Table of contents

Mutations in mammalian Pax-6 homologs: General

The human eye malformation aniridia results from haploinsufficiency of PAX6, a paired box DNA-binding protein. To study this dosage effect, two PAX6 mutations were characterized in a family segregating aniridia and a milder syndrome consisting of congenital cataracts and late onset corneal dystrophy. The nonsense mutations, at codons 103 and 353, truncate PAX6 within the N-terminal paired and C-terminal PST domains, respectively. The wild-type PST domain activates transcription autonomously and the mutant form has partial activity. A compound heterozygote had severe craniofacial and central nervous system defects and no eyes. The pattern of malformations is similar to that in homozygous Sey mice and suggests a critical role for PAX6 in controlling the migration and differentiation of specific neuronal progenitor cells in the brain (Glaser, 1994).

Vertebrate Pax proteins share a conserved 128-amino-acid DNA-binding motif, the paired domain. The PAX6 gene, which is mutated in the murine Small eye and human aniridia developmental defects, also encodes a second protein with a 14-amino-acid insertion in the paired domain. This protein, which arises by alternative mRNA splicing, exhibits unique DNA-binding properties. Unlike other paired domains, which bind DNA predominantly by their amino termini, the extended Pax6 paired domain interacts with DNA exclusively through its carboxyl terminus. This property can be stimulated by deletion of 30 amino-terminal residues from the Pax6 or Pax2 paired domains. Thus, the insertion acts as a molecular toggle to unmask the DNA-binding potential of the carboxyl terminus. The functional nonequivalence of the two Pax6 proteins is underscored by a T-->C mutation at position -3 of the alternative splice acceptor site that changes the ratio of the two isoforms and causes a distinct human ocular syndrome (Epstein, 1994).

Mouse embryos, homozygous for the small eye (Sey) mutation die soon after birth with severe facial abnormalities that result from the failure of the eyes and nasal cavities to develop. Mutations in the Pax6 gene are responsible for the Sey phenotype. As a general disruption of eye and nasal development occurs in the homozygous Sey embryos, it is unclear, from the mutant phenotype alone, which tissues require functional Psx6. Chimeric mouse embryos were created composed of wild-type and Sey mutant cells. In these embryos mutant cells are excluded from both the lens and nasal epithelium. Both of these tissues are smaller, and in some cases absent, in chimeras with high proportions of mutant cells. The morphology of the optic cup is also severely affected in these chimeras; mutant cells were excluded from the retinal pigmented epithelium and did not intermix with wild-type cells in other regions. The evidence shows that Pax6 has distinct roles in the nasal epithelium and the principal tissue components of the embryonic eye, acting directly and cell autonomously in the optic cup and lens. It is suggested that that Pax6 may promote cell surface changes in the optic cup and control the fate of the ectoderm from which the lens and nasal epithelia are derived (Quinn, 1996).

The transcription factor Pax6 is widely expressed throughout the developing nervous system, including most alar regions of the newly formed murine diencephalon. Later in embryogenesis its diencephalic expression becomes more restricted. It persists in the developing anterior thalamus (conventionally termed 'ventral' thalamus) and pretectum but is downregulated in the body of the posterior (dorsal) thalamus. At the time of this downregulation, the dorsal thalamus forms its major axonal efferent pathway via the ventral telencephalon to the cerebral cortex. This pathway is absent in mice lacking functional Pax6 (small eye homozygotes: Sey/Sey). A test was performed to see whether the mechanism underlying this defect includes abnormalities of the dorsal thalamus itself. A new transgenic mouse was used that ubiquitously expresses green fluorescent protein tagged with tau. In this transgenic mouse, axonal tracts are clearly visible. Dorsal thalamic explants from Pax6+/+ or Pax6Sey/Sey embryos carrying the transgene were co-cultured with wild-type tissues from other regions of the forebrain. Whereas Pax6+/+ thalamic explants produce strong innervation of wild-type ventral telencephalic explants in a pattern that mimics the thalamocortical tract in vivo, Pax6Sey/Sey explants do not, indicating a defect in the ability of mutant dorsal thalamic cells to respond to signals normally present in ventral telencephalon. Pax6Sey/Sey embryos also show early alterations in the expression of regulatory genes in the region destined to become dorsal thalamus. Whereas in normal mice Nkx2.2 and Lim1/Lhx1 are expressed ventral to this region, in the mutants their expression domains are throughout the region, suggesting that a primary action of Pax6 is to generate correct dorsoventral patterning in the diencephalon. These results suggest that normal thalamocortical development requires the actions of Pax6 within the dorsal thalamus itself (Pratt, 2000).

Early events in the formation of the thalamocortical tract remain poorly understood. A model to explain how the thalamocortical tract forms postulates a role for transient axons projecting from cells in the medial part of the ventral telencephalon into the dorsal thalamus. Thus thalamocortical axons follow a path pioneered by transient thalamic afferents originating from the medial part of the ventral telencephalon. The development of these transient afferents and the thalamocortical tract were studied in mutant mice lacking transcription factors normally expressed in the dorsal thalamus or ventral telencephalon. Pax6 is expressed in the dorsal thalamus, but not in the medial part of the ventral telencephalon, and the thalamocortical tract fails to form in Pax6-/- embryos. Transient thalamic afferents from the ventral telencephalon do not form in Pax6-/- embryos; this may contribute to the failure of their thalamocortical development. The distribution of Pax6-/- cells in Pax6-/-->Pax6+/+ chimeras supports conclusions drawn from forebrain marker gene expression that Pax6 is not required for the normal development of the medial part of the ventral telencephalon but is required in the dorsal thalamus. Failure of the transient afferent pathway to develop is therefore likely a cell nonautonomous defect reflecting primary defects in the thalamus. The formation of thalamic afferents and efferents were studied in Foxg1-/- embryos, which lack recognizable ventral telencephalic structures. In these embryos thalamic efferents navigate correctly through the thalamus but fail to turn laterally into the telencephalon, whereas other axons are able to cross the diencephalic/telencephalic boundary. These results support a role for the ventral telencephalon in guiding the early development of the thalamocortical tract and identify a new role for the transcription factor Pax6 in regulating the ability of the thalamus to attract ventral telencephalic afferents (Pratt, 2002).

A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing beta-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants: Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitfmi, with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of beta-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitfmi/+ embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quantification and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitfmi/Mitfmi embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration (Hornyak, 2001).

Phenotype-based mutagenesis experiments will increase the mouse mutant resource, generating mutations at previously unmarked loci as well as extending the allelic series at known loci. Mapping, molecular characterization, and phenotypic analysis of nine independent Pax6 mutations of the mouse recovered in mutagenesis experiments are all presented. Seven mutations result in premature termination of translation and all express phenotypes characteristic of null alleles, suggesting that Pax6 function requires all domains to be intact. Of major interest is the identification of two possible hypomorph mutations: Heterozygotes express less severe phenotypes and homozygotes develop rudimentary eyes and nasal processes and survive up to 36 hr after birth. Pax64Neu results in an amino acid substitution within the third helix of the homeodomain. Three-dimensional modeling indicates that the amino acid substitution interrupts the homeodomain recognition alpha-helix, which is critical for DNA binding. Whereas cooperative dimer binding of the mutant homeodomain to a paired-class DNA target sequence is eliminated, weak monomer binding is observed. Thus, a residual function of the mutated homeodomain may explain the hypomorphic nature of the Pax64Neu allele. Pax67Neu is a base pair substitution in the Kozak sequence and results in a reduced level of Pax6 translation product. The Pax64Neu and Pax67Neu alleles may be very useful for gene-dosage studies (Favor, 2001).

The transcription factors Pax2 and Pax6 are co-expressed in the entire optic vesicle (OV) prior and concomitant with the establishment of distinct neuroretinal, retinal, pigmented-epithelial and optic-stalk progenitor domains, suggesting redundant functions during retinal determination. Pax2; Pax6 compound mutants display a dose-dependent reduction in the expression of the melanocyte determinant Mitf, accompanied by transdifferentiation of retinal pigmented epithelium (RPE) into neuroretina (NR) in Pax2-/-; Pax6+/- embryos, which strongly resembles the phenotype of Mitf-null mutants. In Pax2-/-; Pax6-/- OVs Mitf fails to be expressed and NR markers occupy the area that usually represents the Mitf+ RPE domain. Furthermore, both Pax2 and Pax6 bind to and activate a MITF RPE-promoter element in vitro, whereas prolonged expression of Pax6 in the Pax2-positive optic stalk leads to ectopic Mitf expression and RPE differentiation in vivo. Together, these results demonstrate that the redundant activities of Pax2 and Pax6 direct the determination of RPE, potentially by directly controlling the expression of RPE determinants (Bäumer, 2003).

