Glucocorticoid Effects on Cerebellar Development in a Chicken Embryo Model: Exploring Changes in PAX6 and Metalloproteinase-9 After Exposure to Dexamethasone.

The developing cerebellum is vulnerable to effects of glucocorticoids and cerebellar dysfunction is associated with neurodevelopmental disorders (e.g. autism). Transcription factor PAX6 and matrix metalloproteinase-9 (MMP-9) are critical for normal cerebellar development and are highly expressed in migrating neurones. Alterations in MMP-9 and PAX6 are associated with altered cerebellar development. In the present study, we characterised the growth rate and development of the cortical layers, and further investigated how the levels of PAX6 and MMP-9, as well as glucocorticoid receptor (GR) and proliferating cell nuclear antigen (PCNA), change in the cerebellum during the foetal period [embryonic day (E)12-21] in chicken, which corresponds to the human perinatal period. Dexamethasone (DEX) was administered in ovo at E13 and E16, aiming to investigate how prenatal exposure to glucocorticoids interferes with normal development. DEX reduced foetal and cerebellar weight at E17 in a dose-dependent manner linked to a reduced level of PCNA and, over time, down-regulation of GR. We report that promoter activity of PAX6 and MMP-9 increased as a result of GR-stimulation in vitro. Prenatal DEX increased the protein level of PAX6 in a transient manner. PAX6 is reduced in mature granule neurones, and this occurred earlier in embryos exposed to DEX than in non-exposed controls. DEX exposure also led to a slow-onset down-regulation of MMP-9. Taken together, these findings indicate that excess prenatal glucocorticoid stimulation disturbs normal development of the cerebellum through mechanisms associated with reduced proliferation and accelerated maturation where PAX6 and MMP-9 play important roles.

Association of maternal smoking during pregnancy with aerobic fitness of offspring in young adulthood: a prospective cohort study.

OBJECTIVE: We evaluated the association of maternal pre-pregnancy body mass index (BMI), gestational weight gain (GWG), and maternal smoking with aerobic fitness in young men aged 19-20 years. DESIGN: A 19-year prospective cohort study. SETTING: Data from the Northern Finland Birth Cohort 1986 (NFBC 1986) and the Sodankylä Jaeger Brigade, Finland, in 2005-6. POPULATION: Mothers and the 508 offspring in the NFBC 1986 who entered military service at the Sodankylä Jaeger Brigade in 2005. METHODS: Associations of weight, 12-minute running test (Cooper test), and muscle fitness index (MFI) of offspring on entry to military service were evaluated with antenatal factors, including maternal smoking, pre-pregnancy BMI, and GWG. MAIN OUTCOME MEASURES: Aerobic and muscle fitness of the offspring were evaluated by the Cooper test and MFI. RESULTS: Maternal smoking during pregnancy was associated with lower aerobic fitness of male adolescents, measured by the Cooper test (2356 m; 95% confidence interval, 95% CI 2265-2446 m), compared with the offspring of mothers who did not smoke during pregnancy (2537 m, 95% CI 2499-2574 m). This association was independent of the BMIs of both the mother and the offspring, GWG, and the smoking and physical activity of offspring (regression coefficient -140.6 m, 95% CI -273.1 to -8.0 m). High maternal pre-pregnancy BMI and excessive GWG were also associated with lower aerobic fitness of the offspring; however, this association was mediated via the weight of the offspring. CONCLUSIONS: Maternal smoking during pregnancy may have a negative impact on the aerobic fitness of the offspring. TWEETABLE ABSTRACT: Study shows that young men have lower aerobic fitness if their mothers smoked during pregnancy.

Superactivation of Pax6-mediated transactivation from paired domain-binding sites by dna-independent recruitment of different homeodomain proteins.

