Loss- and gain-of-function analyses reveal targets of Pax6 in the developing mouse telencephalon
Introduction
Transcription factors are key regulators of cell fate and differentiation during development and in adult stem cell lineages. Members of the Pax gene family are known as master regulators of organ development influencing cell fate and cell proliferation in various organs (Chi and Epstein, 2002, Eccles et al., 2002). In the developing nervous system, members of this paired-type homeobox transcription factor family act as major determinants of patterning and regionalization, and in addition, regulate cell fate and proliferation (Basch et al., 2006, Dahl et al., 1997, Gehring, 2002, Liu and Joyner, 2001, Mansouri et al., 1999, Nutt et al., 2001, Pichaud and Desplan, 2002). However, little is known about how Pax genes exert their key influence in development at the molecular level.
One of the best known members of the Pax gene family is the transcription factor Pax6, which plays key roles in the development of the eye (Hill et al., 1991, Schedl et al., 1996), pancreas (St-Onge et al., 1997) and nervous system (Schmahl et al., 1993, Stoykova et al., 1996, Warren and Price, 1997). In the developing nervous system the absence of functional Pax6 results in aberrations in proliferation, cell fate and patterning in the brain and spinal cord (Ericson et al., 1997, Osumi et al., 1997, Schmahl et al., 1993, Stoykova et al., 1997, Takahashi and Osumi, 2002). For example, Pax6 is expressed in the developing telencephalon only dorsally, in the anlage of the cerebral cortex, where its function has been analyzed in the Pax6-mutant mice Small Eye (Pax6Sey−/−). These mice express a truncated, non-functional Pax6 protein and represent functional null-mutants with a phenotype similar to Pax6−/− mice (Fuccillo et al., 2006, Götz et al., 1998, Hill et al., 1991, Schmahl et al., 1993) and to mice with a large deletion in the paired domain of the Pax6 gene Pax6Aey18 (Haubst et al., 2004, Hill et al., 1991). The Pax6Sey−/− cortex is morphologically distorted due to migration defects (Caric et al., 1997, Chapouton et al., 1999, Götz et al., 1998, Schmahl et al., 1993, Talamillo et al., 2003) and neurons residing in the cortical plate are reduced by half (Götz et al., 1998, Heins et al., 2002). In addition, loss of functional Pax6 in the developing cortex of Pax6Sey−/− mice leads to patterning defects with higher expression levels of genes that are normally restricted to the ventral telencephalon (Haubst et al., 2004, Heins et al., 2002, Schmahl et al., 1993, Stoykova et al., 1996, Stoykova et al., 2000, Toresson et al., 2000, Yun et al., 2001). In line with their misexpression of ventral telencephalic transcription factors, dorsal cortical precursors in the Pax6-deficient cortex often generate GABAergic neurons instead of glutamatergic neurons. GABAergic neurons normally arise in the ventral telencephalon (for review see: Marin and Rubenstein, 2003) while glutamatergic neurons are generated by dorsal cortical precursors in the presence of Pax6 and its targets neurogenin 1 and 2 (Schuurmans et al., 2004, Kroll and O'Leary, 2005). Moreover, migration of GABAergic neurons from the ventral into the dorsal telencephalon is increased in the Pax6-deficient forebrain due to the loss of the boundary structures delineating the dorsal and ventral telencephalon (Chapouton et al., 1999, Nomura and Osumi, 2004, Stoykova et al., 1996, Stoykova et al., 1997).
Pax6 is also involved in the specification of various neuronal subtypes throughout the developing nervous system (Andrews et al., 2003, Ericson et al., 1997, Osumi et al., 1997, Vitalis et al., 2000) as well as in neurogenesis of olfactory bulb (OB) interneurons in the adult (Hack et al., 2005, Kohwi et al., 2005); the generation of new olfactory bulb neurons can be completely blocked by interference with Pax6 protein levels or function (Hack et al., 2005). Moreover, transduction of Pax6 into postnatal and adult cortical glial cells in vitro (Heins et al., 2002) and in vivo after stab wound injury (Buffo et al., 2005) is sufficient to evoke neurogenesis in some of the transduced cells. A better understanding of the molecular program evoked by Pax6 would better elucidate how neurogenesis can be re-activated following injury.
The potent neurogenic effect of Pax6 seems to be region-specific however. Ectopic expression of Pax6 in embryonic precursors of the ventral telencephalon, which are normally Pax6-negative, has no effect on neurogenesis (Hack et al., 2004). This is consistent with the restricted depletion of forebrain neurons in Pax6-deficient mouse mutants. Moreover, Pax6 exerts almost opposing roles in the developing retina and the developing cortex. While Pax6 inhibits proliferation and promotes neurogenesis in the developing cortex (Hack et al., 2004, Haubst et al., 2004, Heins et al., 2002), it promotes proliferation and an undifferentiated, stem cell phenotype in the developing retina (Marquardt et al., 2001, Philips et al., 2005). Thus, to elucidate the neurogenic program evoked by Pax6 in telencephalic precursor cells, it is crucial to understand the regulation of Pax6 target genes in the telencephalon and compare it to that in the developing eye.
