normally, thus excluding neuronal precocity at this stage. we next sought to relate observed differences in the pax and pax expression profiles to temporal differences in neuronal differentiation. we have shown previously that pax co-localises with βiii-tubulin and ephrin-a in the mouse superior colliculus at p. we therefore used these and other markers of early neuronal differentiation, α-internexin and doublecortin as well as the mature neuronal marker, neun to analyse neuronal differentiation in wildtype and mutant mice. the efficacy of βiii-tubulin to discriminate differentiating neurons and tract formation in the mouse embryo has been demonstrated previously. at e we observed βiii-tubulin+ cells predominantly located within the intermediate zone at the pial surface of the tectum, identifying the emerging stratum profundum containing the first differentiating neurons of the superior colliculus. immunolabelled cells were also observed further ventrally at the rostral tectum, but were reduced in number throughout the mid-caudal region, indicating a rostral to caudal cellular maturation, with the occasional immunoreactive cell seen in more ventral regions. at e the sp is clearly visible in the ventral tectum, and immunolabelled, horizontally-oriented processes are now visible extending throughout the dorsorostral superior colliculus. these processes extend more caudally at e and most likely represent the incoming axons of retinal ganglion cells, the major afferent projection to the superficial superior colliculus. when we compared βiii-tubulin expression in the embryonic superior colliculus of wildtype mice to that of pax mutant mice we could not discern any differences in expression profiles. similar results were obtained at e for α-internexin, and doublecortin, early markers of postmitotic and migrating and differentiating neurons, respectively. results confirm that in mutant mice at e, neuronal differentiation occurs normally within the intermediate zone close to the pial surface in a rostralhigh to mid-caudallow manner. during all embryonic stages investigated, we could not detect any difference in the cellular distribution of neun+ within the superior colliculus of pax mutant mice relative to that of wildtype mice. the tectum of wildtype mice at e contains a small number of postmitotic, neun+ cells within the intermediate zone, distributed in a rostralhigh to caudallow manner. at all other embryonic stages examined, neun+ cells are distributed throughout the superior colliculus from ventral to pial surfaces. by contrast, at p the most superficial region of the superior colliculus of pax mutant mice is almost completely devoid of neun+ cells indicating dorsal neuronal loss at this important developmental time point. neun+ cells can be detected throughout the superior colliculus and are in close proximity to the pial surface in wildtype mice. interestingly, within the adult mesencephalon neun, like pax shows similar expression in both mutant mice and wildtype mice. alterations to superior collicular polarity in pax mutant mice loss of pax7+ cells and neurons in the dorsal superior colliculus at p, when retinal ganglion cell axonal innervation and arborisation is occurring, would be expected to impact on superior collicular polarity and retinotopic mapping. we therefore assessed ephrin-a expression in pax mutant mice relative to expression in wildtype littermates. consistent with the altered pax and neun profile, at p ephrin-a2+ cells are missing in the most superficial region, in contrast to wildtype expression displaying immunoreactivity close to the pial surface, providing further evidence of neuronal loss and changes to polarity. neuronal loss is not due to astrocytic cell-fate switching or transdifferentiation to assess the likelihood of astrocytic cell-fate switching or transdifferentiation, we analysed gfap expression in postnatal stages as the majority of gliogenesis occurs within the first postnatal week. we could not detect co-expression of pax and gfap at p or p in either wildtype or pax mutant mice, consistent with previous results determined in the chick tectum. gfap+ processes can be seen extending from cells located at the pial surface, however the dorsal regions of the superior colliculus in pax mutant mice are similar to wildtype mice and are not populated by gfap+ cells within the region of perturbation at p or p, indicating that cell fate switching or transdifferentiation to the astrocytic lineage is not responsible for the reduced dorsal expression of pax neun and ephrin-a at this stage. neuronal loss may be attributable to cellular regression to evaluate whether neuronal loss was due to apoptosis, we investigated activated caspase expression from e to p however we did not detect either co-expression with pax at any stage, or increased numbers of cells in mutant mice relative to wildtype. this marker, however, proved difficult to analyse as elevated and widespread immunoreactivity detected at the earlier embryonic stages tested may be due to