During development, Pax6 is expressed in a rostrolateral-high to caudomedial-low gradient in the majority of the cortical radial glial progenitors and endows them with neurogenic properties. Using a Cre/loxP-based approach, the effect was studied of conditional activation of two Pax6 isoforms, Pax6 and Pax6-5a, on the corticogenesis of transgenic mice. It was found that activation of either Pax6 or Pax6-5a inhibits progenitor proliferation in the developing cortex. Upon activation of transgenic Pax6, specific progenitor pools with distinct endogenous Pax6 expression levels at different developmental stages show defects in cell cycle progression and in the acquisition of apoptotic or neuronal cell fate. The results provide new evidence for the complex role of Pax6 in mammalian corticogenesis (Berger, 2007).

Dual requirement for Pax6 in retinal progenitor cells

Throughout the developing central nervous system, pre-patterning of the ventricular zone into discrete neural progenitor domains is one of the predominant strategies used to produce neuronal diversity in a spatially coordinated manner. In the retina, neurogenesis proceeds in an intricate chronological and spatial sequence, yet it remains unclear whether retinal progenitor cells (RPCs) display intrinsic heterogeneity at any given time point. A detailed study was performed of RPC fate upon temporally and spatially confined inactivation of Pax6. Timed genetic removal of Pax6 appeared to unmask a cryptic divergence of RPCs into qualitatively divergent progenitor pools. In the more peripheral RPCs under normal circumstances, Pax6 seemed to prevent premature activation of a photoreceptor-differentiation pathway by suppressing expression of the transcription factor Crx. More centrally, Pax6 contributed to the execution of the comprehensive potential of RPCs: Pax6 ablation resulted in the exclusive generation of amacrine interneurons. Together, these data suggest an intricate dual role for Pax6 in retinal neurogenesis, while pointing to the cryptic divergence of RPCs into distinct progenitor pools (Oron-Karni, 2008).

Recent studies have shown that in the developing neocortex there are several distinct neurogenic progenitor cells that are multipotent, including the radial glia and intermediate progenitor cells. Within these populations, Pax6 is expressed and plays different roles, depending on the temporal and spatial context. Moreover, a dual role for Pax6 has been reported in the generation of neurons of the adult olfactory bulb, where Pax6 was found to initially regulate the establishment of the neuronal lineages and, subsequently, their specification toward a periglumerular cell fate. In contrast to the developing neocortex, differences among RPCs have not yet been recognized in the developing retina. However, there are several lines of evidence supporting distinct transient states of these cells: first, considering the central-peripheral pattern of differentiation, it is likely that the RPCs located adjacent to differentiating neurons at the central OC are exposed to different cues from the RPCs located far from the differentiation front, at the OC periphery. Second, recent findings have shown the differential expression of genes in the central versus peripheral regions. Finally, in this study, two distinct phenotypes of RPCs were identified after Pax6 inactivation in the OC, including differences in the expression Crx, the expression profile of proneural bHLH genes, proliferation index and neurogenic potential. Moreover, these different phenotypes were correlated to the location of the cells along the central-peripheral axis of the OC. Together, these findings indicate an important distinction between Pax6 activities within adjacent RPC pools, suggesting an inherent difference between RPC populations. Considering that all retinal cell types eventually populate both central and peripheral retina in the adult, it seems likely that the differences documented in this study between central and peripheral OC RPCs primarily reflect distinct differentiation stages of the multipotent progenitor pools, similar to the transient states observed in cortical neurogenesis rather than differences in cell specification. In this case, the role of Pax6 is to promote the maturation of progenitor cells and their eventual differentiation to all of the retinal cell types (Oron-Karni, 2008).

The iterative deployment of Pax6 in the process of eye formation in evolutionarily distant organisms, suggests that there are common transcriptional targets for Pax6 in the different species, such as the regulation of opsin gene expression. In support of this idea, eyeless, the fly homolog of Pax6, was found to be expressed in photoreceptors and was subsequently shown to regulate the expression of the Drosophila rhodopsin genes in these cells. In vertebrates, however, this role for Pax6 does not appear to be conserved, in line with the rapid downregulation of Pax6 expression in differentiating photoreceptors during vertebrate retinogenesis. Moreover, ChIP data indicate selective binding of Pax6 protein to the Crx promoter region, supporting its role as a direct transcriptional repressor of photoreceptor fate. The current study reveals the complex involvement of Pax6 in the transcriptional network leading to photoreceptor differentiation in mammals. Surprisingly, although in both regions Pax6 is essential for completion of the photoreceptor-differentiation program, its regulation of the genes involved in the photoreceptor lineage is different in the two regions of the OC: in region 1 it plays a role in inhibiting the onset of Crx expression, whereas in region 2 it is required for the expression of Crx. Thus, based on these findings, the ancestral role of Pax6 in regulating opsin expression appears to have switched to a different, more complex, level of control over key retinogenic programs (Oron-Karni, 2008).

Mutations in mammalian Pax-6 homologs: Pax-6 role in cortical and sub-cortical development

To evaluate the role of Pax6 in forebrain development, mouse Small eye/Pax6 mutant brains were studied in detail. This analysis revealed severe defects in forebrain regions where Pax6 is specifically expressed. The establishment of some expression boundaries along the dorsoventral axis of the secondary prosencephalon is distorted and the specification of several ventral structures and nuclei is abolished. Specifically, the development of the hypothalamo-telencephalic transition zone and the ventral thalamus is distorted. The detailed analysis included a comparison of the expression of Pax6, Dlx1 (Drosophila homolog: Distal-less) and several other genes during embryonic mouse brain development in wild-type and in the mutant Small eye (Sey) brain. The results from the analysis of normal brain development show that the restricted expression of Pax6 and Dlx1 at E12.5 dpc respect domains within the forebrain, consistent with the implications of the prosomeric model for the organization of the forebrain. Furthermore, the is an early restriction of Pax6 and Dlx1 expression into presumptive histogenetic fields that correlate with the formation of distinct forebrain structures and nuclei. The lack of the restrictive capacity at the border between neocortex and lateral ganglionic eminence in Sey mutants may be explained by a loss of adhesive barrier properties in the Pax6 mutant. These adhesive differences decline at later developmental stages, correlating with the decreased abundance of Pax 6 transcripts in the cortical region. The close correlation between the region-specific expression of adhesion molecules such as R-cadherin or a selectin ligand in the forebrain with the Pax6 expression domains further supports a link between adhesive events and the patterning by Pax6 (Stoykova, 1996).

Mutations in the gene for the transcription factor Pax6 induce marked developmental abnormalities in the CNS and the eye, but the cellular mechanisms that underlie the phenotype are unknown. The adhesive properties of cells from the developing forebrain in Small eye, the Pax6 mutant mouse, have been examined. The segregation normally observed in aggregates of cortical and striatal cells in an in vitro assay is lost in Small eye. This correlates with an alteration of in vivo expression of the homophilic adhesion molecule, R-cadherin, which is expressed exclusively in the cortex. Moreover, the boundary between cortical and striatal regions of the telencephalon is dramatically altered in Small eye: radial glial fascicles do not form at the border, and the normal expression of R-cadherin and tenascin-C at the border is lost. These data suggest links between the transcription factor Pax6 and R-cadherin expression, cellular adhesion and boundary formation between developing forebrain regions (Stoykova, 1997).

Pax-6 is one of the earliest regulatory genes to be expressed in the diencephalon: Pax-6 protein is required for early diencephalic development. In Pax-6 mutant Small-eye mice, diencephalic morphology was abnormal at all the embryonic ages studied (days 10.5, 12.5 and 14.5). Regional differences in diencephalic cell density are lost, the diencephalon/mesencephalon boundary is unclear and the third ventricle is enlarged. Diencephalic proliferative rates are abnormally low in mutants as early as embryonic day 10.5. In older mutants, the diencephalon contained fewer cells than normal (Warren, 1997).