During eye development, Pax6 plays important roles in the induction of the lens placode (Chow and Lang, 2001, Grindley et al., 1995, van Raamsdonk and Tilghman, 2000), in lens differentiation (Duncan et al., 2004, Kondoh et al., 2004, Reza and Yasuda, 2004) and in the retina (Ashery-Padan et al., 2000). Homozygous Pax6Sey mice completely lack eyes, whereas Pax6Sey heterozygote mice have abnormally small lenses due to fewer rounds of cell division (van Raamsdonk and Tilghman, 2000). Studies of global gene expression changes as a result of altered Pax6 levels in the eye have provided a more comprehensive picture of the role of Pax6 in this system. Eyes from Pax6Sey heterozygote mice (Chauhan et al., 2002) as well as from transgenic mice over-expressing Pax6 in lens fibre cells (Chauhan et al., 2002) were examined for gene expression alterations using microarray analysis. This approach allowed the identification of a large number of direct and indirect targets of Pax6-mediated transcription in the eye, very much aiding this field of research (Davis et al., 2003, Tyas et al., 2003).
Here, we pursued a similar approach in the developing telencephalon to identify some of the key molecular programs regulated by Pax6 in this region. We took advantage of the region-specific function of Pax6 by comparing gene expression changes in the developing ventral telencephalon, the ganglionic eminence (GE), and the dorsal telencephalon, cerebral cortex, of wild-type (WT) and Pax6Sey−/− mice. Pax6 is expressed at high levels in the dorsal telencephalon, but at comparatively very low levels in the ventral telencephalon where overexpression of the protein has minor effects, as mentioned above. Gene expression changes in the ventral telencephalon are likely to largely be indirect consequences of alterations in telencephalic patterning and signalling. By contrast, gene expression changes in the dorsal telencephalon should at least include some direct targets of Pax6. We chose to study two different stages of development in order to distinguish between the more direct effects of the loss of Pax6 function at earlier developmental time points and those alterations that occur at later developmental stages that may result in part by mispatterning and altered tangential cell migration.
Section snippets
Experimental set up of microarray gene expression analysis in wild-type and Pax6Sey−/− embryonic telencephalon
To assess gene expression differences during telencephalic neurogenesis in the functional null mutant Pax6Sey we compared tissue that was isolated at the onset of neurogenesis (E12) and at mid-neurogenesis (E15) from the dorsal (cortex) and the ventral (GE) telencephalon of WT and Pax6Sey−/− littermates. RNA preparation was performed as described in the Experimental methods and used for hybridization of Affymetrix microarray (MOE430A). The material was collected in three sets (A, B, and C),
Technical and experimental considerations
This study provides further insights into the genes regulated by Pax6 in the developing telencephalon by a combination of loss- and gain-of-function analysis using microarray, Q-PCR and in situ expression analysis. We decided to use the entire cortex and GE as mRNA sources for the microarray experiments to obtain general information about the role of Pax6 in cortical development, even though Pax6 is only expressed in ventricular zone precursor cells (apical precursors) and in the earliest
Animals
Pax6Sey mice were maintained as heterozygotes on a mixed C57BL/6JxDBA/2J background. Electroporation experiments were done with C57BL/6J mice. The day of vaginal plug was considered to be embryonic day 0 (E0).
Tissue preparation and electroporation
Pregnant Pax6Sey heterozygous mice were sacrificed at E12 or E15 by exposure to CO2 and cervical dislocation. Embryos were harvested and transferred to Hanks’ buffered salt solution (HBSS; GIBCO) supplemented with 10 mM HEPES (GIBCO) where they were genotyped based on eye anatomy (Hill et
Acknowledgments
We thank Joakim Lundeberg and Robert Månsson for assistance with microarray analysis, Marcus Körbs for excellent technical assistence and Leanne Godinho for excellent suggestions on the manuscript. PH was supported by an EMBO long-term fellowship, MG by the DFG and BMBF.
References (123)
- et al.
Dlx transcription factors regulate differentiation of dopaminergic neurons of the ventral thalamus
Mol. Cell. Neurosci.
(2003) - et al.
Characterization of Lhx9, a novel LIM/homeobox gene expressed by the pioneer neurons in the mouse cerebral cortex
Mech. Dev.
(1999) - et al.
Getting your Pax straight: Pax proteins in development and disease
Trends Genet.
(2002) - et al.
Characterisation of the Wnt antagonists and their response to conditionally activated Wnt signalling in the developing mouse forebrain
Brain Res. Dev. Brain Res.
(2004) - et al.
SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation
Cell
(2006) - et al.
Transcription factor AP-2 regulates human insulin-like growth factor binding protein-5 gene expression
J. Biol. Chem.
(1995) - et al.
Altered retinoid signaling in the heads of small eye mouse embryos
Dev. Biol.
(2000) - et al.
Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling
Cell
(1997) Roles of the NFI/CTF gene family in transcription and development
Gene
(2000)- et al.
Pax6 controls radial glia differentiation in the cerebral cortex
Neuron
(1998)
Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6
Mol. Cell. Neurosci.
Mxi1 is essential for neurogenesis in Xenopus and acts by bridging the pan-neural and proneural genes
Dev. Biol.
Complex regulatory element within the gammaE- and gammaF-crystallin enhancers mediates Pax6 regulation and is required for induction by retinoic acid
Gene
Cadherin-4 expression in the zebrafish central nervous system and regulation by ventral midline signaling
Brain Res. Dev. Brain Res.
Pax6 is required for the multipotent state of retinal progenitor cells
Cell
A screen for downstream effectors of Neurogenin2 in the embryonic neocortex
Dev. Biol.
Novel essential DNA repair proteins Nse1 and Nse2 are subunits of the fission yeast Smc5–Smc6 complex
J. Biol. Chem.
Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons
Neuron
Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2
Neuron
Neural and head induction by insulin-like growth factor signals
Dev. Cell
Precocious retinal neurons: Pax6 controls timing of differentiation and determination of cell type
Dev. Biol.
Pax genes and eye organogenesis
Curr. Opin. Genet. Dev.
Basic helix–loop–helix factors in cortical development
Neuron
Influence of PAX6 gene dosage on development: overexpression causes severe eye abnormalities
Cell
R-cadherin is a Pax6-regulated, growth-promoting cue for pioneer axons
J. Neurosci.
Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex
J. Neurosci.
Neurohypophysial dysmorphogenesis in mice lacking the homeobox gene Uncx4.1
J. Mol. Endocrinol.
Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye
Genes Dev.
A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes
Bioinformatics
Specification of the neural crest occurs during gastrulation and requires Pax7
Nature
Differentiation of mouse embryonic stem cells into a defined neuronal lineage
Nat. Neurosci.
A comparison of normalization methods for high density oligonucleotide array data based on variance and bias
Bioinformatics
Polycomb complexes repress developmental regulators in murine embryonic stem cells
Nature
Novel transcription factor Satb2 interacts with matrix attachment region DNA elements in a tissue-specific manner and demonstrates cell-type-dependent expression in the developing mouse CNS
Eur. J. Neurosci.
Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair
Proc. Natl. Acad. Sci. U. S. A.
Vertebrate neurogenesis is counteracted by Sox1–3 activity
Nat. Neurosci.
Determination of the migratory capacity of embryonic cortical cells lacking the transcription factor Pax-6
Development
The role of Pax6 in restricting cell migration between developing cortex and basal ganglia
Development
The transcription factor neurogenin 2 restricts cell migration from the cortex to the striatum
Development
Expression patterns of the four nuclear factor I genes during mouse embryogenesis indicate a potential role in development
Dev. Dyn.
A comparative cDNA microarray analysis reveals a spectrum of genes regulated by Pax6 in mouse lens
Genes Cells
Early eye development in vertebrates
Annu. Rev. Cell Dev. Biol.
The vertebrate ortholog of Aristaless is regulated by Dlx genes in the developing forebrain
J. Comp. Neurol.
Regulation of gene expression by Pax6 in ocular cells: a case of tissue-preferred expression of crystallins in lens
Int. J. Dev. Biol.
Pax genes and organogenesis
BioEssays
Requirement for Pax6 in corneal morphogenesis: a role in adhesion
J. Cell Sci.
C-terminal activating and inhibitory domains determine the transactivation potential of BSAP (Pax-5), Pax-2 and Pax-8
EMBO J.
Dual roles for Pax-6: a transcriptional repressor of lens fiber cell-specific beta-crystallin genes
Mol. Cell. Biol.
Ectopic Pax6 expression disturbs lens fiber cell differentiation
Invest. Ophthalmol. Visual Sci.
Pax6 is required for delta-catenin/neurojugin expression during retinal, cerebellar and cortical development in mice
Dev. Biol.
Cited by (102)
Multiple roles of Pax6 in postnatal cornea development
2022, Developmental BiologyFunctional role of pax6 during eye and nervous system development in the annelid Capitella teleta
2019, Developmental BiologyA retinal-binding protein mediates olfactory attraction in the migratory locusts
2019, Insect Biochemistry and Molecular BiologyCitation Excerpt :The transcription factor pax6 has been reported to mediate cralbp mRNA transcription in mice (Boppana et al., 2012). PaX6 regulates morphogenesis and neurogenesis in olfactory epithelium and bulb and is coexpressed with cralbp in nervous system (Holm et al., 2007; Nomura et al., 2007). cralbp may be the direct downstream target of PaX6 in the development of mouse forebrain (Collinson et al., 2003).
Mechanisms of Cortical Differentiation
2018, International Review of Cell and Molecular BiologyTranscriptional regulation of Nfix by NFIB drives astrocytic maturation within the developing spinal cord
2017, Developmental Biology