the emerging role of caspase in neuronal differentiation. we therefore utilised hoechst as an indicator of pyknotic nuclei, and assessed co-expression with activated caspase as confirmation of the apoptotic status of the cells, but only a few apoptotic cells were detected at any given time point. the identification of apoptotic cells would, however, require knowledge of the exact timeframe in which the neurons were lost, due to rapid neuronal degeneration noted previously. mesencephalic boundary formation to explore the effects of perturbed pax expression on formation of superior collicular boundaries, ie between the diencephalon and mesencephalon and between the superior colliculus and tegmentum, we assessed pax expression at the rostral and ventral boundaries of the superior colliculus. at e, there are a few weakly-stained pax6+ cells superior to the interpeduncular fossa. at e in wildtype mice, pax6+ cells can be detected dorsally in the pretectum sometimes spreading to the rostral margin of the ventricular zone. pax immunoreactivity could not be detected within the pretectum at e in either wildtype or mutant mice. we could not detect any abnormality in the expression domain of pax in pax mutant mice at these midgestational stages. to investigate alterations to mesencephalic/metencephalic boundary formation, we assessed the expression of the marker en- from e to p. en-1+ cells can be detected at the midbrain/hindbrain boundary, from the interpeduncular fossa to the rhombencephalic isthmus and expression wanes as development proceeds until p, when en- expression in the midbrain is usually restricted to a cluster of cells superior to the interpeduncular fossa. throughout all stages examined, we could not detect any difference between wildtype and mutant mice. discussion in order to understand the role of pax in superior collicular development we investigated the superior colliculus of pax mutant mice relative to wildtype at a variety of developmental stages. whilst both pax and pax are known to define the early tectum subsequent to mesencephalic determination, and the ability to form ectopic tectum subsequent to misexpression within the diencephalon and ventral mesencephalon testifies to their critical nature in specification of tectal identity, individual roles during further development have remained elusive. here we show that loss of pax dramatically alters pax expression domains and disturbs the neuronal profile in the dorsal layers of the developing superior colliculus. pax and pax have separate roles during neuronal differentiation in the superior colliculus since pax and pax have similar expression domains in the developing superior colliculus at mid-gestation, we undertook a thorough, comparative analysis of their expression patterns in wildtype and mutant mice. it was anticipated that this analysis would highlight any changes caused by loss of pax and shed light on their individual roles during superior collicular development. results indicate that as the superior colliculus emerges and the dorsal laminae are formed by cellular migration and differentiation, pax expression becomes progressively more refined towards the upper, developing strata whereas pax expression becomes increasingly restricted towards the proliferative region. at e, pax7- and pax3-expressing cells overlap in the ventricular and subventricular zones, but not in the emerging, differentiating intermediate zone. at e tectal pax expression within the ventricular zone decreases, and is no longer evident at e, with expression persisting within the upper developing primary layers. however, pax expression becomes restricted to the ventricular zone, with a few scattered cells within the intermediate region at e decreasing at e. postnatally, expression of pax is detected throughout the superior colliculus, numerically declining with maturation yet persisting at all developmental stages examined, whilst pax expression is extinguished. the divergent pax3/pax expression profiles, as demonstrated in this paper, suggest a dichotomy of function with respect to neuronal differentiation. the pax expression profile, without co-expression of pax appears spatially associated with neuronal differentiation and maturation of the superior colliculus, whilst pax expression appears to be negatively associated with these characteristics. a role for pax in maintaining the undifferentiated phenotype has been demonstrated previously, both in vitro and in vivo for neuronal cells, for schwann cells and for melanoblasts. conversely, transfection of pax into p mouse embryonal cells in vitro directs these cells along a neuronal pathway. this dichotomy may, therefore, represent an important functional divergence between paralogues which may have implications for future stem cell therapies designed to treat midbrain disorders. to investigate this further, we performed a comparative assessment of βiii-tubulin, α-internexin, doublecortin and neun expression at e, just prior to the normal birthdate of the superficially located