In wild-type E14.5 diencephalon, Pax-6, Dlx-2 and Wnt-3 are expressed in discrete regions along the rostrocaudal and dorsoventral axes. In situ hybridizations for these genes in E14.5 Small-eye mice reveal discrete zones of diencephalic expression that have similar relative positions to those in wild-type mice. Some differences of detail in their expression are seen: Pax-6 has an expanded rostral domain of expression and an abnormally indistinct caudal boundary; Dlx-2 has a diffuse, rather than a sharp, caudal boundary of expression; the normally high dorsal midline expression of Wnt-3 is lost. It is concluded that normal expression of Pax-6 is required for the correct regulation of diencephalic precursor proliferation. Pax-6 may also control some aspects of diencephalic differentiation, but its mutation in Small-eye mice does not preclude the development of a degree of diencephalic regionalization resembling that in normal mice. The alteration in the level of Pax-6 transcripts in Small-eye embryos suggests that a functional Pax-6 protein is require (directly or indirectly) for the expression of its own gene in the forebrain: in vitro binding studies have demonstrated that Pax-6 can recognize sites within its own promoter. A degree of diencephalic regionalization can occur without normal Pax-6 expression. Despite the early proliferative defects, distinct and recognizable dienchephalic regions (ventral thalamus, dorsal thalamus and pretectum), corresponding to their counterparts in wild-type embryos, do emerge in Small-eye embryos. Thus, if there is a master regulator of diencephalic regionalization, it is unlikely to be Pax-6. It is suggested that the major roles of Pax-6 in the diencepahlon are in the control of cell proliferation and the later differentiation of some features of its three main regions (Warren, 1997).

The Pax-6 gene encodes a transcription factor that is expressed in regionally restricted patterns in the developing brain and eye. Pax-6 expression is described in the early forebrain (prosencephalon) from embryonic day 9.5 (E9.5) to E10.5. There is a close correlation between Pax-6+ domains and initial neural patterning, and these domains correspond to those showing defects in embryos homozygous for the Pax-6 allele, Small eye (Sey). Pax-6 expression defines the prosencephalon-mesencephalon boundary. Mutant embryos lack this morphological boundary. In mutants, markers for the caudal prosencephalon are lost (Pax-6, Lim-1, Gsh-1) and a homoeodomain marker for mesencephalon is expanded rostrally into the prosencephalon. It is concluded that the caudal prosencephalon (prosomere 1) is at least partially transformed to a mesencephalic fate. This transformation results in a specific deficit of posterior commissure axons. Sey/Sey mutant embryos also exhibit an axon pathfinding defect specific to the first longitudinal tract in the prosencephalon (tpoc, tract of the postoptic commissure). In wild type, tpoc axons fan out on coming in contact with a superficial patch of Pax-6+ neuron cell bodies. In the mutant, the tpoc axons have normal initial projections, but make dramatic errors where they contact the neuron cell bodies, and fail to pioneer this first tract. Thus Pax-6 is required for local navigational information used by axons passing through its domain of expression. It is concluded that Pax-6 plays multiple roles in forebrain patterning, including boundary formation, regional patterning, neuron specification and axon guidance (Mastick, 1997).

The cerebral cortex forms by the orderly migration and subsequent differentiation of neuronal precursors generated in the proliferative ventricular zone. The role of the transcription factor Pax-6, which is expressed in the ventricular zone, was studied in cortical development. Embryos homozygous for a mutation of Pax-6known as Small eye (Sey) have abnormalities suggesting defective migration of late-born cortical precursors. Despite their apparent inability to exit from the subventricular zone, the later-born cells in Sey/Sey embryos do express the neuron-specific marker, TuJ1. Mutant cortical precursors born early in neurogenesis migrate into the cortical plate and adopt positions very similar to those adopted by wild-type cells. Radial glia are present in E16 Sey/Sey embryos, although few of them are in the heterotopic clusters. It is not yet clear whether this is a primary cause of the failure of clustered cells to migrate or whether radial glia are simply displaced by an accumulation of cells that do not migrate for some other reason. When late-born Sey/Sey precursors are transplanted into wild-type embryonic rat cortex, they showed similar integrative, migrational and differentiative abilities to those of transplanted wild-type mouse precursors. These results suggest that postmitotic cortical cells do not need Pax-6 to acquire the capacity to migrate and differentiate, but that Pax-6 generates a cortical environment that permits later-born precursors to express their full developmental potential (Caric, 1997).

The contribution of extrinsic and genetic mechanisms in determining areas of the mammalian neocortex has been a contested issue. This study analyzes the roles of the regulatory genes Emx2 and Pax6, which are expressed in opposing gradients in the neocortical ventricular zone. Emx2 is expressed in low rostral to high caudal and low lateral to high medial gradients, whereas Pax6 is expressed in low caudal to high rostral and low medial to high lateral gradients. Changes in the patterning of molecular markers and area-specific connections between the cortex and thalamus suggest that arealization of the neocortex is disproportionately altered in Emx2 and Pax6 mutant mice in opposing manners predicted from their countergradients of expression: rostral areas expand and caudal areas contract in Emx2 mutants, whereas the opposite effect is seen in Pax6 mutants. These findings suggest that Emx2 and Pax6 cooperate to regulate arealization of the neocortex and to confer area identity to cortical cells (Bishop, 2000).

The type II classical cadherins, Cadherin6 (Cad6) and Cadherin8 (Cad8), are expressed in areal patterns in the late embryonic mouse neocortex: Cad8 has been reported to mark the rostrally located motor area, and Cad6 marks the somatosensory area, located immediately caudal to the motor area, and the auditory area, located in the caudolateral neocortex. Because the cortex is reduced in size in both Emx2 and Pax6 mutants, as compared to wild type, the proportion of the cortical surface covered by domains of cadherin expression is determined as well as absolute domain size. No change in proportional sizes of cadherin expression domains would indicate that arealization per se has not been affected, suggesting that the full range of putative area identities is present in the smaller cortex and that all areas are uniformly reduced in size. A change in proportional sizes would indicate that areas are disproportionately affected in the mutant neocortex and therefore that Emx2 and/or Pax6 has a role in regulating arealization of the neocortex. This finding and interpretation would be most strongly supported by a change in absolute sizes of cadherin expression domains (Bishop, 2000).

The areal pattern of cadherin expression in the Emx2 homozygous mutant cortex is substantially different from that in wild type. The domain of Cad8 expression is expanded caudally. This expansion appears to be greater along the medial edge of the cortex than laterally, farther on, suggesting that Cad8 expression may also be expanded medially. The domain of Cad6 expression is shifted caudally and medially, as seen on the dorsal and lateral surfaces of the cortex. No changes in the cortical patterns of cadherin expression are observed in Emx2 heterozygous mice. The domain of Cad8 expression is significantly larger in the Emx2 homozygous mutant neocortex, in both proportional and absolute area, as well as in its proportional and absolute linear extent across the cortical surface. In fact, the absolute area of the Cad8 expression domain in Emx2 homozygous mutants is almost double that in wild type, even though the surface area of the dorsal cortex is reduced by one-third. In Emx2 homozygous mutants, the domain of Cad6 expression on the dorsal cortical surface is significantly increased in proportional area and in both proportional and absolute width, compared to that in wild type. In contrast, the area and length of the Cad6 expression domain on the lateral cortical surface of Emx2 homozygous mutants each exhibit both proportional and absolute reductions compared to that in wild type. These results suggest that areas located in rostral and lateral parts of the neocortex are expanded and shifted caudally and medially in the Emx2 mutant neocortex (Bishop, 2000).

Because Pax6 is expressed in a countergradient to Emx2, it was predicted that changes in domains of cadherin expression in Pax6 mutant mice (Sey/Sey mutants) would be in the opposite direction of those observed in Emx2 mutants. The domain of Cad8 expression is contracted rostrally in the Sey/Sey cortex compared to that in the wild type. The Cad6 expression domain is contracted both laterally and rostrally in the Sey/Sey cortex. The domains of Cad8 and Cad6 expression on the dorsal surface of the Sey/Sey cortex, which is about three-quarters the size of that in the wild type, show both proportional and absolute reductions in area. Heterozygous mice (Sey/+) show no changes in cortical cadherin expression compared to wild-type mice. These results suggest that areas located in rostral and lateral parts of the neocortex contract rostrally and laterally in Sey/Sey mutants (Bishop, 2000).

