Bringing Onco-Innovation to Europe's Healthcare Systems: The Potential of Biomarker Testing, Real World Evidence, Tumour Agnostic Therapies to Empower Personalised Medicine.

Rapid and continuing advances in biomarker testing are not being matched by uptake in health systems, and this is hampering both patient care and innovation. It also risks costing health systems the opportunity to make their services more efficient and, over time, more economical. The potential that genomics has brought to biomarker testing in diagnosis, prediction and research is being realised, pre-eminently in many cancers, but also in an ever-wider range of conditions-notably BRCA1/2 testing in ovarian, breast, pancreatic and prostate cancers. Nevertheless, the implementation of genetic testing in clinical routine setting is still challenging. Development is impeded by country-related heterogeneity, data deficiencies, and lack of policy alignment on standards, approval-and the role of real-world evidence in the process-and reimbursement. The acute nature of the problem is compellingly illustrated by the particular challenges facing the development and use of tumour agnostic therapies, where the gaps in preparedness for taking advantage of this innovative approach to cancer therapy are sharply exposed. Europe should already have in place a guarantee of universal access to a minimum suite of biomarker tests and should be planning for an optimum testing scenario with a wider range of biomarker tests integrated into a more sophisticated health system articulated around personalised medicine. Improving healthcare and winning advantages for Europe's industrial competitiveness and innovation require an appropriate policy framework-starting with an update to outdated recommendations. We show herein the main issues and proposals that emerged during the previous advisory boards organised by the European Alliance for Personalized Medicine which mainly focus on possible scenarios of harmonisation of both oncogenetic testing and management of cancer patients.

A European survey on the insights of patients living with metastatic colorectal cancer: the patient journey before, during and after diagnosis - an Eastern European perspective.

BACKGROUND: Despite being highly preventable and treatable if diagnosed early, colorectal cancer (CRC) remains the second leading cause of cancer-related death in Europe. Limited information is available from the patient perspective on the persisting unmet needs of the journey of the patient with CRC. OBJECTIVE: To capture European metastatic CRC (mCRC) patients' insights during the patient journey (prediagnosis; diagnosis; postdiagnosis) through a patient survey. METHODS: In total, 883 patients from 15 European countries participated. Participants were divided into four groups from Hungary, Poland, Serbia and 'other European countries' (n=103, 163, 170 and 447 patients, respectively). RESULTS: General awareness of CRC and its symptoms prediagnosis varied among groups, with patients from Poland recording the lowest levels. Screening practices and attitudes also varied; while more patients from Serbia had been invited to CRC screening (~15%) compared with the other groups, the ones not invited claimed mostly (~20%) that would not have attended if they had been invited. Whereas most patients were diagnosed within a month after the first consultation/positive screening, the percentages varied substantially being lowest among patients in Poland (~30%) and Serbia (~25%). Although CRC-related information provision varied, with most informed patients from Hungary (~90%) and least from Serbia (~50%), all groups requested an easier-to-understand language by the healthcare team. Approximately 50% of patients from Eastern Europe had to wait longer than a month to receive treatment, in contrast to ~30% from other European countries. All groups emphasised the unmet need for support from psychologists and other patients. CONCLUSIONS: Our survey reveals the key aspects of the journey of the patient with mCRC and highlights the areas of similarities and differences between patients with mCRC from Eastern Europe versus those from other European countries as well as among patients from different Eastern European countries, calling for improvement particularly around awareness, screening, treatment availability, communication and support networks.

Bringing Greater Accuracy to Europe's Healthcare Systems: The Unexploited Potential of Biomarker Testing in Oncology.

