Pax6 limits the competence of developing cerebral cortical cells to respond to inductive intercellular signals.

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors' competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6's main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.

Gli3 is required autonomously for dorsal telencephalic cells to adopt appropriate fates during embryonic forebrain development.

The Gli3 zinc finger transcription factor is expressed in developing forebrain, with the highest levels of expression in dorsal telencephalon. In Gli3(-/-) embryos the dorsal telencephalon is abnormally small and fails to develop dorsomedial telencephalic structures, including hippocampus and cortical hem, while the ventral telencephalon appears to expand. A hurdle to understanding the underlying mechanisms is that abnormalities of developing Gli3(-/-) telencephalic cells in Gli3(-/-) mutants result from a combination of their own cell autonomous defects and defects in the Gli3(-/-) cells that surround them. Here we used chimeras to identify some of the defects of Gli3(-/-) telencephalic cells that are likely to be autonomous by studying how Gli3(-/-) cells develop when surrounded by a majority of wild-type cells. We found that Gli3(-/-) cells are present in all components of the Gli3(-/-)<-->Gli3(+/+) chimeric forebrain, including dorsomedial structures, in proportions that either equal or exceed proportions found elsewhere in the embryo. Gli3(-/-) cells segregate from Gli3(+/+) cells to form many abnormal structures particularly in dorsal telencephalon. Gli3(-/-) cells in some locations are misspecified: in those parts of the dorsal telencephalon near to its boundaries with the diencephalon and the ventral telencephalon, mutant cells express sets of transcription factors expressed by wild-type cells on the other side of the boundary. Elsewhere in the dorsal telencephalon, in the diencephalon and in the ventral telencephalon, mutant cells express sets of transcription factors similar to those expressed by their immediately surrounding wild-type cells. We propose that an important cell autonomous action of Gli3 is to regulate the competence of dorsal telencephalic cells, preventing cells near to its boundaries expressing regulatory factors normally restricted to adjacent tissues.

Novel lines of Pax6-/- embryonic stem cells exhibit reduced neurogenic capacity without loss of viability.

BACKGROUND: Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells died rapidly after neuronal differentiation in vitro. RESULTS: Here we report the derivation of new lines of Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- <--> Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- <--> Pax6+/+ chimeras made by embryo aggregation. CONCLUSIONS: We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development.

Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids.

Although there have been extensive studies on the effects of gonadotrophins and steroids on follicular development, less is known as to the effects these hormones have on the acquisition of oocyte developmental competence. This study investigates the effect of altering the gonadotrophin or steroidal environment on follicular development and on oocyte viability and DNA methylation. Oocytes were obtained from pre-ovulatory follicles after individual follicle culture from the pre-antral stage; gonadotrophin or steroid levels were manipulated during the culture period. Oocytes obtained from follicles grown in gonadotrophin free conditions were able to fertilize and develop to the blastocyst stage despite their impaired follicle development. There was no effect of luteinizing hormone or steroids on follicular growth. Altering the steroidal environment did, however, affect oocyte development. The oocytes of follicles exposed to high estrogen levels had lower fertilization rates, regardless of the presence or absence of high androgen levels. The combined presence of high levels of both steroids altered the level of global methylation. This study demonstrates that gonadotrophins and steroids influence the acquisition of developmental competence of the oocyte and suggests that optimal steroid exposure during follicle development is required for the oocyte to mature correctly.

Pax6 is required intrinsically by thalamic progenitors for the normal molecular patterning of thalamic neurons but not the growth and guidance of their axons.

BACKGROUND: In mouse embryos, the Pax6 transcription factor is expressed in the progenitors of thalamic neurons but not in thalamic neurons themselves. Its null-mutation causes early mis-patterning of thalamic progenitors. It is known that thalamic neurons generated by Pax6 (-/-) progenitors do not develop their normal connections with the cortex, but it is not clear why. We investigated the extent to which defects intrinsic to the thalamus are responsible. RESULTS: We first confirmed that, in constitutive Pax6 (-/-) mutants, the axons of thalamic neurons fail to enter the telencephalon and, instead, many of them take an abnormal path to the hypothalamus, whose expression of Slits would normally repel them. We found that thalamic neurons show reduced expression of the Slit receptor Robo2 in Pax6 (-/-) mutants, which might enhance the ability of their axons to enter the hypothalamus. Remarkably, however, in chimeras comprising a mixture of Pax6 (-/-) and Pax6 (+/+) cells, Pax6 (-/-) thalamic neurons are able to generate axons that exit the diencephalon, take normal trajectories through the telencephalon and avoid the hypothalamus. This occurs despite abnormalities in their molecular patterning (they express Nkx2.2, unlike normal thalamic neurons) and their reduced expression of Robo2. In conditional mutants, acute deletion of Pax6 from the forebrain at the time when thalamic axons are starting to grow does not prevent the development of the thalamocortical tract, suggesting that earlier extra-thalamic patterning and /or morphological defects are the main cause of thalamocortical tract failure in Pax6 (-/-) constitutive mutants. CONCLUSIONS: Our results indicate that Pax6 is required by thalamic progenitors for the normal molecular patterning of the thalamic neurons that they generate but thalamic neurons do not need normal Pax6-dependent patterning to become competent to grow axons that can be guided appropriately.

Cell-autonomous repression of Shh by transcription factor Pax6 regulates diencephalic patterning by controlling the central diencephalic organizer.