Changes in the expression domains of Cad6 and Cad8 in Emx2 and Pax6 mutants suggest corresponding changes in neocortical arealization. The primary neocortical areas (motor, visual, somatosensory, and auditory) receive area-specific inputs from the principal motor and sensory thalamic nuclei [ventrolateral (VL), dorsal lateral geniculate (dLG), ventroposterior (VP), and medial geniculate (MG), respectively]. During normal development, thalamocortical axons target and invade their neocortical areas in a precise manner. Therefore, as an additional assay for changes in area identity in the Emx2 mutant neocortex, retrograde and anterograde axon tracing were used to map thalamocortical projections. This analysis was not done in Sey/Sey mutants because thalamic axons do not reach the cortex in these mice. Retrograde labeling from the cortex of Emx2 homozygous mutants indicates a caudal shift in the border between the somatosensory and visual areas compared to that of the wild type. Injections confined to the cortical plate of the occipital cortex, the location of the primary visual area, normally backlabel neurons in the dLG nucleus. However, in Emx2 mutants, similarly placed injections label cells in the VP nucleus, which normally projects to the primary somatosensory area located rostral to the visual area. Deeper injections made into the subplate of the occipital neocortex in Emx2 mutants backlabel neurons in both dLG and VP nuclei, indicating that dLG thalamic axons extend through the subplate below the occipital cortex but fail to invade the overlying cortical plate. Retrograde tracing from anterior and posterior portions of the occipital cortex reveals the expected topography in wild-type mice but again indicates a caudal shift in the border between the somatosensory and visual areas in Emx2 mutants. In wild-type mice, injections into the anterior occipital cortex backlabel cells in the posterior dLG nucleus, and injections into the posterior occipital cortex backlabel cells in the anterior dLG nucleus. In contrast, in Emx2 mutants, injections into the anterior occipital cortex do not label cells in the dLG nucleus but instead label cells in the VP nucleus; injections in the very posterior occipital cortex do label cells in the dLG nucleus, but their number is significantly reduced compared to the wild type. These findings suggest that the visual area in Emx2 mutants is contracted and restricted to the extreme caudal part of the occipital cortex (Bishop, 2000).

Anterograde tracing of thalamocortical projections is consistent with the retrograde tracing results. Injections into the dLG nucleus of Emx2 mutants label axons in the subplate beneath the caudal occipital cortex, but in comparison to the wild type, few invade the cortical plate. Injections into the VP nucleus of Emx2 mutants label axons that extend farther caudally than in the wild type and aberrantly invade the cortical plate of occipital cortex, whereas in the wild type, VP axons invade the cortical plate of the more rostrally located parietal cortex (the location of the primary somatosensory area). Thalamocortical projections in heterozygous Emx2 mutants resemble those in the wild type. Overall, anterograde and retrograde tracing of thalamocortical projections in Emx2 mutants provides evidence for a contraction of the visual area and a caudal shift in the border between the somatosensory and visual areas (Bishop, 2000).

Area-specific connections between thalamic nuclei and neocortical areas are reciprocal. Injections into thalamic nuclei backlabel cortical neurons in wild-type mice and Emx2 mutants. Injections into the dLG nucleus backlabel significantly fewer cells in the occipital cortex of Emx2 mutants compared to the wild type. The few labeled cells in Emx2 mutants are restricted to the very caudal cortex. Injections into the VP nucleus in wild-type mice backlabel cells in the parietal cortex but backlabel none in the occipital cortex. In contrast, VP injections in Emx2 mutants backlabel a substantial number of cells in the occipital cortex. These findings suggest that, in Emx2 mutants, corticothalamic neurons located in the occipital cortex have acquired a somatosensory area identity instead of their usual visual area identity. These changes in area-specific corticothalamic projections in Emx2 mutants are consistent with the changes observed in area-specific thalamocortical projections. Together, they suggest that the primary visual area is substantially reduced and restricted to the very caudal part of the neocortex, with a corresponding caudal shift in the border between visual and somatosensory areas (Bishop, 2000).

Emx2 is reported to be expressed in a small patch of neuroepithelium in the ventral-most part of the dorsal thalamus. Although this part does not generate cells of the principal sensory and motor thalamic nuclei, several markers were used to confirm the normal development and organization of the dorsal thalamus in Emx2 mutants. Patterns of acetylcholinesterase (AChE) staining and Gbx2 expression and general morphology revealed by nuclear 4',6-diamidino-2-phenylindole staining are all normal in the dorsal thalamus of Emx2 mutants. Thus, alterations in thalamocortical and corticothalamic projections in Emx2 mutants can be presumed to be due to changes intrinsic to the neocortex (Bishop, 2000).

These findings implicate Emx2 and Pax6 in the genetic control of neocortical arealization. They cannot be explained by a potential delay in neocortical development, nor are the expansions and contractions of the cadherin expression domains secondary to a loss of thalamocortical input. In addition, the observed changes in cadherin expression domains are not simply a by-product of the reduced overall size of the cerebral cortex in Emx2 and Pax6 mutants, because the changes are disproportionate in each mutant and opposing in the two mutants as predicted from the countergradients of Emx2 and Pax6 expression. Similarly, the observed changes in thalamocortical and corticothalamic projections in the Emx2 mutants are not due simply to a caudal truncation of the neocortex with an associated loss of the visual area. Arguing against this possibility are the changes in cadherin expression in Emx2 mutants, especially the expansion of the Cad8 expression domain in the frontal cortex (the motor area), in both proportional and absolute size. Instead, the findings presented here indicate a disproportional, but orderly, arealization of the Emx2 mutant neocortex reflected by an expansion of rostral areas and a contraction of caudal areas, and an opposite effect on arealization in the Pax6 mutant neocortex (Bishop, 2000).

Changes in the areal expression patterns of the cadherins and the area-specific distribution of corticothalamic neurons in the mutants suggest that Emx2 and Pax6 confer area identities to cortical cells, including projection neurons. The changes in cadherin expression and, presumably, receptors for axon guidance molecules that control corticothalamic axon targeting may be indicative of a direct role for Emx2 and Pax6 in their regulation, or they may be an indirect effect of the regulation of area identity. Similarly, the changes in area-specific thalamocortical projections suggest that Emx2 and Pax6 are involved either directly or indirectly in the regulation of axon guidance molecules within the cortex that control thalamocortical axon targeting. The restricted cortical expression of Eph receptor tyrosine kinases and their ligands, the ephrins, which act as axon guidance molecules in several systems, makes these candidates for controlling the development of area-specific projections between the thalamus and cortex (Bishop, 2000).

Emx2 and Pax6 may act independently or in a combinatorial manner (possibly with other transcription factor genes) to specify neocortical areas. Because areas in the neocortex have sharp borders, it is likely (but not required) that the graded expression patterns of Emx2 and Pax6 are translated to regulate some downstream genes in restricted patterns with abrupt borders that relate to areas. Although the downstream targets of Emx2 and Pax6 in the cortex have yet to be identified, transcription factors such as T-brain1 and Id2 are expressed in the neocortex in discrete patterns with abrupt borders that may be controlled by upstream regulatory genes expressed in gradients (Bishop, 2000).

Emx2 and Pax6 appear to be independently regulated. The opposing gradients of Emx2 and Pax6 may be induced by signals secreted from the poles of the cortex. Several secreted proteins are candidates for these inductive signals, including the BMP, WNT (2b, 3a, 5a, and 7a), and FGF8 proteins. In addition, cortical expression of the transcription factor Gli3 is required for Emx2 expression. Thus, combinations of inductive signals and upstream transcription factors may specify gradients of Emx2 and Pax6. A better understanding of the roles of Emx2 and Pax6 in regulating neocortical arealization will require identifying the patterning mechanisms that establish their differential expression, identifying downstream targets, and defining the mechanisms by which they, in combination with other factors, intrinsic and extrinsic, control the process of arealization of the neocortex (Bishop, 2000).