Rapid and continuing advances in biomarker testing are not being matched by take-up in health systems, and this is hampering both patient care and innovation. It also risks costing health systems the opportunity to make their services more efficient and, over time, more economical. This paper sets out the potential of biomarker testing, the unfolding precision and range of possible diagnosis and prediction, and the many obstacles to adoption. It offers case studies of biomarker testing in breast, ovarian, prostate, lung, thyroid and colon cancers, and derives specific lessons as to the potential and actual use of each of them. It also draws lessons about how to improve access and alignment, and to remedy the data deficiencies that impede development. And it suggests solutions to outstanding issues - notably including funding and the tangled web of obtaining reimbursement or equivalent coverage that Europe's fragmented health system implies. It urges a European evolution towards an initial minimum testing scenario, which would guarantee universal access to a suite of biomarker tests for the currently most common conditions, and, further into the future, to an optimum testing scenario in which a much wider range of biomarker tests would be introduced and become part of a more sophisticated health system articulated around personalised medicine. For exploiting genomics to the full, it argues the need for a new policy framework for Europe. Biomarker testing is not an issue that can be treated in isolation, since the purpose of testing is to improve health. Its use is therefore always closely linked to specific health challenges and needs to be viewed in the broader policy context in the EU and more widely. The paper is the result of extensive engagement with experts and decision makers to develop the framework, and consequently represents a wide consensus of views on how healthcare systems should respond from push and pull factors at local, national and cross-border and EU level. It contains strong views and clear recommendations springing from the convictions of patients, clinicians, academics, medicines authorities, HTA bodies, payers, the diagnostic, pharmaceutical and ICT industries, and national policy makers.

The Transcription Factor Foxg1 Promotes Optic Fissure Closure in the Mouse by Suppressing Wnt8b in the Nasal Optic Stalk.

During vertebrate eye morphogenesis, a transient fissure forms at its inferior part, known as the optic fissure. This will gradually close, giving rise to a healthy, spherical optic cup. Failure of the optic fissure to close gives rise to an ocular disorder known as coloboma. During this developmental process, Foxg1 is expressed in the optic neuroepithelium, with highest levels of expression in the nasal optic stalk. Foxg1-/- mutant mice have microphthalmic eyes with a large ventral coloboma. We found Wnt8b expression upregulated in the Foxg1-/- optic stalk and hypothesized that, similar to what is observed in telencephalic development, Foxg1 directs development of the optic neuroepithelium through transcriptional suppression of Wnt8b To test this, we generated Foxg1-/-;Wnt8b-/- double mutants of either sex and found that the morphology of the optic cup and stalk and the closure of the optic fissure were substantially rescued in these embryos. This rescue correlates with restored Pax2 expression in the anterior tip of the optic fissure. In addition, although we do not find evidence implicating altered proliferation in the rescue, we observe a significant increase in apoptotic cell density in Foxg1-/-;Wnt8b-/- double mutants compared with the Foxg1-/- single mutant. Upregulation of Wnt/ß-catenin target molecules in the optic cup and stalk may underlie the molecular and morphological defects in the Foxg1-/- mutant. Our results show that proper optic fissure closure relies on Wnt8b suppression by Foxg1 in the nasal optic stalk to maintain balanced apoptosis and Pax2 expression in the nasal and temporal edges of the fissure.SIGNIFICANCE STATEMENT Coloboma is an ocular disorder that may result in a loss of visual acuity and accounts for ∼10% of childhood blindness. It results from errors in the sealing of the optic fissure (OF), a transient structure at the bottom of the eye. Here, we investigate the colobomatous phenotype of the Foxg1-/- mutant mouse. We identify upregulated expression of Wnt8b in the optic stalk of Foxg1-/- mutants before OF closure initiates. Foxg1-/-;Wnt8b-/- double mutants show a substantial rescue of the Foxg1-/- coloboma phenotype, which correlates with a rescue in molecular and cellular defects of Foxg1-/- mutants. Our results unravel a new role of Foxg1 in promoting OF closure providing additional knowledge about the molecules and cellular mechanisms underlying coloboma formation.

Elevated FOXG1 and SOX2 in glioblastoma enforces neural stem cell identity through transcriptional control of cell cycle and epigenetic regulators.