neurons. at this timepoint, the intermediate zone appears as an expanded region rostrally diminishing to a thin region at the pial surface mid-caudally, indicating a rostral to caudal maturity, and is characterised by the expression of pax βiii-tubulin, α-internexin, doublecortin and neun, and morphologically by increased internuclear distances. by contrast, pax expression cannot be detected within the intermediate zone. we could not discern any evidence of precocious neuronogenesis at this time, a function previously demonstrated for pax within the eye of sey mutant mice. however, a more thorough investigation at closer temporal increments from e would be required to thoroughly preclude this possibility, as precocious or more rapid neuronogenesis may occur between the time frames investigated in this study. furthermore, the perturbation to pax expression within the ventricular zone at the later embryonic stages investigated, suggests that the early processes of specification and/or migration may be accelerated somewhat with insufficient pax levels. the unaltered proliferation/specification of tectal cells does not preclude a role for pax in these processes per se, but may indicate compensation via pax the observed perturbations in pax expression in pax mutant mice may indicate that pax acts to limit the expression domain of pax this is supported by the rostral, ventral and caudal expansion of pax3+ cells in the presence of reduced or absent pax expression. these regions of expanded expression occur where pax expression would normally be encountered, and suggests that pax acts to dorsalise pax expression. a cell autonomous relationship between pax and pax has been determined whereby misexpression of one paralogue represses expression of the other, and a balancing mechanism may exist to produce correct total expression levels. the exact mechanism behind this relationship is ambiguous, however it may be significant that the expression of pax precedes that of pax within the headfold and primitive fold ectoderm in the developing chick. moreover, it is clear that this relationship is also dosage-sensitive, as perturbations occur in the haploinsufficient state. role for pax in timing of neuronal specification expression analysis and cellular quantification have demonstrated that pax haploinsufficiency does not alter the initial proliferation steps in pax7+ cells during mid embryonic stages. the only notable disparity to pax expression occurs at p where a subpopulation of pax7+ cells, situated in the dorsal half of the superior colliculus, is no longer detectable, presenting with an obvious margin lacking pax7+ cells superficially. sagittal and coronal sections have identified that this perturbation occurs across the dorsal surface of the entire rostrocaudal axis and most of the mediolateral axis. to understand the significance of this anomaly we reviewed previous studies, which indicate that the majority of superior collicular neurons are born throughout the period e to e and distinct temporal and spatial migration patterns culminate in dictation of the correct cytoarchitecture of the developing superior colliculus. cells generated at e initially migrate to populate the superior colliculus, and are subsequently divided dorsoventrally by cells generated at e. the cells that eventually reside within the most superficial region of the superior colliculus undergo their final postmitotic division at e while in the ventricular/subventricular zones, and migrate to subdivide their predecessors in the intermediate zone at e thereafter, a subpopulation assume their final position superficially at e which coincides with retinal ganglion cell innervation of both superficial and intermediate zones. the stratum superficiale thus contains cells generated at both e and e with the latter cells being more superficially placed. in contrast, the ventral regions of the superior colliculus, associated with auditory and motor responses, undergo earlier development than the superficial regions and are thus populated by earlier-born neurons. this temporal specification of cells is similarly observed in the chick tectum, where two populations of cells are generated, each with distinct laminar fates, with later born neurons expressing pax fated to reside in the more dorsal laminae. it is therefore evident that the timely migration of cells from the ventricular zone contributes to the correct placement of cells within the superficial laminae, and this temporal-based specification of neurons ensures that placement of cells within laminae coincides with their maturation and initiation of circuitry. overall, our results tend to suggest that pax may partially contribute to the correct timing of neuronogenesis by regulating migration of pax3+ cells from the ventricular zone into the mid-collicular region in a timely and orderly fashion, a characteristic that is lost in pax7-/- mice between e and e, at the time when wildtype cells destined to reside in the more superficial strata are migrating and assuming