The Pax6 genes encode evolutionary conserved transcription factors that act high up in the regulatory hierarchy controlling development of central organs such as the eyes and the central nervous system. These proteins contain two DNA-binding domains. The N-terminal paired domain is separated from a paired-type homeodomain by a linker region, and a transactivation domain is located C-terminal to the homeodomain. Vertebrate Pax6 genes express a paired-less isoform of Pax6 (Pax6DeltaPD) from an internal start codon in the coding region between the paired domain and homeodomain. We now provide evidence for an interaction between the full-length isoform and Pax6DeltaPD, which enhances the transactivation activity of Pax6 from paired domain-binding sites. The paired-like homeodomain protein Rax behaved similarly to Pax6DeltaPD. Both Pax6DeltaPD and Rax bound to the homeodomain of Pax6 in vitro in the absence of specific DNA binding. Coimmunoprecipitation experiments following cotransfection confirmed the existence of complexes between Pax6 and Pax6DeltaPD, Pax6 and Rax, and Pax6DeltaPD and Rax in vivo. Interestingly, the C-terminal subdomain of the paired domain and the homeodomain can interact with each other. The paired domain can also interact with itself. Surprisingly, GST pull-down assays revealed that the homeodomains of such diverse proteins as Chx10, Six3, Lhx2, En-1, Prep1, Prox1, and HoxB1 could all bind to Pax6, and several of these enhanced Pax6-mediated transactivation upon coexpression. Since many homeodomain proteins are coexpressed with Pax6 in several tissues during development, our results indicate the existence of novel regulatory interactions that may be important for fine tuning of gene regulation.

Phosphorylation of the transactivation domain of Pax6 by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase.

The transcription factor Pax6 is required for normal development of the central nervous system, the eyes, nose, and pancreas. Here we show that the transactivation domain (TAD) of zebrafish Pax6 is phosphorylated in vitro by the mitogen-activated protein kinases (MAPKs) extracellular-signal regulated kinase (ERK) and p38 kinase but not by Jun N-terminal kinase (JNK). Three of four putative proline-dependent kinase phosphorylation sites are phosphorylated in vitro. Of these sites, the serine 413 (Ser413) is evolutionary conserved from sea urchin to man. Ser413 is also phosphorylated in vivo upon activation of ERK or p38 kinase. Substitution of Ser413 with alanine strongly decreased the transactivation potential of the Pax6 TAD whereas substitution with glutamate increased the transactivation. Reporter gene assays with wild-type and mutant Pax6 revealed that transactivation by the full-length Pax6 protein from paired domain-binding sites was strongly enhanced (16-fold) following co-transfection with activated p38 kinase. This enhancement was largely dependent on the Ser413 site. ERK activation, however, produced a 3-fold increase in transactivation which was partly independent of the Ser413 site. These findings provide a starting point for further studies aimed at elucidating a post-translational regulation of Pax6 following activation of MAPK signaling pathways.

Zebrafish contains two pax6 genes involved in eye development.

The Pax6 genes of both vertebrates and invertebrates are expressed in the developing eye and in the central nervous system. These genes encode transcription factors with two DNA-binding domains, an N-terminal paired domain and a homeodomain separated by a flexible linker region. Ectopic eye structures are obtained upon targeted expression of Drosophila, squid, ascidian or mouse Pax6 genes in various imaginal disc primordia of Drosophila. We have previously cloned a Pax6 cDNA from zebrafish. Here we report the cloning of a novel Pax6 homolog from zebrafish denoted Pax6.2. The coding sequences of the two genes show 82% identity whereas the deduced amino acid sequences are 95% identical with complete conservation of the paired- and homeodomains. The embryonic expression patterns of Pax6.1 and Pax6.2 reveal both overlapping and discrete expression domains suggesting a division of labor between these two very similar gene products during development of brain and eye structures. Both Pax6.1 and Pax6.2 can act as transcriptional activators with Pax6.2 being more efficient than Pax6.1. Both Pax6.1 and Pax6.2 are able to induce ectopic eyes in Drosophila, while Pax2 is not, suggesting that eye induction is not a general feature of Pax family genes but a distinct characteristic of Pax6 and its direct homologs. Attempts to detect Pax6. 2 homologs in chick, mice or humans proved unsuccessful suggesting that this gene either was lost during evolution of higher vertebrates or, more likely, arose as part of a larger scale duplication of chromosome segments occurring in the zebrafish lineage.

Zebrafish Pax9 encodes two proteins with distinct C-terminal transactivating domains of different potency negatively regulated by adjacent N-terminal sequences.

We describe the isolation of cDNA clones for zebrafish Pax9. Pax9 expression was initiated at the end of the segmentation period in mesenchymal sclerotome cells on both sides of the notochord similarly to the corresponding mouse and chick genes. Two transcripts, Pax9a and -b, are generated by alternative splicing. The gene contains 4 exons with exon 3 being included in the Pax9a transcript and spliced out in the Pax9b transcript. The Pax9a and -b proteins are identical for 212 amino acids from the N terminus but contain distinct C-terminal regions of 131 and 58 amino acids, respectively. The paired domain of Pax9 displayed a binding-site specificity distinct from Pax6 but similar to Pax1 and -2. Both Pax9a and -b activated a promoter containing a paired domain binding site. However, this activation was observed when low amounts of Pax9 expression vectors were used. Higher amounts led to a sharp decrease in the activation and even turned into repression. Both the distinct C-terminal regions of Pax9a and -b harbored transcriptional activating domains of different potency not revealed in the context of the full-length proteins due to a negative influence of the N-terminal region including the paired domain.