The genetic mechanisms that regulate dorsal-ventral identity in the embryonic mouse telencephalon and, in particular, the specification of progenitors in the cerebral cortex and striatum have been examined. The respective roles of Pax6 and Gsh2 in cortical and striatal development were studied in single and double loss-of-function mouse mutants. Gsh2 gene function is essential to maintain the molecular identity of early striatal progenitors and in its absence the ventral telencephalic regulatory genes Mash1 and Dlx are lost from most of the striatal germinal zone. In their place, the dorsal regulators Pax6, neurogenin 1 and neurogenin 2 are found ectopically. Conversely, Pax6 is required to maintain the correct molecular identity of cortical progenitors. In its absence, neurogenins are lost from the cortical germinal zone and Gsh2, Mash1 and Dlx genes are found ectopically. These reciprocal alterations in cortical and striatal progenitor specification lead to the abnormal development of the cortex and striatum observed in Pax6 (small eye) and Gsh2 mutants, respectively. In support of this, double homozygous mutants for Pax6 and Gsh2 exhibit significant improvements in both cortical and striatal development compared with their respective single mutants. Taken together, these results demonstrate that Pax6 and Gsh2 govern cortical and striatal development by regulating genetically opposing programs that control the expression of each other as well as the regionally expressed developmental regulators Mash1, the neurogenins and Dlx genes in telencephalic progenitors (Toresson, 2000).

The Gsh1/2 homolog, intermediate neuroblasts defective (ind), is expressed in selected areas of the fly CNS including the intermediate region of the ventral nerve cord. ind mutants show a reduction in the number of neuroblasts in this region of the ventral nerve cord. The remaining neuroblasts, by all markers tested, appear to have acquired a dorsal fate. This phenotype is reminiscent of the mouse Gsh2 mutant phenotype where the progenitors in the LGE (i.e. the intermediate region of the telencephalon) have lost the expression of certain ventral genes and instead express the dorsal genes Pax6, neurogenin 1 and neurogenin 2. These findings therefore indicate that at least some aspects of Gsh/ind function in dorsal-ventral specification of the developing nervous system have been conserved throughout evolution (Toresson, 2000).

The expression domains of genes implicated in forebrain patterning often share borders at specific anteroposterior positions. This observation lies at the heart of the prosomeric model, which proposes that such shared borders coincide with proposed compartment boundaries and that specific combinations of genes expressed within each compartment are responsible for its patterning. Thus, genes such as Emx1, Emx2, Pax6, and qin (Bf1) are seen as being responsible for specifying different regions in the forebrain (diencephalon and telencephalon). However, the early expression of these genes, before the appearance of putative compartment boundaries, has not been characterized. In order to determine whether they have stable expression domains before this stage, mRNA expression of each of the above genes, relative both to one another and to morphological landmarks, has been compared in closely staged chick embryos. Between HH stage 8 and HH stage 13, each of the genes has a dynamic spatial and temporal expression pattern. To test for autonomy of gene expression in the prosencephalon, tissue was grafted from this region to more caudal positions in the neural tube and expresssion of Emx1, Emx2, qin, or Pax6 was analyzed. Gene expression is autonomous in prosencephalic tissue from as early as HH stage 8. In the case of Emx1, the data suggest that, from as early as stage 8, presumptive telencephalic tissue also is committed to express this gene. It is proposed that early patterning along the anteroposterior axis of the presumptive telencephalon occurs across a field that is subdivided by different combinations of genes, with some overlapping areas, but without either sharp boundaries or stable interfaces between expression domains (Bell, 2001).

In the proliferative zone of the developing cerebral cortex, multipotential progenitors predominate early in development and divide to increase the progenitor pool. As corticogenesis progresses, proportionately fewer progenitors are produced and, instead, cell divisions yield higher numbers of postmitotic neurons or glial cells. As the switch from the generation of progenitors to that of differentiated cells occurs, the orientation of cell division alters from predominantly symmetrical to predominantly asymmetrical. It has been hypothesised that symmetrical divisions expand the progenitor pool, whereas asymmetrical divisions generate postmitotic cells, although this remains to be proved. The molecular mechanisms regulating these processes are poorly understood. The transcription factor Pax6 is highly expressed in the cortical proliferative zone and there are morphological defects in the Pax6Sey/Sey (Pax6 null) cortex, but little is known about the principal cellular functions of Pax6 in this region. The cell-cycle kinetics, the progenitor cleavage orientation and the onset of expression of differentiation markers has been examined in Pax6Sey/Sey cortical cells in vivo and in vitro. Early in corticogenesis at embryonic day (E) 12.5, the absence of Pax6 accelerates cortical development in vivo, shortening the cell cycle and the time taken for the onset of expression of neural-specific markers. This also occurs in dissociated culture of isolated cortical cells, indicating that the changes are intrinsic to the cortical cells. From E12.5 to E15.5, proportions of asymmetrical divisions increase more rapidly in mutant than in wild-type embryos. By E15.5, interkinetic nuclear migration during the cell cycle is disrupted and the length of the cell cycle is significantly longer than normal in the Pax6Sey/Sey cortex, with a lengthening of S phase. Together, these results show that Pax6 is required in developing cortical progenitors to control the cell-cycle duration, the rate of progression from symmetrical to asymmetrical division and the onset of expression of neural-specific markers (Estivill-Torrus, 2002).

The transcription factor PAX6 has been implicated in forebrain patterning, cerebral cortical arealization and in development of thalamocortical connections. Using a Pax6/lacZ knockout mouse, in which the endogenous Pax6 expression is reflected by ß-galactosidase activity, the consequences of the loss of Pax6 function on thalamocortical (TCA) and corticofugal axon (CFA) pathfinding was studied during the period of embryonic day (E) 14.5 to E18.5. Carbocyanine dye tracing in Pax6 heterozygotes (Pax6+/-) and Pax6 wild-type (Pax6+/+) brains revealed that CFAs and TCAs temporarily arrested their growth at E14.5 at the border of the ß-galactosidase-positive region at the pallial/subpallial boundary (PSPB), before they continued towards their targets. However, in Pax6 homozygous (Pax6-/-) embryos, CFAs and TCAs are unable to encounter each other at the PSPB and reach their final targets. Instead of crossing the PSPB, they had the tendency to descend into the ventral pallium in large aberrant fascicles. In addition, cells with a presumptive guide-post function, which are normally situated in the ventral thalamus, internal capsule and hypothalamus, are more dispersed in the hypothalamus and ventral pallium. These pathfinding defects were confirmed by immunohistochemistry for L1 and TAG1, markers of the early axonal connections. The aberrant development of axonal connections in the absence of Pax6 function appears to be related to ultrastructural defects of cells along the PSPB, as well as to a failure of axonal guidance molecule expression, including Sema3c and Sema5a (Jones, 2002).

The mammalian neocortex is organized into subdivisions referred to as areas that are distinguished from one another by differences in architecture, axonal connections, and function. The transcription factors EMX1, EMX2, and PAX6 have been proposed to regulate arealization. Emx1 and Emx2 are expressed by progenitor cells in a low rostrolateral to high caudomedial gradient across the embryonic neocortex, and Pax6 is expressed in a high rostrolateral to low caudomedial gradient. Recent evidence has suggested that EMX2 and PAX6 have a role in the genetic regulation of arealization. A panel of seven genes (Cad6, Cad8, Id2, RZRß, p75, EphA7, and ephrin-A5), representative of a broad range of proteins has been used as complementary markers of positional identity to obtain a more thorough assessment of the suggested roles for EMX2 and PAX6 in arealization, and in addition, to assess the proposed but untested role for EMX1 in arealization. Orderly changes in the size and positioning of domains of marker expression in Emx2 and Pax6 mutants strongly imply that rostrolateral areas (motor and somatosensory) are expanded, whereas caudomedial areas (visual) are reduced in Emx2 mutants and that opposite effects occur in Pax6 mutants, consistent with their opposing gradients of expression. In contrast, patterns of marker expression, as well as the distribution of area-specific thalamocortical projections, appear normal in Emx1 mutants, indicating that these mutants do not exhibit changes in arealization. This lack of a defined role for EMX1 in arealization is supported by finding of similar shifts in patterns of marker expression in Emx1; Emx2 double mutants as in Emx2 mutants. Thus, these findings indicate that EMX2 and PAX6 regulate, in opposing manners, arealization of the neocortex and impart positional identity to cortical cells, whereas EMX1 appears not to have a role in this process (Bishop, 2002).

