Pax7 is requisite for maintenance of a subpopulation of superior collicular neurons and shows a diverging expression pattern to Pax3 during superior collicular development
© Thompson et al; licensee BioMed Central Ltd. 2008
Received: 19 February 2008
Accepted: 30 May 2008
Published: 30 May 2008
Pax7 encodes a transcription factor well-established as an important determinant of mesencephalic identity and superior collicular development. Pax7 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 Pax3. Here we utilise Pax7 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 Pax3 during embryonic development.
Analysis of the superior colliculus of Pax7 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 Pax3 and Pax7 expression profiles indicate that Pax3 expression overlaps extensively with that of Pax7 initially, but their expression domains increasingly diverge as development progresses, coinciding spatiotemporally with neuronal differentiation and maturation of the tissue. Furthermore, Pax3 expression is perturbed within the CNS of embryonic Pax7 mutant mice.
In summary, these results demonstrate that during superior collicular development, Pax7 is required to maintain a subpopulation of dorsal, mesencephalic neurons and partially regulates, spatiotemporally, Pax3 expression within the CNS. The differential nature of Pax7 and Pax3 with respect to neuronal differentiation may have implications for future stem cell therapies aimed at exploiting their developmental capabilities.
It is evident that Pax7 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. Pax7 expression initially occurs in the neural tube and mesencephalon from very early stages [1, 2] 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 [4–7]. In the developing superior colliculus, graded expression of Pax7 establishes rostrocaudal and dorsoventral polarity. Expression of Pax7 localises within superior collicular neurons as development proceeds. This expression is upregulated during retinal innervation and axonal arborisation but reduced in Pax6 (Sey) mutant mice , with reduced (20–30%) retinal innervation , confirming that Pax7-expressing cells are responsive to retinal input. Demonstrated colocalisation in superior collicular neurons with the mapping marker ephrin-A2 validates Pax7 participation in retinotopic mapping . Continued, graded expression into adulthood is thought to maintain a small population of dorsal neurons in the mature colliculus [7, 9], although the functional requirement for this feature remains obscure.
Given the aforementioned importance of Pax7 in mesencephalic and superior collicular development, the lack of gross defects in this region in Pax7 mutant mice is surprising, and points to rescue by the paralogous Pax3 gene which has overlapping expression domains [9, 10]. Here, within the superior colliculus, we seek to determine the developmental role of Pax7 in specification of neurons, and assess its spatiotemporal relationship with Pax3. We have analysed Pax7 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 Pax7 mutant mice to maintain a subpopulation of dorsal superior collicular neurons.
Furthermore, analysis of Pax3 expression in embryonic wildtype and Pax7 mutant mice indicates crossregulation between paralogues, and illustrates a functional divergence during superior collicular development. We propose that within the superior colliculus initial overlapping Pax3 expression ensures correct neuronal specification, and temporospatial separation of expression patterns leads to solitary expression of Pax7 during a critical period of neuronal maturation, which abrogates the ability of Pax3 to compensate, revealing the aberrant phenotype.
Paxgene expression patterns
The Pax7 antibody has previously been demonstrated to be suitable for use in mouse tissue by Western Blot analysis . The Pax3 antibody has been tested in vitro and in vivo for specificity in recognition of mouse Pax3 by Western Blot analysis .
Pax7expression in wildtype mice
Pax7+ cell distribution in the superior colliculus of wildtype and Pax7 mutant mice at P5.
Pax7+ Cell Distribution
117.50 ± 8.29
311.50 ± 60.88
551.25 ± 106.79
126.25 ± 34.97
257.75 ± 56.98
317.25 ± 85.55
Pax7 expression in Pax7+/-mutant mice
In Pax7+/- embryos, the Pax7 expression profile is grossly indistinguishable from that of wildtype mice (Fig 1d,f); cell counts, wt = 2248.67 ± 94.75; Pax7+/- = 2373.67 ± 206.88 (p > 0.05; n = 3; Fig 2a). However, at P5, 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 (Table 1; Fig 1h; Fig 2b–e; n = 4 each). This manifests as a distinct area near the pial surface that is almost devoid of Pax7+ cells uniformly across the entire rostrocaudal (Fig 2d) and mediolateral (data not shown) 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 P5 does not reach statistical significance, this phenomenon results in the loss of graded cellular distribution across the dorsoventral axis (Table 1; Fig 2e; p > 0.05). Concomitantly there is some variation in cellular protein levels ventrally creating a lack of graded expression medially (Fig 2g). Interestingly, at P18.5 we did not discern any differences in Pax7 expression between heterozygous and wildtype mice; cell numbers, wt = 828.5 ± 108.97; Pax7+/- = 913.60 ± 69.77 (p > 0.05, Table 1; Fig 1i–j; Fig 2a; n = 3–5 at all stages).