During development, region-specific patterns of regulatory gene expression are controlled by signaling centers that release morphogens providing positional information to surrounding cells. Regulation of signaling centers themselves is therefore critical. The size and the influence of a Shh-producing forebrain organizer, the zona limitans intrathalamica (ZLI), are limited by Pax6. By studying mouse chimeras, we find that Pax6 acts cell autonomously to block Shh expression in cells around the ZLI. Immunoprecipitation and luciferase assays indicate that Pax6 can bind the Shh promoter and repress its function. An analysis of chimeras suggests that many of the regional gene expression pattern defects that occur in Pax6(-/-) diencephalic cells result from a non-cell-autonomous position-dependent defect of local intercellular signaling. Blocking Shh signaling in Pax6(-/-) mutants reverses major diencephalic patterning defects. We conclude that Pax6's cell-autonomous repression of Shh expression around the ZLI is critical for many aspects of normal diencephalic patterning.

Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous mechanism.

Many cerebral cortical neurons and glia are produced by apical progenitors dividing at the ventricular surface of the embryonic dorsal telencephalon. Other neurons are produced by basal progenitor cells, which are derived from apical progenitors, dividing away from the ventricular surface. The transcription factor Pax6 is expressed in apical progenitors and is downregulated in basal progenitors, which upregulate the transcription factor Tbr2. Here we show that Pax6(-/-) cells are under-represented in the cortex of Pax6(+/+)<-->Pax6(-/-) chimeras early in corticogenesis, indicating that Pax6 is required for the production of normal numbers of cortical cells. We provide evidence that this underproduction is attributable to an early depletion of the progenitor pool caused by greater than normal proportions of newly divided cells exiting the cell cycle. We show that most progenitor cells dividing away from the ventricular surface in Pax6(-/-) embryos fail to express the transcription factor Tbr2 and that Pax6 is required cell autonomously for Tbr2 expression in the developing cortex of Pax6(+/+)<-->Pax6(-/-) chimeras. Transcription factors normally expressed ventrally in the telencephalic ganglionic eminences (Mash1, Dlx2 and Gsh2) are upregulated cell autonomously in mutant cells in the developing cortex of Pax6(+/+)<-->Pax6(-/-) chimeras; Nkx2.1, which is expressed only in the medial ganglionic eminence, is not. These data indicate that early functions of Pax6 in developing cortical cells are to repress expression of transcription factors normally found in the lateral ganglionic eminence, to prevent precocious differentiation and depletion of the progenitor pool, and to induce normal development of cortical basal progenitor cells.

Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization.

Levels of expression of the transcription factor Pax6 vary throughout corticogenesis in a rostro-lateral(high) to caudo-medial(low) gradient across the cortical proliferative zone. Previous loss-of-function studies have indicated that Pax6 is required for normal cortical progenitor proliferation, neuronal differentiation, cortical lamination and cortical arealization, but whether and how its level of expression affects its function is unclear. We studied the developing cortex of PAX77 YAC transgenic mice carrying several copies of the human PAX6 locus with its full complement of regulatory regions. We found that PAX77 embryos express Pax6 in a normal spatial pattern, with levels up to three times higher than wild type. By crossing PAX77 mice with a new YAC transgenic line that reports Pax6 expression (DTy54), we showed that increased expression is limited by negative autoregulation. Increased expression reduces proliferation of late cortical progenitors specifically, and analysis of PAX77<---->wild-type chimeras indicates that the defect is cell autonomous. We analyzed cortical arealization in PAX77 mice and found that, whereas the loss of Pax6 shifts caudal cortical areas rostrally, Pax6 overexpression at levels predicted to shift rostral areas caudally has very little effect. These findings indicate that Pax6 levels are stabilized by autoregulation, that the proliferation of cortical progenitors is sensitive to altered Pax6 levels and that cortical arealization is not.

The role of neurotrophin receptors in female germ-cell survival in mouse and human.

During mammalian ovary formation, the production of ovarian follicles is accompanied by an enormous loss of germ cells. It is not known how this loss is regulated. We have investigated the role of the Trk tyrosine kinase receptors, primarily TrkB, in this process. The ovaries of TrkB-/- and TrkC-/- mice with a mixed (129Sv x C57BL/6) genetic background were examined shortly after birth. Around 50% of TrkB-/- mice had grossly abnormal ovaries that contained greatly reduced numbers of follicles. No defects were found in the ovaries of TrkC-/- mice. Congenic TrkB-/- mice were generated on 129Sv and C57BL/6 backgrounds: whereas the former had a mixed ovarian phenotype similar to that of the original colony of mice, the ovaries of all offspring of the C57BL/6 congenic line contained reduced numbers of follicles. RT-PCR showed that mRNA encoding TrkB and its two ligands, neurotrophin 4 (NT4) and brain-derived neurotrophic factor (BDNF), were present throughout the period of follicle formation in the mouse. In situ hybridisation showed that TrkB was expressed primarily in the germ cells before and after follicle formation. Mouse neonatal and fetal ovaries and human fetal ovaries were cultured in the presence of K252a, a potent inhibitor of all Trk receptors. In mice, K252a inhibited the survival of germ cells in newly formed (primordial) follicles. This effect was rescued by the addition of basic fibroblast growth factor (bFGF) to the culture medium. Combined addition of both BDNF and NT4 blocking antibodies lowered germ-cell survival, indicating that these TrkB ligands are required in this process. The results indicate that signalling through TrkB is an important component of the mechanism that regulates the early survival of female germ cells.