The role of Tlx, an orphan nuclear receptor, has been examined in dorsal-ventral patterning of the mouse telencephalon. Tlx is expressed broadly in the ventricular zone, with the exception of the dorsomedial and ventromedial regions. The expression spans the pallio-subpallial boundary, which separates the dorsal (i.e. pallium) and ventral (i.e. subpallium) telencephalon. Despite being expressed on both sides of the pallio-subpallial boundary, Tlx homozygous mutants display alterations in the development of this boundary. These alterations include a dorsal shift in the expression limits of certain genes that abut at the pallio-subpallial boundary as well as the abnormal formation of the radial glial palisade that normally marks this boundary. The Tlx mutant phenotype is similar to, but less severe than, that seen in Small eye (i.e. Pax6) mutants. Interestingly, removal of one allele of Pax6 in the homozygous Tlx mutant background significantly worsens the phenotype. Thus Tlx and Pax6 cooperate genetically to regulate the establishment of the pallio-subpallial boundary. The patterning defects in the Tlx mutant telencephalon result in a loss of region-specific gene expression in the ventral-most pallial region. This correlates well with the malformation of the lateral and basolateral amygdala in Tlx mutants, both of which have been suggested to derive from ventral portions of the pallium (Stenman, 2002).

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wild-type cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. It is suggested that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras (Talamillo, 2003).

Three basic aspects of cerebral cortex development have been recently investigated, specification of cortical versus ganglionic identity, regionalization of the early cortical primordium and arealization of the developing cortex. Emx2 and Pax6 promote development of caudal-medial and rostral-lateral cortex, respectively, by properly shaping the early cortical protomap and possibly modulating the tangential growth ratio between medial and lateral cortical anlagen. By analyzing the brains of embryos bearing mutations for Emx2 and Pax6 in different combinations, it was found that both genes are necessary and sufficient for a more basic developmental choice, i.e. the specification of neuroblasts in the dorsal telencephalon as cortical versus ganglionic neuroblasts. The possible roles of the Emx2 paralog, Emx1, in these processes was investigated. By looking at embryos mutant for Emx1, Emx2 and Pax6 in various combinations, no evidence was found of Emx1 involvement in the process of cortical specification; conversely, this gene appeared to be involved to some extent in the process of regionalization of the cortical primordium along the medial-lateral axis, as a promoter of medial fates (Muzio, 2003).

Successful brain development requires tight regulation of sequential symmetric and asymmetric cell division. Although Pax6 is known to exert multiple roles in the developing nervous system, its role in the regulation of cell division is unknown. This study demonstrates profound alterations in the orientation and mode of cell division in the cerebral cortex of mice deficient in Pax6 function (Pax6Sey/Sey) or after acute induced deletion of Pax6. Live imaging revealed an increase in non-vertical cellular cleavage planes, resulting in an increased number of progenitors with unequal inheritance of the apical membrane domain and adherens junctions in the absence of Pax6 function. This phenotype appears to be mediated by the direct Pax6 target Spag5, a microtubule-associated protein, reduced levels of which result in the replication of the Pax6 phenotype of altered cell division orientation. In addition, lack of Pax6 also results in premature delamination of progenitor cells from the apical surface due to an overall decrease in proteins mediating anchoring at the ventricular surface. Moreover, continuous long-term imaging in vitro revealed that Pax6-deficient progenitors generate daughter cells with asymmetric fates at higher frequencies. These data demonstrate a cell-autonomous role for Pax6 in regulating the mode of cell division independently of apicobasal polarity and cell-cell interactions. Taken together, this work reveals several direct effects that the transcription factor Pax6 has on the machinery that mediates the orientation and mode of cell division (Asami, 2011).

Identification of positionally distinct astrocyte subtypes whose identities are specified by a homeodomain code

Astrocytes constitute the most abundant cell type in the central nervous system (CNS) and play diverse functional roles, but the ontogenetic origins of this phenotypic diversity are poorly understood. This study investigated whether positional identity, a fundamental organizing principle governing the generation of neuronal subtype diversity, is also relevant to astrocyte diversification. Three positionally distinct subtypes of white-matter astrocytes (WMA) were identified in the spinal cord, that can be distinguished by the combinatorial expression of Reelin and Slit1. These astrocyte subtypes derive from progenitor domains expressing the homeodomain transcription factors Pax6 and Nkx6.1, respectively. Loss- and gain-of-function experiments indicate that the positional identity of these astrocyte subtypes is controlled by Pax6 and Nkx6.1 in a combinatorial manner. Thus, positional identity is an organizing principle underlying astrocyte, as well as neuronal, subtype diversification and is controlled by a homeodomain transcriptional code whose elements are reutilized following the specification of neuronal identity earlier in development (Hochstim, 2008).

Reelin and Slit1 were not expressed by all astrocytes, but rather by positionally distinct subsets in the ventral white matter. Reelin was expressed in the dorsolateral and ventrolateral white matter, but not in astrocytes close to the ventral midline. Slit1, conversely, was expressed in astrocytes in the ventromedial and ventrolateral white matter, but not in the dorsolateral white matter. Double labeling for Reelin and Slit1 revealed the existence of three adjacent domains of WMAs: a dorso-lateral domain of Reelin+, Slit1 cells; a ventro-lateral domain of Reelin+, Slit1+ cells; and a ventro-medial domain of Slit1+, Reelin cells. For convenience, these subpopulations henceforth are referred to as ventral astrocyte subtypes 1, 2, and 3 (VA1, VA2, and VA3, respectively. Quantification indicated that each of these three subpopulations is present in roughly equal numbers (Hochstim, 2008).

In principle, VA1-VA3 phenotypes could be established after astrocyte precursors migrate to the WM, under the influence of local environmental cues, or could be specified by positional mechanisms prior to emigration from the VZ. As a first step toward addressing this question, it was asked whether Reelin and Slit1 were expressed by positionally distinct subsets of astrocyte precursors within the neuroepithelium. Examination of spinal cord sections at E13.5, a stage when most astrocyte precursors have been specified in the ventral VZ, revealed that Reelin and Slit1 are expressed in cells within the germinal layer. Triple labeling for Reelin, Slit1-GFP, and NFIA indicated that Reelin and Slit1 are expressed by NFIA+ glial precursors and that the domains of their expression partially overlap. This partial overlap subdivides the ventral-most VZ into three domains: a dorsal-most Reelin+, Slit1 domain; a more ventral Reelin+, Slit1+ domain; and a ventro-medial Reelin, Slit1+ domain. The spatial organization of these progenitor domains, which are referred to as pA1, pA2, and pA3, respectively, therefore mirrors that of the VA1, VA2, and VA3 domains in the WM (Hochstim, 2008).

Mutations in mammalian Pax-6 homologs: Pax-6 role in eye development

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance. The expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, it has been demonstrated that the addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, the expression of several early lens placode-specific markers were examined in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway that is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. It is concluded that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation (Wawersik 1999).

Aniridia in man and Small eye in mice are semidominant developmental disorders caused by mutations within the paired box gene PAX6. Whereas heterozygotes suffer from iris hypoplasia, homozygous mice lack eyes and nasal cavities and exhibit brain abnormalities. Yeast artificial chromosome transgenic mice have been created carrying the human PAX6 locus. When crossed onto the Small eye background, the transgene rescues the mutant phenotype. Strikingly, mice carrying multiple copies on a wild-type background show specific developmental abnormalities of the eye, but not of other tissues expressing the gene. Thus, at least five different eye phenotypes are associated with changes in PAX6 expression. Thus, not only reduced, but also increased levels of transcriptional regulators can cause developmental defects (Schedl, 1996).

Math5, a mouse basic helix-loop-helix (bHLH) gene that is closely related to Drosophila atonal and Xenopus Xath5, is largely restricted to the developing eye. Math5 retinal expression precedes differentiation of the first neurons and persists within progenitor cells until after birth. To position Math5 in a hierarchy of retinal development, Math5 and Hes1 expression were compared in wild-type and Pax6-deficient (Sey) embryos. Math5 expression is downregulated in Sey/+ eyes and abolished in Sey/Sey eye rudiments, whereas the bHLH gene Hes1 is upregulated in a similar dose-dependent manner. These results link Pax6 to the process of retinal neurogenesis and provide the first molecular correlate for the dosage-sensitivity of the Pax6 phenotype. During retinogenesis, Math5 is expressed significantly before NeuroD, Ngn2 or Mash1. To test whether these bHLH genes influence the fates of distinct classes of retinal neurons, Math5 and Mash1 were ectopically expressed in Xenopus retinal progenitors. Unexpectedly, lipofection of either mouse gene into the frog retina causes an increase in differentiated bipolar cells. Directed expression of Math5, but not Xath5, in Xenopus blastomeres produces an expanded retinal phenotype. It is proposed that Math5 acts as a proneural gene, but has properties different from its most closely related vertebrate family member, Xath5 (Brown, 1998).