Glioblastoma multiforme (GBM) is an aggressive brain tumor driven by cells with hallmarks of neural stem (NS) cells. GBM stem cells frequently express high levels of the transcription factors FOXG1 and SOX2. Here we show that increased expression of these factors restricts astrocyte differentiation and can trigger dedifferentiation to a proliferative NS cell state. Transcriptional targets include cell cycle and epigenetic regulators (e.g., Foxo3, Plk1, Mycn, Dnmt1, Dnmt3b, and Tet3). Foxo3 is a critical repressed downstream effector that is controlled via a conserved FOXG1/SOX2-bound cis-regulatory element. Foxo3 loss, combined with exposure to the DNA methylation inhibitor 5-azacytidine, enforces astrocyte dedifferentiation. DNA methylation profiling in differentiating astrocytes identifies changes at multiple polycomb targets, including the promoter of Foxo3 In patient-derived GBM stem cells, CRISPR/Cas9 deletion of FOXG1 does not impact proliferation in vitro; however, upon transplantation in vivo, FOXG1-null cells display increased astrocyte differentiation and up-regulate FOXO3. In contrast, SOX2 ablation attenuates proliferation, and mutant cells cannot be expanded in vitro. Thus, FOXG1 and SOX2 operate in complementary but distinct roles to fuel unconstrained self-renewal in GBM stem cells via transcriptional control of core cell cycle and epigenetic regulators.

The molecular and cellular signatures of the mouse eminentia thalami support its role as a signalling centre in the developing forebrain.

The mammalian eminentia thalami (EmT) (or thalamic eminence) is an embryonic forebrain structure of unknown function. Here, we examined the molecular and cellular properties of the mouse EmT. We first studied mRNA expression of signalling molecules and found that the EmT is a structure, rich in expression of secreted factors, with Wnts being the most abundantly detected. We then examined whether EmT tissue could induce cell fate changes when grafted ectopically. For this, we transplanted EmT tissue from a tau-GFP mouse to the ventral telencephalon of a wild type host, a telencephalic region where Wnt signalling is not normally active but which we showed in culture experiments is competent to respond to Wnts. We observed that the EmT was able to induce in adjacent ventral telencephalic cells ectopic expression of Lef1, a transcriptional activator and a target gene of the Wnt/ß-catenin pathway. These Lef1-positive;GFP-negative cells expressed the telencephalic marker Foxg1 but not Ascl1, which is normally expressed by ventral telencephalic cells. These results suggest that the EmT has the capacity to activate Wnt/ß-catenin signalling in the ventral telencephalon and to suppress ventral telencephalic gene expression. Altogether, our data support a role of the EmT as a signalling centre in the developing mouse forebrain.

Loss of functional Dicer in mouse radial glia cell-autonomously prolongs cortical neurogenesis.

Radial glia of the mouse cerebral cortex emerge from neuroepithelial stem cells around embryonic day 11 and produce excitatory cortical neurons until a few days before birth. The molecular mechanisms that regulate the end of cortical neurogenesis remain largely unknown. Here we investigated if the Dicer-dependent microRNA (miRNA) pathway is involved. By electroporating a cre-recombinase expression vector into the cortex of E13.5 embryos carrying a conditional allele of Dicer1, we induced mosaic recombination causing Dicer1 deletion and reporter activation in a subset of radial glia. We analysed the long-term fates of their progeny. We found that mutant radial glia produced abnormally large numbers of Cux1-positive neurons, many of which populated the superficial cortical layers. Injections of the S-phase marker bromodeoxyuridine between postnatal days 3 and 14 showed that much of this population was generated postnatally. Our findings suggest a role for Dicer-dependent processes in limiting the timespan of cortical neurogenesis.

Foxg1 is required to limit the formation of ciliary margin tissue and Wnt/ß-catenin signalling in the developing nasal retina of the mouse.