their final destination. a putative anti-apoptotic role for pax in postnatal superior collicular development as earlier quantification did not detect any numerical changes in the pax cellular profile in pax7+/- mice, it became apparent that these cells had either altered their differentiation profile or had regressed within the first postnatal week. we assessed gfap expression postnatally and as there was no change in the cellular distribution of gfap+ cells in pax mutant mice we excluded cell fate switching or transdifferentiation to the astrocytic lineage as a causative factor for the reduced number of pax7+ cells dorsally. we were, however, unable to detect increased apoptosis within the superior colliculus using activated caspase cresyl violet or hoechst staining. a more detailed examination commencing from birth would be required to determine the exact time interval when perturbation occurs in order to conclusively demonstrate apoptotic mechanisms. apoptotic labelling methods such as tunel or caspase concomitant with pax immunolabelling, would be required to address this issue, however rapid neuronal degeneration coupled with a progressive rostrocaudal maturation to the tissue which is likely to result in a punctuated apoptotic phenomenon, will likely impede the quantitative nature of the analysis required to differentiate between normal and increased apoptosis. however, the loss of neurons evidenced in pax mutant mice indicates a loss of cells, rather than simply extinction of pax expression. relating phenotype to the specification of cells during superior collicular development suggests that the second population of neurons, generated at e may be less capable of long-term survival than their earlier-produced counterparts, and as such are sensitive to gene dosage. whilst the differential placement of late-born neurons identified in previous studies coincides spatially with the perturbed neuronal profile of pax mutant mice further experimentation is required to distinguish a link between the generation of subpopulations of cells and their relationship with pax and postnatal cytoarchitecture. furthermore, differential sensitivity to neuron death has been suggested for different functional populations of neurons within the superior colliculus. anti-apoptotic roles for pax genes are well-documented to-date and, importantly, the ciliary neurotrophic factor receptor has recently been demonstrated as a downstream target of pax within the mouse embryo. apparent neuronal recovery at p we have demonstrated altered pax7+ and neuronal profiles in pax mutant mice at p however at p we can no longer detect these variations between pax mutant and wildtype mice. whilst it is feasible that the similar number of pax7+ cells in pax7+/- mice relative to wildtype at p occurs because pax7+ cells in the adult do not normally include the subpopulation of cells which are missing at p in pax mutant mice, the apparent recovery of the neuronal profile by p has proven enigmatic. we have drawn from established studies of cns and superior collicular development to address this issue. it is clear that early postnatal stages of superior collicular development are tumultuous. the colliculus becomes invaded by retinal afferents just prior to birth and this continues within the first postnatal week, with formation of collaterals giving rise to axonal arborisation and commencement of synaptogenesis. during this process, a dramatic transformation occurs in which almost half of all fibre bundles are removed from the upper half of the stratum superficiale. this process coincides with naturally occurring neuron death in the superior colliculus, indicating neuronal formation in excess of adult requirements. excessive neuron formation is a recurrent theme in cns development. therefore it is likely that excessive neuron formation masks the loss of the more susceptible, pax7-deficient neurons, with sufficient cells remaining to satisfy functional requirements. subsequently, displacement of cells due to fibre invasion and the ensuring maturation of the tissue could possibly account for the apparent phenotypic equivalence of neun+ cells noted at p. it should be noted that functional studies have not been performed on pax mutant mice to-date to assess visual acuity. further studies a possible cause for the loss of superficial neurons in early postnatal stages could be altered circuitry. neurons of the superficial superior colliculus communicate with the deeper collicular regions through superficial neuronal axons, or via dendrites from neurons of the deep layers which in turn contain a variety of efferent projections from regions such as the cortex, retina, zona incerta and indirectly from the subthalamus via the substantia nigra pars reticulata or enteropeduncular nucleus. taking this into consideration, an interesting finding from this research is the persistent pax7+/pax3- expression profile of the subthalamus throughout all timepoints examined. providing pax is not expressed in this region at an earlier