Molecular dissection of Pax6 function: the specific roles of the paired domain and homeodomain in brain development

The transcription factor Pax6 plays a key role during development of various organs, including the brain where it affects cell fate, cell proliferation and patterning. To understand how Pax6 coordinates these diverse effects at the molecular level, the role was examined of distinct DNA-binding domains of Pax6, the homeodomain (HD), the paired domain (PD) and its splice variant (5a), using loss- and gain-of-function approaches. The PD is necessary for the regulation of neurogenesis, cell proliferation and patterning effects of Pax6, since these aspects are severely affected in the developing forebrain of the Pax6Aey18 mice with a deletion in the PD but intact homeo- and transactivation domains. In contrast, a mutation of the HD lacking DNA-binding (Pax64Neu) resulted in only subtle defects of forebrain development. Distinct roles of the two splice variants of the PD have been demonstrated. Retrovirally mediated overexpression of Pax6 containing exon 5a inhibits cell proliferation without affecting cell fate, while Pax6 containing the canonical form of the PD lacking exon 5a simultaneously affects cell fate and proliferation. Therefore, these results demonstrate a key role of the PD in brain development and implicate splicing as a pivotal factor regulating the potent neurogenic role of Pax6 (Haubst, 2004).

Functional analysis of the distinct DNA-binding domains of Pax6 shows that the canonical form of the PD coordinately regulates, neurogenesis, cell proliferation and patterning in the developing telencephalon, while the spliced form of the PD [Pax6(5a)] specifically affects cell proliferation without any effects on cell fate and regionalization. In contrast, mutation of the HD affected only subtle aspects of the boundary delineating the dorsal and ventral telencephalon. Since the canonical form of the PD binds to both Pax6 consensus sites (P6CON, 5aCON) and the Pax6(5a) isoform binds exclusively to the 5aCON site, these results suggest that the regulation of neurogenesis and the forebrain patterning is mediated via target genes containing P6CON sites, whereas the regulation of proliferation is mediated by target genes containing 5aCON sites (Haubst, 2004).

Analysis of the Pax64Neu–/– mutant, which carries a point mutation in the third helix of the HD that abolishes its DNA binding, reveals that DNA binding of the HD plays no role in the regulation of neurogenesis and cell proliferation in the developing forebrain, but is required together with the PD for the differentiation of one aspect of boundary formation in the forebrain, namely the expression of SFRP2. SFRP2 is a wnt-inhibitor expressed at the border between the dorsal and the ventral telencephalon. In both, the PD (Pax6Aey18–/–) and HD (Pax64Neu–/–) mutant mice SFRP2 fails to be expressed at this position, while it is apparent in other parts of the brain. Other aspects of boundary differentiation, such as reticulon-1 expression or high BLBP-content in fasciculating radial glia fibers, are normal in the HD mutant Pax64Neu–/–, suggesting that DNA binding of the HD is required only for some aspects of boundary specification. Since both HD and PD mutations fail to regulate SFRP2, either both DNA-binding domains may need to bind cooperatively to mediate transcription of SFRP2, or an intact PD is required for the appropriate modulation of HD DNA binding as previously demonstrated in vitro. These results further support the multitude of regulatory mechanisms contributing to the boundary formation at this position that may also act as an organizing centre in the developing telencephalon (Haubst, 2004).

The novel Pax6 mutation Pax6Aey18 with a PD lacking most of its DNA-binding domain but intact HD and TA results in the same telencephalic phenotype as in the Pax6 functional null allele Pax6Sey–/–. Even at the quantitative level, no differences in neurogenesis and cell proliferation were detectable between Pax6Aey18–/– and Pax6Sey–/– cortices, suggesting that the PD alone is necessary and sufficient for all of these aspects of cortical development. In addition, both genotypes express similar severity in the disturbance of patterning in the telencephalon. The results therefore demonstrate that targets of the PD are necessary and sufficient to regulate cell proliferation and cell fate, comprising neurogenesis and patterning, in the developing forebrain (Haubst, 2004).

Given the predominant role of the PD in exerting the Pax6 functions in forebrain development, the specific role of the 5a splice insert into the PD that shifts DNA binding from the N-terminal to the C-terminal domain of the PD was determined by analysis of the Pax6(5a)–/– mice. No changes were detected in neurogenesis, cell proliferation and regionalization in the Pax6(5a)–/–. This finding has to be interpreted with caution, since the mRNA of the canonical form of Pax6 was upregulated (1.4-fold) in amounts sufficient to compensate for the lack of Pax6(5a) in Pax6(5a)–/– mice. Messenger RNA of Pax6(5a) comprises about 10%-20% of the total Pax6 mRNA (E10-E12), a ratio previously observed to be rather effective for transcriptional regulation. From the in vivo analysis it can be concluded that there are no specific roles of the Pax6(5a) isoform that could not also be exerted by the canonical PD in the developing forebrain. Indeed, Pax6(5a) affects solely a subset of the functions of the canonical form of Pax6. Overexpression of Pax6(5a) in individual cortical precursor cells shows a specific and cell-autonomous effect on the number of progeny of a single precursor, without affecting cell fate even in the absence of functional Pax6 in the Pax6Sey–/– background. This phenotype, the reduction of clone size, can be due to three mechanisms: an increase in cell death, an increase in postmitotic cells or an increase in asymmetric rather than symmetric cell divisions. No difference was observed in cell death as analyzed by DAPI staining, but a significant reduction of proliferating cells was seen in the Pax6-transduced clones already 2 days after infection (71% proliferating cells among all control virus infected cells compared to 53% after Pax6 overexpression). Thus, Pax6 and Pax6(5a) overexpression increases the number of cells leaving the cell cycle either due to an increase in asymmetric cell division or a lengthening of cell cycle. Both effects could also explain the increase in the number of precursors seen in the Pax6 loss-of-function mutations. Further experiments are needed to clarify the exact role of Pax6 on cell cycle length or the mode of cell division in proliferating precursors. Since the Pax6(5a) form is sufficient to induce these changes in proliferation and binds exclusively to the 5aCON site, these data demonstrate that targets of the 5aCON site are specifically involved in the regulation of cell proliferation, while targets of the P6CON site seemingly regulate neurogenesis. The latter is consistent with the role of Ngn2, a target containing the P6CON site, in neurogenesis (Haubst, 2004).

The effect on cell proliferation depends on the CNS region – while Pax6 negatively regulates proliferation in the telencephalon, it promotes proliferation in the vertebrate and invertebrate eye. This is also the case for the Pax6(5a) isoform. The deletion of exon 5a results in a reduced number of iris and lens fiber cells in the eye, consistent with its proliferation promoting effect. Similarly, in the Drosophila eye, eyegone and the murine Pax6(5a) positively affect cell proliferation. Thus, the positive effect of the 5aCON targets on cell proliferation in the eye is widespread across vertebrates and invertebrates, while the Pax6(5a)-mediated reduction of cell proliferation observed in precursors of the developing mouse telencephalon seems to be more specific and may have evolved more recently. The HD of Pax6 plays an important role in the eye, but not in the developing forebrain. The Pax64Neu mutation leads to severe defects in eye formation with homozygous mice developing no eye, except a remnant of the retinal neuroepithelium (pseudo-optic cup), suggesting that targets of the HD are important for both the early role of Pax6 in the surface ectoderm for lens formation, and later processes during retina specification. This analysis has further shown reduced proliferation and Ngn2 immunoreactivity in the remnant of the retinal neuroepithelium in the Pax64Neu–/–, similar to the phenotype in the PD mutant Pax6Aey18–/–. Both the PD and HD are thus important for the regulation of proliferation and cell fate in the retina, while the HD plays no role in these aspects in the telencephalon. In conclusion these results thus imply the selective use of PD, PD5a and HD targets as one mechanism that may contribute to the region-specific differences in Pax6 function within the CNS as well as in different organs (Haubst, 2004).