Pax3 expression in wildtype and Pax7mutant 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 Pax7 mutant mice was due to altered proliferation we explored Ki67 expression at E12.5. We could not detect any variation in expression patterns between wildtype and mutant mice (data not shown), indicating that cellular proliferation proceeds normally, thus excluding neuronal precocity at this stage (n = 3–4).
We next sought to relate observed differences in the Pax7 and Pax3 expression profiles to temporal differences in neuronal differentiation. We have shown previously that Pax7 co-localises with βIII-tubulin and ephrin-A2 in the mouse superior colliculus at P5 . 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.
At E15.5 the SP is clearly visible in the ventral tectum, and immunolabelled, horizontally-oriented processes are now visible extending throughout the dorsorostral superior colliculus (see Additional file 1). These processes extend more caudally at E18.5 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 Pax7 mutant mice we could not discern any differences in expression profiles (Fig 4a–b) (n = 2–3 at all embryonic stages).
Similar results were obtained at E12.5 for α-internexin (Fig 4c–d; n = 3), and doublecortin (Fig 4e–f; n = 3), early markers of postmitotic  and migrating and differentiating [17, 18] neurons, respectively. Results confirm that in mutant mice (n = 3 each) at E12.5, neuronal differentiation occurs normally within the intermediate zone close to the pial surface in a rostralhigh to mid-caudallow manner.
By contrast, at P5, the most superficial region of the superior colliculus of Pax7 mutant mice is almost completely devoid of NeuN+ cells (Fig 5f) 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 (Fig 5e) (n = 3–6). Interestingly, within the adult mesencephalon (P18.5) NeuN, like Pax7, shows similar expression in both mutant mice and wildtype mice (n = 2–5).
Alterations to superior collicular polarity in Pax7mutant mice
Loss of Pax7+ cells and neurons in the dorsal superior colliculus at P5 (Fig 1g–h; Fig 2a–e; Fig 5e–f), 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-A2 expression in Pax7 mutant mice relative to expression in wildtype littermates. Consistent with the altered Pax7 and NeuN profile, at P5 ephrin-A2+ cells are missing in the most superficial region (Fig 5h,j), in contrast to wildtype expression displaying immunoreactivity close to the pial surface (Fig 5g,i), providing further evidence of neuronal loss and changes to polarity.
Neuronal loss is not due to astrocytic cell-fate switching or transdifferentiation
Neuronal loss may be attributable to cellular regression
To evaluate whether neuronal loss was due to apoptosis, we investigated activated caspase 3 expression from E15.5 to P5, however we did not detect either co-expression with Pax7 at any stage, or increased numbers of cells in mutant mice relative to wildtype (data not shown). 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 3 in neuronal differentiation . We therefore utilised Hoechst as an indicator of pyknotic nuclei, and assessed co-expression with activated caspase 3 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 Pax7 expression on formation of superior collicular boundaries, ie between the diencephalon and mesencephalon and between the superior colliculus (dorsal) and tegmentum (ventral mesencephalon), we assessed Pax6 expression at the rostral and ventral boundaries of the superior colliculus [4, 9, 23]. At E12.5, there are a few weakly-stained Pax6+ cells superior to the interpeduncular fossa. At E15.5 in wildtype mice, Pax6+ cells can be detected dorsally in the pretectum (Fig 6e,f) sometimes spreading to the rostral margin of the ventricular zone. Pax6 immunoreactivity could not be detected within the pretectum at E18.5 in either wildtype or mutant mice. We could not detect any abnormality in the expression domain of Pax6 in Pax7 mutant mice at these midgestational stages (n = 3).