The ciliary margin (CM) develops in the peripheral retina and gives rise to the iris and the ciliary body. The Wnt/ß-catenin signalling pathway has been implicated in ciliary margin development. Here, we tested the hypothesis that in the developing mouse retina Foxg1 is responsible for suppressing the Wnt/ß-catenin pathway and restricting CM development. We showed that there is excess CM tissue in Foxg1(-/-) null embryos and this expansion is more pronounced in the nasal retina where Foxg1 normally shows its highest expression levels. Results on expression of a reporter allele for Wnt/ß-catenin signalling and of Lef1, a target of Wnt/ß-catenin signalling, displayed significant upregulation of this pathway in Foxg1(-/-) nulls at embryonic days 12.5 and 14.5. Interestingly, this upregulation was observed specifically in the nasal retina, where normally very few Wnt-responsive cells are observed. These results indicate a suppressive role of Foxg1 on this signalling pathway. Our results reveal a new role of Foxg1 in limiting CM development in the nasal peripheral retina and add a new molecular player in the developmental network involved in CM specification.

MicroRNA-92b regulates the development of intermediate cortical progenitors in embryonic mouse brain.

Cerebral cortical neurons arise from radial glia (direct neurogenesis) or from intermediate progenitors (indirect neurogenesis); intriguingly, the sizes of intermediate progenitor populations and the cortices they generate correlate across species. The generation of intermediate progenitors is regulated by the transcription factor Tbr2, whose expression marks these cells. We investigated how this mechanism might be controlled. We found that acute blockade of mature microRNA biosynthesis in murine cortical progenitors caused a rapid cell autonomous increase in numbers of Tbr2-expressing cells. Acute microRNA-92b (miR-92b) gain of function caused rapid reductions in numbers of Tbr2-expressing cells and proliferating intermediate progenitors. Acute miR-92b loss of function had opposite effects. These findings indicate that miR-92b limits the production of intermediate cortical progenitors.

Wnt/ß-catenin signaling is disrupted in the extra-toes (Gli3(Xt/Xt) ) mutant from early stages of forebrain development, concomitant with anterior neural plate patterning defects.

The zinc finger transcription factor Gli3 is essential for normal development of the forebrain. Mutant mice with no functional Gli3 (extra-toes, Gli3(Xt/Xt) mutants) display a massive reduction in the size of the telencephalic lobes and absence of dorsomedial telencephalic structures, including the cortical hem, which normally expresses a number of Wnt molecules essential for patterning the hippocampus. Dorsomedial telencephalic Wnt activity, transduced through the Wnt/ß-catenin signaling pathway, is also required for hippocampal specification and dorsoventral telencephalic patterning. Wnts whose normal expression is restricted to the cortical hem are completely absent in Gli3(Xt/Xt) embryos, but some expression of those Wnts with a broader expression domain persists, raising the possibility that Wnt/ß-catenin signaling may still be active in this mutant. We examined whether the Wnt expression that persists in the Gli3(Xt/Xt) mutant neocortex activates Wnt/ß-catenin signaling, using the BAT-gal transgenic reporter. We found Wnt/ß-catenin signaling consistently decreased in the forebrains of Gli3(Xt/Xt) mutants, even prior to the formation of the cortical hem. This is accompanied by a severe reduction in expression of Wnt7b and Wnt8b at the lateral edges of the anterior neural plate that will give rise to the pallium. In addition, we found a significant increase in the expression of rostroventral markers of the anterior neural plate that will give rise to the basal forebrain. Our data reveal that Gli3 is required at the neural plate stage to regulate Wnt expression and Wnt/ß-catenin signaling in the presumptive forebrain and confirm its previously proposed role in patterning the anterior neural plate.

Loss of Wnt8b has no overt effect on hippocampus development but leads to altered Wnt gene expression levels in dorsomedial telencephalon.

Wnt signalling proteins regulate many aspects of animal development. We have investigated the function of mouse Wnt8b during forebrain development. Wnt8b is expressed in a highly restricted pattern including the prospective hippocampus and hypothalamus. Mutant mice lacking Wnt8b are viable and healthy. The size and morphology of the hippocampus appeared normal in mutant embryos and adults, and we found no evidence of hypothalamic defects in mutants. Wnt8b is also expressed in the neurogenic region of the adult dentate gyrus, however, cell proliferation was unchanged in Wnt8b(-/-) mutants. Mutant embryos did, however, display altered levels of expression of other Wnt genes normally expressed in forebrain. The spatial expression patterns of other Wnt genes and the overall level of canonical Wnt activity were indistinguishable from wild-types. Thus, loss of Wnt8b does not give rise to an overt morphological phenotype, but does affect expression levels of other Wnts in developing forebrain.