Mutations in mammalian Pax-6 homologs: Pax-6 role in cerebellar development

The cerebellum develops as part of the rhombencephalon at a region of incomplete closure of the dorsal neural tube. The rim of the 'open' neural tube is often referred to as the rhombic lip. Upper (rostral) and lower (caudal) lips are brought into close apposition when the pontine flexure develops. The rostral lip gives rise only to cerebellar granule cell precursors. The caudal lip undergoes a series of morphological transformations to form the highly proliferative precerebellar neuroepithelium (pcn) in the dorsal medulla. Neurons generated by the pcn migrate out sequentially, along divergent pathways, to form the precerebellar nuclei, all of which project to the cerebellum. These nuclei come to lie in the pontine and medullary regions of the hindbrain. The five pre-cerebellar nuclei are: the inferior olive, the external cuneate and lateral reticular nuclei in the medulla and the pontine and reticulotegmental nuclei in the pons. Three distinct migration streams carry cells from the pcn to the five nuclei. Cells of the anterior extramural stream (aes) cross several rhombomere boundaries before settling ipsilaterally in the pontine and reticulotegmental nuclei. Cells in the posterior extramural stream (pes) cross the midline and settle contralaterally in the lateral reticular and external cuneate nuclei in the medulla. The aes and pes encircle the hindbrain using pathways perpendicular to the glial fibers which radiate across the medulla. The inferior olive, in contrast, is populated by cells using a third, intramural, migration path. Formation of the pontine, external cuneate and lateral reticular nuclei is disrupted in Pax6Sey/Pax6Sey mice (Engelkamp, 1999 and references).

Post-mitotic neurons generated at the rhombic lip undertake long distance migration to widely dispersed destinations, giving rise to cerebellar granule cells and the precerebellar nuclei. Pax6, a key regulator in CNS and eye development, is strongly expressed in rhombic lip and in cells migrating away from it. Development of some structures derived from these cells is severely affected in Pax6-null Small eye (Pax6Sey/Pax6Sey) embryos. Cell proliferation and initial differentiation seem unaffected, but cell migration and neurite extension are disrupted in mutant embryos. Three of the five precerebellar nuclei fail to form correctly. In the cerebellum the pre-migratory granule cell sub-layer and fissures are absent. Some granule cells are found in ectopic positions in the inferior colliculus which may result from the complete absence of Unc5h3 expression in Pax6Sey granule cells. These results suggest that Pax6 plays a strong role during hindbrain migration processes and at least part of its activity is mediated through regulation of the netrin receptor Unc5h3 (Engelkamp, 1999).

Pax6 appears to fulfil different roles in the development of the forebrain and cerebellum. In the telencephalon, the Pax6+ ventricular layer gives rise to virtually all cortical cells, including glia and neurons, whereas in cerebellum, Pax6+ rhombic lip precursors give rise solely to granule cells: other cell types (Purkinje cells, interneurons and radial glia) are of Pax6- neuroepithelial origin. In Pax6Sey forebrain proliferation defects have been reported that are not observed in rhombic lip-derived structures. In the telencephalon, Pax6 controls the differentiation of radial glia, whereas, in Pax6Sey cerebellum, granule cell behaviour is altered. In addition, marked morphological differences exist between cortical and cerebellar radial glia: the former are bipolar, the latter monopolar. Even among rhombic lip derivatives, Pax6 appears to function upstream of different genetic pathways: while Unc5h3 is still expressed in mutant pontine and reticulotegmental nuclei remnants, it is completely absent in the mutant granule layer, suggesting a loss of responsiveness to some essential signaling cues (Engelkamp, 1999 and references).

A possible unifying role for Pax6 may be in the control of interkinetic nuclear migration, which is altered in Pax6Sey forebrain. Cortical cells derived from ventricular germinal zones undergo this type of fluctuating movement as an essential component of the cell division process at the spatially limited columnar ventricular surface. Impairment of this process would lead to altered proliferation rates. Pax6-expressing hindbrain neurons, however, are generated at the pcn and the external granular layer (EGL) with virtually no spatial limitation. These germinal zones are not organized as columnar epithelia and the typical 'up-and-down' migration of nuclei has not been observed during cell division. Proliferative activity would therefore remain undisturbed in the Pax6Sey pcn and EGL. Long distance migration of granule cells and of neurons derived from the pcn do, however, involve a similar process: neurite extension in the direction of future movement and subsequent perikaryal translocation of the nucleus. Disturbance of this process in postmitotic granule cells may underlie the failure to form a distinct pre-migratory layer, and the altered explant culture behavior. Comparative analysis of cerebellar system development in normal and mutant mice, offers a potent opportunity for further dissection of the role of Pax6 and its direct and indirect downstream targets, a role which may well be different in each Pax6- expressing tissue. Future identification of Pax6 target genes will provide molecular insight into how different mutant phenotypes are produced (Engelkamp, 1999 and references).

The molecular mechanisms that govern the coordinated programs of axonogenesis and cell body migration of the cerebellar granule cell are not well understood. In Pax6 mutant rats (rSey2/rSey2), granule cells in the external germinal layer (EGL) fail to form parallel fiber axons and to migrate tangentially along these fibers despite normal expression of differentiation markers. In culture, mutant cells sprout multiple neurites with enlarged growth cones, suggesting that the absence of Pax6 function perturbs cytoskeletal organization. Some of these alterations are cell-autonomous and rescuable by ectopic expression of Pax6 but not by co-culture with wild-type EGL cells. Cell-autonomous control of cytoskeletal dynamics by Pax6 is independent of the ROCK-mediated Rho small GTPase pathway. It is proposed that in addition to its roles during early patterning of the CNS, Pax6 is involved in a novel regulatory step of cytoskeletal organization during polarization and migration of CNS neurons (Yamasaki, 2001).

To verify that Pax6 function is truly involved in the genetic cascade of granule cell differentiation in normal developing cerebellum, a dominant-negative form of Pax6 was misexpressed in granule cell precursors in situ as well as in vitro. The DNA-binding domain of Pax6 was fused to the repressor domain of Drosophila Engrailed (EnR), producing a fusion protein that should block transcriptional activation by intrinsic Pax6. Misexpression of the Pax6-EnR cDNA in the microexplant culture of wild-type cells results in multipolar morphology similar to rSey2/rSey2 cells Thus, perturbation in the maintenance of Pax6 function to activate transcription of the downstream target gene(s) is sufficient to cause the morphological deficits observed in Pax6 mutant EGL cells, even within the wild-type genetic background (Yamasaki, 2001).

Meis2 is a Pax6 co-factor in neurogenesis and dopaminergic periglomerular fate specification in the adult olfactory bulb

Meis homeodomain transcription factors control cell proliferation, cell fate specification and differentiation in development and disease. Previous studies have largely focused on Meis contribution to the development of non-neuronal tissues. By contrast, Meis function in the brain is not well understood. This study provides evidence for a dual role of the Meis family protein Meis2 in adult olfactory bulb (OB) neurogenesis. Meis2 is strongly expressed in neuroblasts of the subventricular zone (SVZ) and rostral migratory stream (RMS) and in some of the OB interneurons that are continuously replaced during adult life. Targeted manipulations with retroviral vectors expressing function-blocking forms or with small interfering RNAs demonstrated that Meis activity is cell-autonomously required for the acquisition of a general neuronal fate by SVZ-derived progenitors in vivo and in vitro. Additionally, Meis2 activity in the RMS is important for the generation of dopaminergic periglomerular neurons in the OB. Chromatin immunoprecipitation identified doublecortin and tyrosine hydroxylase as direct Meis targets in newly generated neurons and the OB, respectively. Furthermore, biochemical analyses revealed a previously unrecognized complex of Meis2 with Pax6 and Dlx2, two transcription factors involved in OB neurogenesis. The full pro-neurogenic activity of Pax6 in SVZ derived neural stem and progenitor cells requires the presence of Meis. Collectively, these results show that Meis2 cooperates with Pax6 in generic neurogenesis and dopaminergic fate specification in the adult SVZ-OB system (Agoston, 2014).

Table of contents

eyeless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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