Midline signalling is required for Pax gene regulation and patterning of the eyes.

Pax6 and Pax2 are members of the Pax family of transcription factors that are both expressed in the developing visual system of zebrafish embryos. Pax6 protein is present in all cells that form the neural retina and pigment epithelium, whereas Pax2 is located primarily in cells that will give rise to the optic stalk. In this study, we have addressed the role of midline signalling in the regulation of Pax2 and Pax6 distributions and in the subsequent morphogenesis of the eyes. Midline signalling is severely perturbed in cyclops mutant embryos resulting in an absence of ventral midline CNS tissue and fusion of the eyes. Mutant embryos ectopically express Pax6 in a bridge of tissue around the anterior pole of the neural keel in the position normally occupied by cells that form the optic stalks. In contrast, Pax2 protein is almost completely absent from this region in mutant embryos. Concommitant with the changes in Pax protein distribution, cells in the position of the optic stalks differentiate as retina. These results suggest that a signal emanating from the midline, which is absent in cyclops mutant embryos, may be required to promote Pax2 and inhibit Pax6 expression in cells destined to form the optic stalks. Sonic hedgehog (Shh also known as Vhh-1 and Hhg-1) is a midline signalling molecule that is absent from the neuroepithelium of cyclops mutant embryos at early developmental stages. To test the possibility that Shh might be able to regulate the spatial expression of Pax6 and Pax2 in the optic primordia, it was overexpressed in the developing CNS. The number of cells containing Pax2 was increased following shh overexpression and embryos developed hypertrophied optic stalk-like structures. Complimentary to the changes in Pax2 distribution, there were fewer Pax6-containing cells and pigment epithelium and neural retina were reduced. Our results suggest that Shh or a closely related signalling molecule emanating from midline tissue in the ventral forebrain either directly or indirectly induces the expression of Pax2 and inhibits the expression of Pax6 and thus may regulate the partitioning of the optic primordia into optic stalks and retinal tissue.

Regulatory gene expression boundaries demarcate sites of neuronal differentiation in the embryonic zebrafish forebrain.

During development of the zebrafish forebrain, a simple scaffold of axon pathways is pioneered by a small number of neurons. We show that boundaries of expression domains of members of the eph, forkhead, pax, and wnt gene families correlate with the positions at which these neurons differentiate and extend axons. Analysis of genetically or experimentally altered forebrains indicates that if a boundary is maintained, there is appropriate neural differentiation with respect to the boundary. Conversely, in the absence of a boundary, there is concomitant disruption of neural patterning. We also show that a strip of cells within the dorsal diencephalon shares features with ventral midline cells. This strip of cells fails to develop in mutant fish in which specification of the ventral CNS is disrupted, suggesting that its development may be regulated by the same inductive pathways that pattern the ventral midline.

The paired domain-containing nuclear factor pax[b] is expressed in specific commissural interneurons in zebrafish embryos.

The zebrafish paired box (Pax) genes are expressed in the early neural tube and are thought to be transcription factors that regulate the differentiation of cells in the central nervous system (CNS). The protein product of one of these Pax genes, pax[b], is detectable as a nuclear antigen in all the regions of the embryo that transcribe the gene including the posterior midbrain, the nephritic primordium, the Wolffian duct, the optic stalk, and, in specific neurons, in the hindbrain and spinal cord. The timing and pattern of axonal outgrowth by the early pax[b]-positive neurons suggest that they are the commissural secondary ascending (CoSA) interneurons in the spinal cord; the primary commissural interneurons (MiD2c and MiD3c) in hindbrain rhombomeres mi2 and mi3; and a previously unclassified set of commissural interneurons that we termed the commissural caudalrhombomere ascending (CoCaA) interneurons in the caudal hindbrain. In contrast, the Mauthner interneurons do not express pax[b] early in development. Shortly after the appearance of the first pax[b]-positive interneurons, additional nuclei adjacent to the first pax[b]-positive interneurons become pax[b] positive. This pattern of expression suggests that the pax[b] protein may be involved in determining the identity of specific commissural interneurons.