Analysis of early ventral telencephalic defects in mice lacking functional Gli3 protein.

The transcription factor Gli3 is expressed throughout developing telencephalon. Previous studies have focused on Gli3's role in dorsal telencephalon, which is greatly reduced in size in Gli3(Xt/Xt) mutants. We examined the effects of loss of Gli3 on early development of ventral telencephalon. Ventral telencephalon was defined in both wildtypes and Gli3(Xt/Xt) mutants on the basis of its expression of Olig2, Nkx2.1, Mash1, and Foxg1 and its lack of expression of Pax6. We found that at embryonic day (E)10.5 the volume of the ventral telencephalon is about 50% greater in Gli3(Xt/Xt) mutants than in wildtypes. By E12.5, however, the volume of the ventral telencephalon is about 20% lower in Gli3(Xt/Xt) mutants than in wildtypes. We observed a significant increase in the number of both apoptotic cells and newly differentiated neurons in the E10.5 Gli3(Xt/Xt) ventral telencephalon, suggesting that increased cell death and withdrawal of cells from the cell cycle might account for the failure of the Gli3(Xt/Xt) ventral telencephalon to grow normally by E12.5. We found no changes in the lengths of the cell cycles of proliferating ventral telencephalic cells at E10.5. We used marker analysis and optical projection tomography to assess the Gli3(Xt/Xt) forebrain in three dimensions and found that the Gli3(Xt/Xt) diencephalon is shifted relatively rostrally. We conclude that in the absence of Gli3 an abnormally large portion of the newly formed telencephalon is specified to a ventral fate but this then suffers impaired growth, due to defects of cell differentiation and death, contributing to severe distortion of the forebrain.

The protein kinase DYRK1A regulates caspase-9-mediated apoptosis during retina development.

The precise regulation of programmed cell death is critical for the normal development of the nervous system. We show here that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. We provide evidence that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. We show that DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. We demonstrate that DYRK1A phosphorylates caspase-9 on threonine residue 125, and that this phosphorylation event is crucial to protect retina cells from apoptotic cell death. Our data suggest a model in which dysregulation of the apoptotic response in differentiating neurons participates in the neuropathology of diseases that display DYRK1A gene-dosage imbalance effects, such as Down's syndrome.

Newly identified patterns of Pax2 expression in the developing mouse forebrain.

BACKGROUND: The availability of specific markers expressed in different regions of the developing nervous system provides a useful tool for the study of mouse mutants. One such marker, the transcription factor Pax2, is expressed at the midbrain-hindbrain boundary and in the cerebellum, spinal cord, retina, optic stalk, and optic chiasm. We recently described a group of diencephalic cells that express Pax2 as early as embryonic day (E) 10.5, and become part of the eminentia thalami by E11.5. The discovery of this previously undescribed cell population prompted us to examine Pax2 protein expression in the developing mouse forebrain in more detail. RESULTS: We determined the expression pattern of Pax2 in the forebrain of wild type mouse embryos between E10.5 and postnatal day (P) 15. Pax2 expression was detected in the septum of the basal forebrain, hypothalamus, eminentia thalami and in the subfornical organ. To evaluate Pax2 as a marker for septal cells, we examined Pax2 expression in Pax6Sey/Sey mutants, which have an enlarged septum. We found that Pax2 clearly marks a population of septal cells equivalent to that seen in wild types, indicating its utility as a marker of septal identity. These cells did not express the GABAergic marker calbindin nor the cholinergic marker choline acetyltransferase and were not detectable after P15. CONCLUSION: Pax2 is expressed in populations of cells within the developing septum, hypothalamus, and eminentia thalami. It seems especially useful as a marker of the telencephalic septum, because of its early, strong and characteristic expression in this structure. Further, its expression is maintained in the enlarged septum of Pax6Sey/Sey mutants.