To investigate alterations to mesencephalic/metencephalic boundary formation, we assessed the expression of the marker En-1  from E15.5 to P18.5. En-1+ cells can be detected at the midbrain/hindbrain boundary, from the interpeduncular fossa to the rhombencephalic isthmus (Fig 6g,h) and expression wanes as development proceeds until P18.5, when En-1 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 (n = 2–3).
In order to understand the role of Pax7 in superior collicular development we investigated the superior colliculus of Pax7 mutant mice relative to wildtype at a variety of developmental stages. Whilst both Pax3 and Pax7 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 Pax7 dramatically alters Pax3 expression domains and disturbs the neuronal profile in the dorsal layers of the developing superior colliculus.
Pax7 and Pax3have separate roles during neuronal differentiation in the superior colliculus
Since Pax7 and Pax3 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 Pax7 and shed light on their individual roles during superior collicular development.
At E15.5 tectal Pax7 expression within the ventricular zone decreases, and is no longer evident at E18.5, with expression persisting within the upper developing primary layers (strata profundum, intermedium and superficiale). However, Pax3 expression becomes restricted to the ventricular zone, with a few scattered cells within the intermediate region at E15.5 decreasing at E18.5. Postnatally, expression of Pax7 is detected throughout the superior colliculus, numerically declining with maturation yet persisting at all developmental stages examined, whilst Pax3 expression is extinguished.
The divergent Pax3/Pax7 expression profiles, as demonstrated in this paper, suggest a dichotomy of function with respect to neuronal differentiation. The Pax7 expression profile, without co-expression of Pax3, appears spatially associated with neuronal differentiation and maturation of the superior colliculus, whilst Pax3 expression appears to be negatively associated with these characteristics. A role for Pax3 in maintaining the undifferentiated phenotype has been demonstrated previously, both in vitro and in vivo for neuronal cells [25, 26], for Schwann cells  and for melanoblasts [28, 29]. Conversely, transfection of Pax7 into P19 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 E12.5, just prior to the normal birthdate of the superficially located neurons. At this timepoint, the intermediate zone appears as an expanded region rostrally (which varies slightly at different positions across the mediolateral axis) diminishing to a thin region at the pial surface mid-caudally, indicating a rostral to caudal maturity, and is characterised by the expression of Pax7, βIII-tubulin (somatic staining), α-internexin, doublecortin and NeuN, and morphologically by increased internuclear distances. By contrast, Pax3 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 Pax6 within the eye of Sey mutant mice . However, a more thorough investigation at closer temporal increments from E13 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 Pax3 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 Pax7 levels. The unaltered proliferation/specification of tectal cells does not preclude a role for Pax7 in these processes per se, but may indicate compensation via Pax3.
The observed perturbations in Pax3 expression in Pax7 mutant mice may indicate that Pax7 acts to limit the expression domain of Pax3. This is supported by the rostral, ventral and caudal expansion of Pax3+ cells in the presence of reduced or absent Pax7 expression. These regions of expanded expression occur where Pax7 expression would normally be encountered, and suggests that Pax7 acts to dorsalise (or at least positionally constrain) Pax3 expression. A cell autonomous relationship between Pax7 and Pax3 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 Pax7 precedes that of Pax3 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 Pax7in timing of neuronal specification
Expression analysis and cellular quantification have demonstrated that Pax7 haploinsufficiency does not alter the initial proliferation steps in Pax7+ cells during mid embryonic stages. The only notable disparity to Pax7 expression occurs at P5, 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.
Overall, our results tend to suggest that Pax7 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 E15.5 and E18.5, 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 Pax7in postnatal superior collicular development
As earlier quantification did not detect any numerical changes in the Pax7 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 Pax7 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 3, 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 3, concomitant with Pax7 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 (wildtype) and increased (mutant) apoptosis. However, the loss of neurons evidenced in Pax7 mutant mice indicates a loss of cells, rather than simply extinction of Pax7 expression.
