Pax7 is requisite for maintenance of a subpopulation of superior collicular neurons and shows a diverging expression pattern to Pax3 during superior collicular development

BMC Developmental Biology Pax7 is requisite for maintenance of a subpopulation of superior collicular neurons and shows a diverging expression pattern to Pax3 during superior collicular development Jennifer A Thompson 2 Andreas Zembrzycki 0 1 Ahmed Mansouri 1 Mel Ziman 2 0 The Salk Institute for Biological Studies, Molecular Neurobiology Laboratory , La Jolla, CA , USA 1 Max Planck Institute for Biophysical Chemistry , Goettingen , Germany 2 School of Exercise, Biomedical and Health Science, Edith Cowan University , Joondalup Drive, Joondalup, Western Australia 6027 , Australia Background: 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. Results: 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. Conclusion: 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. - Background 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 [3] 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 [7], with reduced (2030%) retinal innervation [8], 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 [7]. 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 [10] 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. Results Pax gene expression patterns The Pax7 antibody has previously been demonstrated to be suitable for use in mouse tissue by Western Blot analysis [11]. The Pax3 antibody has been tested in vitro and in vivo for specificity in recognition of mouse Pax3 by Western Blot analysis [12]. Pax7 expression in wildtype mice Within the anlage of the tectum, at the earliest embryonic stage examined (E12.5), Pax7 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 (Fig 1ab). Pax7 expression can also be detected within the subthalamus, pretectum, pons, and in the ventricular zones of the cerebellar primordium, pons and myelencephalon (4th ventricle). This profile concurs with in situ hybridisation results reported previously at E13 [9], with the exception of expression detected at the 4th ventricle. At E15.5, a large number of immunoreactive cells are detected (Fig 2a) 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 (Fig 1c). Expression remains robust within the pons. Pax7 expression at E18.5 is similar to that noted at E15.5 (Fig 1e), however immunoreactivity is no (...truncated)