developmental stage than examined in this study, the subthalamus may represent a novel opportunity to further dissect the functional repertoire of pax from that of pax additional work would therefore be required to characterize the subthalamus of pax mutant mice to address this issue, as this important brain region is a target of deep brain stimulation to treat disorders such as parkinson's disease and epilepsy. further studies on the role of pax in superior collicular development would benefit from investigation into the axonal projection and circuity of pax7+ cells, which may be elaborated by the generation of pax7/tau-labelled x pax mutant mice. cell lineage tracing experiments in pax mutant mice would be required to trace the progression of cells generated at e and e to conclusively resolve the questions related to the early postnatal perturbation and the apparent neuronal recovery in the adult. moreover, functional studies assessing the ability of pax mutant mice to evoke a co-ordinated response to stimuli will be required to assess the integrity of the mature superior colliculus.  in summary pax while not required for neuronogenesis or neuronal differentiation at early stages of superior collicular development, is absolutely requisite in a dosage-dependent manner for long term maintenance of a subpopulation of dorsal mesencephalic neurons. this characteristic may well impart prophylactic properties to stem cells utilised in future replacement therapies to enhance treatment of neurodegenerative diseases of the midbrain. furthermore, comparative expression analyses indicate a functionally divergent role for pax and pax during neuronal differentiation within the superior colliculus. 
pax encodes a transcription factor well-established as an important determinant of mesencephalic identity and superior collicular development. this finding is paradoxical and has been attributed to functional redundancy afforded by its paralogue pax here we utilise pax mutant mice to investigate the precise role of this important developmental regulator during superior collicular development and neuronal specification/differentiation. in summary, these results demonstrate that during superior collicular development, pax is required to maintain a subpopulation of dorsal, mesencephalic neurons and partially regulates, spatiotemporally, pax expression within the cns. analysis of the superior colliculus of pax mutant and wildtype mice at a variety of developmental timepoints revealed that whilst correct initial specification is maintained, a subpopulation of dorsal mesencephalic neurons is lost at early postnatal stages. the differential nature of pax and pax with respect to neuronal differentiation may have implications for future stem cell therapies aimed at exploiting their developmental capabilities. we also assess its spatiotemporal relationship with pax during embryonic development. moreover, a comparative analysis of embryonic pax and pax expression profiles indicate that pax expression overlaps extensively with that of pax initially, but their expression domains increasingly diverge as development progresses, coinciding spatiotemporally with neuronal differentiation and maturation of the tissue. pax mutant mice, however, present with no obvious morphological impairments to the superior colliculus.
 pax encodes a transcription factor well-established as an important determinant of mesencephalic identity and superior collicular development. pax mutant mice, however, present with no obvious morphological impairments to the superior colliculus. this finding is paradoxical and has been attributed to functional redundancy afforded by its paralogue pax here we utilise pax mutant mice to investigate the precise role of this important developmental regulator during superior collicular development and neuronal specification/differentiation. we also assess its spatiotemporal relationship with pax during embryonic development.  analysis of the superior colliculus of pax mutant and wildtype mice at a variety of developmental timepoints revealed that whilst correct initial specification is maintained, a subpopulation of dorsal mesencephalic neurons is lost at early postnatal stages. moreover, a comparative analysis of embryonic pax and pax expression profiles indicate that pax expression overlaps extensively with that of pax initially, but their expression domains increasingly diverge as development progresses, coinciding spatiotemporally with neuronal differentiation and maturation of the tissue. furthermore, pax expression is perturbed within the cns of embryonic pax mutant mice.  in summary, these results demonstrate that during superior collicular development, pax is required to maintain a subpopulation of dorsal, mesencephalic neurons and partially regulates, spatiotemporally, pax expression within the cns. the differential nature of pax and pax with respect to neuronal differentiation may have implications for future stem cell therapies aimed at exploiting their developmental capabilities. 