Apparent neuronal recovery at P18.5
We have demonstrated altered Pax7+ and neuronal profiles in Pax7 mutant mice at P5, however at P18.5 we can no longer detect these variations between Pax7 mutant and wildtype mice. Whilst it is feasible that the similar number of Pax7+ cells in Pax7+/- mice relative to wildtype at P18.5 occurs because Pax7+ cells in the adult do not normally include the subpopulation of cells which are missing at P5 in Pax7 mutant mice, the apparent recovery of the neuronal profile by P18.5 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 [15, 43, 44]. 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 (the region including the perturbation where Pax7+ cells are essentially absent) [15, 44]. This process coincides with naturally occurring neuron death in the superior colliculus [36, 45], indicating neuronal formation in excess of adult requirements. Excessive neuron formation is a recurrent theme in CNS development [22, 46, 47]. 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 P18.5. It should be noted that functional studies have not been performed on Pax7 mutant mice to-date to assess visual acuity.
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 [48, 49], 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 (region surrounding subthalamus) [32, 51–53] and indirectly from the subthalamus via the substantia nigra pars reticulata or enteropeduncular nucleus [54–56]. 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 Pax3 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 Pax7 from that of Pax3. Additional work would therefore be required to characterize the subthalamus of Pax7 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 [56, 57].
Further studies on the role of Pax7 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 Pax7 mutant mice. Cell lineage tracing experiments in Pax7 mutant mice would be required to trace the progression of cells generated at E11 and E13 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 Pax7 mutant mice to evoke a co-ordinated response to stimuli will be required to assess the integrity of the mature superior colliculus.
In summary Pax7, 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 Pax7 and Pax3 during neuronal differentiation within the superior colliculus.
The generation and genotyping of Pax7 mutant mice has been described previously . Principles of laboratory animal care (NIH publication No. 86-23, revised 1985) were followed, and animal experimental procedures conformed to National Health and Medical Research Council of Australia guidelines, with approval by the Animal Ethics Committee of Edith Cowan University. The production of transgenic animals at the Max Planck Institute for Biophysical Chemistry was performed with the approval of LAVES (Landesamt für Verbraucherschutz und Lebensmittelsicherheit) in Oldenburg.
Whole embryos (E12.5) or embryonic brains (>E15.5) from mutant and wildtype littermates were isolated and postfixed in 4% paraformaldehyde. Postnatal mice were deeply anaesthetised with Avertin and intracardially perfused with fixative. Brains were isolated and postfixed with 4% paraformaldehyde. Tissue was cryoprotected in 20% or 30% sucrose/PBS prior to sectioning. Embryos at E12.5 were sectioned whole. For all other stages, brain hemispheres were sectioned in the sagittal (left) or coronal (right) planes at 10–20 μm, and slides were stored at -80°C until required. A minimum of three animals per group (+/+; +/-; -/-) per timepoint were analysed (with the exception of Pax7-/- animals at E18.5, where n = 2).
Whole embryo tissue from E12.5 mice was subjected to antigen retrieval by microwave heating in sodium citrate buffer pH6.0 prior to processing. All tissue sections were treated with 0.2% Triton-X100/PBS (10 min), 1.5% H2O2/PBS (2–3 × 10 min), blocked with 10% fetal calf serum/PBS (30 min) and incubated overnight at 4°C with primary antibodies; Pax7 (1:20 or 1:10 (E12.5), mouse, monoclonal, DSHB, Iowa City, IA, USA); Pax3 (1:100 or 1:50 (E12.5), mouse, monoclonal, DSHB, Iowa City, IA, USA); Pax6 (1:100, mouse, monoclonal, DSHB, Iowa City, IA, USA); En-1 (1:50, mouse, monoclonal, DSHB, Iowa City, IA, USA); ephrin-A2 (1:500, rabbit, polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA); NeuN (1:100, mouse, monoclonal, Chemicon/Millipore, Billerica, MA, USA). Sections were then incubated with biotinylated anti-mouse/anti-rabbit IgG (1:3, Dako, Sydney, NSW, Australia) followed by streptavidin/HRP complex (1:3, Dako, Sydney, NSW, Australia) (20 mins each at room temperature). Visualization with diaminobenzidine (3% in substrate buffer, Dako, Sydney, NSW, Australia) preceeded processing through an ethanol series/xylene, and mounting in DePex. Control slides without primary antibodies were immunonegative.