Impaired spatial learning strategies and novel object recognition in mice haploinsufficient for the dual specificity tyrosine-regulated kinase-1A (Dyrk1A).

BACKGROUND: Pathogenic aneuploidies involve the concept of dosage-sensitive genes leading to over- and underexpression phenotypes. Monosomy 21 in human leads to mental retardation and skeletal, immune and respiratory function disturbances. Most of the human condition corresponds to partial monosomies suggesting that critical haploinsufficient genes may be responsible for the phenotypes. The DYRK1A gene is localized on the human chromosome 21q22.2 region, and has been proposed to participate in monosomy 21 phenotypes. It encodes a dual-specificity kinase involved in neuronal development and in adult brain physiology, but its possible role as critical haploinsufficient gene in cognitive function has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: We used mice heterozygous for a Dyrk1A targeted mutation (Dyrk1A+/-) to investigate the implication of this gene in the cognitive phenotypes of monosomy 21. Performance of Dyrk1A+/- mice was assayed 1/ in a navigational task using the standard hippocampally related version of the Morris water maze, 2/ in a swimming test designed to reveal potential kinesthetic and stress-related behavioral differences between control and heterozygous mice under two levels of aversiveness (25 degrees C and 17 degrees C) and 3/ in a long-term novel object recognition task, sensitive to hippocampal damage. Dyrk1A+/- mice showed impairment in the development of spatial learning strategies in a hippocampally-dependent memory task, they were impaired in their novel object recognition ability and were more sensitive to aversive conditions in the swimming test than euploid control animals. CONCLUSIONS/SIGNIFICANCE: The present results are clear examples where removal of a single gene has a profound effect on phenotype and indicate that haploinsufficiency of DYRK1A might contribute to an impairment of cognitive functions and stress coping behavior in human monosomy 21.

Pax3 regulates Wnt1 expression via a conserved binding site in the 5' proximal promoter.

The development of the neural crest is orchestrated by a complex interplay between intercellular signalling molecules and transcription factors. Here, we demonstrate a direct interaction between two such factors, the paired-type transcription factor Pax3 and the secretory glycoprotein Wnt1. We found that the Wnt1 promoter can be regulated by Pax3 in a dose-dependent manner. Sequence analysis predicted a conserved binding site for Pax3 within the Wnt1 promoter region. Deletion or mutation of this sequence abolished the promoter response to Pax3. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated that Pax3 interacts with the Wnt1 promoter in vivo. These data indicate that Pax3 directly regulates the expression of Wnt1 in the developing embryo.

Abnormal positioning of diencephalic cell types in neocortical tissue in the dorsal telencephalon of mice lacking functional Gli3.

The transcription factor Gli3 (glioma-associated oncogene homolog) is essential for normal development of the mammalian forebrain. One extreme requirement for Gli3 is at the dorsomedial telencephalon, which does not form in Gli3(Xt/Xt) mutant mice lacking functional Gli3. In this study, we analyzed expression of Gli3 in the wild-type telencephalon and observed a (high)dorsal-to-(low)ventral gradient of Gli3 expression and predominance of the cleaved form of the Gli3 protein dorsally. This graded expression correlates with the (severe)dorsal-to-(mild)ventral telencephalic phenotype observed in Gli3(Xt/Xt) mice. We characterized the abnormal joining of the telencephalon to the diencephalon and defined the medial limit of the dorsal telencephalon in Gli3(Xt/Xt) mice early in corticogenesis. Based on this analysis, we concluded that some of the abnormal expression of ventral telencephalic markers previously described as being in the dorsal telencephalon is, in fact, expression in adjacent diencephalic tissue, which expresses many of the same genes that mark the ventral telencephalon. We observed occasional cells with diencephalic character in the Foxg1 (forkhead box)-expressing Gli3(Xt/Xt) telencephalon at embryonic day 10.5, a day after the anatomical subdivision of the forebrain vesicle. Large clusters of such cells appear in the Gli3(Xt/Xt) neocortical region at later ages, when the neocortex becomes highly disorganized, forming rosettes comprising mainly neural progenitors. We propose that Gli3 is indispensable for formation of an intact telencephalic-diencephalic boundary and for preventing the abnormal positioning of diencephalic cells in the dorsal telencephalon.