Immunohistochemical quantification and analysis
Images were captured with a Leica DC300 camera attached to an Olympus BX41 microscope and analysed using Optimas 6.5 Digital Image Analysis software (Media Cybernetics, Bethesda, MD, USA). For cellular quantification at E15.5 and P18.5 images were captured within Optimas (100× magnification) and Pax7+ cells were marked and counted. For optical density and cell distribution measurements at P5, the microscope was equilibrated prior to analysis, and microscope and image software settings were standardised. Measurements of Pax7 immunostaining were obtained on a frame-by-frame basis (400× magnification), and encompassed the entire Pax7+ portion of the superior colliculus, producing a serial reconstruction of the region (Coggeshall and Lekan 1996). Each individual cell measurement was topographically replotted within Excel and values were normalised against immunonegative adjacent tissue. Results were subsequently analysed producing maps of mean cellular distribution, or graphed as either mean cellular protein level or mean cell number at each axis point. Standard errors are shown.
Tissue sections were blocked with 3% normal goat serum (NGS)/0.2% Triton-X100/TBS (TX-TBS) for one hour at room temperature and incubated overnight at 4°C with primary antibody diluted in 1% NGS/TX-TBS. Primary antibodies included Pax7 (1:10); Pax3 (1:50); activated-Caspase 3 (1:100, rabbit, polyclonal, Promega, Sydney, NSW, Australia); alpha-internexin (1:150, rabbit, polyclonal, Chemicon/Millipore, Billerica, MA, USA); βIII tubulin (TuJ1)(1:500, rabbit, polyclonal, Abcam, Cambridge, MA, USA); Doublecortin (1:500, rabbit, polyclonal, Abcam, Cambridge, MA, USA); GFAP (1:1000, rabbit, polyclonal, Chemicon/Millipore, Billerica, MA, USA); Ki67 (1:150, rabbit, polyclonal, Abcam, Cambridge, MA, USA). Primary antibodies were visualised with conjugated anti-rabbit IgG-AlexaFluor488 (1:500; Molecular Probes-Invitrogen, Melbourne, VIC, Australia), or biotinylated goat anti-mouse IgG (1:500; Abcam, Cambridge, MA, USA), incubated overnight followed by streptavidin-AlexaFluor546 (1:500; Molecular Probes-Invitrogen, Melbourne, VIC, Australia), incubated for 2 hours at room temperature. Slides were counterstained with Hoechst (1/5000; Sigma Aldrich, Sydney NSW, Australia) and mounted in Fluorsave (Calbiochem, La Jolla CA, USA). Controls with primary antibodies omitted were immunonegative. Fluorescent micrographs and the confocal z-stack image (see Additional file 1) were obtained using Confocal Assistant software (Version 4.02) and the Biorad MRC 1000/1024 Confocal Microscope or Olympus BX41 microscope equipped with an Olympus DP71 camera.
Statistical analysis (Table 1)
To assess variation between wildtype and mutant mice at E15.5, P5 and P18.5, we compared the number of Pax7+ cells in wildtype mice with those in Pax7+/- mice using paired t test (two-tailed). For determination of graded expression at P5, mean values at three points across each axis (rostral, mid and caudal, or ventral, mid and dorsal) were calculated by averaging cell numbers in two frames at each axis position. Graded expression across each axis was assessed by analysis of significant variation between axis points using the paired t test (two-tailed): rostral vs mid (R-M), mid vs caudal (M-C), rostral vs caudal (R-C) or ventral vs mid (V-M), mid vs dorsal (M-D), ventral vs dorsal (V-D). Corresponding significance values are reported in Table 1. All statistical analyses were conducted utilising SPSS Version 13.0 Statistical Software, with a 95% confidence interval.
The authors would like to thank Robert White for thoughtful discussions, Judith Blake for assistance with BioRad Microscopy, and Carole Bartlett for the kind gift of Ki67 antibody and for technical assistance. The Pax7 monoclonal antibody, developed by Atsushi Kawakami, and the Pax3 monoclonal antibody, developed by C.P. Ordahl, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. BioRad microscopy was carried out using facilities at the Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, which is supported by University, State and Federal Government funding. This project was supported by a grant to JT from the Karrakatta Club. AZ was supported by the Max Planck Society and the DFG Center for Molecular Physiology of the Brain (CMPB). The authors gratefully acknowledge the assistance of the Max Planck Society and Dr. Helmut Storz Stiftung.
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