Loss of Gli3 enhances the viability of embryonic telencephalic cells in vitro.

The transcription factor Gli3 is important for brain and limb development. Mice homozygous for a mutation in Gli3 (Gli3Xt/Xt) have severe abnormalities of telencephalic development and previous studies have suggested that aberrant cell death may contribute to the Gli3Xt/Xt phenotype. We demonstrate that telencephalic cells from embryonic Gli3Xt/Xt embryos survive better and are more resistant to death induced by cytosine arabinoside, a nucleoside analogue that induces death in neuronal progenitors and neurons, than are control counterparts in vitro. Culture medium conditioned by Gli3Xt/Xt cells is more effective at enhancing the viability of control telencephalic cells than medium conditioned by control cells, indicating that Gli3Xt/Xt cells release a factor or factors which enhance telencephalic cell viability. Gli3(Xt/Xt) cells are also more sensitive to released factors present in conditioned media. These data suggest that Gli3 plays both cell-autonomous and cell-nonautonomous roles in mediating telencephalic cell viability.

Dyrk1A expression pattern supports specific roles of this kinase in the adult central nervous system.

Dyrk1A and its Drosophila orthologue, the protein minibrain (mnb), belong to a family of serine/threonine kinases involved in the development of the central nervous system (CNS). However, additional roles for Dyrk1A have to be proposed, as its expression is still prominent in the adult brain. To gain insight into Dyrk1A physiological roles we have studied the distribution of this kinase in the CNS of mice in adulthood. A homogeneous diffuse immunostaining of variable intensity was detected throughout the neuropile, with the white matter displaying faint Dyrk1A immunoreactivity. Dyrk1A immunostaining was strong in the olfactory bulb, the cerebellar cortex and functionally related structures, the spinal cord and most of the motor nuclei of the midbrain and brain stem. These data agree with a possible implication of this kinase in the physiology of olfaction and motor functions. Cellular and subcellular localisation of Dyrk1A was also studied in primary cell culture of cerebellum, one of the structures showing significant Dyrk1A immunostaining in the adult. The distribution of Dyrk1A in primary cell culture showed the presence of this protein in the nucleus and the cytoplasm of both neurons and astrocytes. Moreover, studies on the subcellular distribution of Dyrk1A in whole brain homogenates of adult mice showed the presence of this protein both in nuclear and cytoplasm-enriched fractions, thus supporting selective functions of this kinase in these two subcellular compartments. The present results showing the distribution of Dyrk1A in widespread areas of the adult CNS and in different subcellular compartments, together with previous reports demonstrating its implication in developmental events concur with the idea of several spatio-temporal functional profiles.

Dyrk1A haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice.

DYRK1A is the human orthologue of the Drosophila minibrain (mnb) gene, which is involved in postembryonic neurogenesis in flies. Because of its mapping position on chromosome 21 and the neurobehavioral alterations shown by mice overexpressing this gene, involvement of DYRK1A in some of the neurological defects of Down syndrome patients has been suggested. To gain insight into its physiological role, we have generated mice deficient in Dyrk1A function by gene targeting. Dyrk1A(-/-) null mutants presented a general growth delay and died during midgestation. Mice heterozygous for the mutation (Dyrk1A(+/-)) showed decreased neonatal viability and a significant body size reduction from birth to adulthood. General neurobehavioral analysis revealed preweaning developmental delay of Dyrk1A(+/-) mice and specific alterations in adults. Brains of Dyrk1A(+/-) mice were decreased in size in a region-specific manner, although the cytoarchitecture and neuronal components in most areas were not altered. Cell counts showed increased neuronal densities in some brain regions and a specific decrease in the number of neurons in the superior colliculus, which exhibited a significant size reduction. These data provide evidence about the nonredundant, vital role of Dyrk1A and suggest a conserved mode of action that determines normal growth and brain size in both mice and flies.