Françoise Jamen

PAC1 receptor–deficient mice display impaired insulinotropic response to glucose and reduced glucose tolerance

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a ubiquitous neuropeptide of the vasoactive intestinal peptide (VIP) family that potentiates glucose-stimulated insulin secretion. Pancreatic beta cells express two PACAP receptor subtypes, a PACAP-preferring (PAC1) and a VIP-shared (VPAC2) receptor. We have applied a gene targeting approach to create a mouse lacking the PAC1 receptor (PAC1(-/-)). These mice were viable and normoglycemic, but exhibited a slight feeding hyperinsulinemia. In vitro, in the isolated perfused pancreas, the insulin secretory response to PACAP was reduced by 50% in PAC1(-/-) mice, whereas the response to VIP was unaffected. In vivo, the insulinotropic action of PACAP was also acutely reduced, and the peptide induced impairment of glucose tolerance after an intravenous glucose injection. This demonstrates that PAC1 receptor is involved in the insulinotropic action of the peptide. Moreover, PAC1(-/-) mice exhibited reduced glucose-stimulated insulin secretion in vitro and in vivo, showing that the PAC1 receptor is required to maintain normal insulin secretory responsiveness to glucose. The defective insulinotropic action of glucose was associated with marked glucose intolerance after both intravenous and gastric glucose administration. Thus, these results are consistent with a physiological role for the PAC1 receptor in glucose homeostasis, notably during food intake.

Dissociation between Light-Induced Phase Shift of the Circadian Rhythm and Clock Gene Expression in Mice Lacking the Pituitary Adenylate Cyclase Activating Polypeptide Type 1 Receptor

The circadian clock located in the suprachiasmatic nucleus (SCN) organizes autonomic and behavioral rhythms into a near 24 hr time that is adjusted daily to the solar cycle via a direct projection from the retina, the retinohypothalamic tract (RHT). This neuronal pathway costores the neurotransmitters PACAP and glutamate, which seem to be important for light-induced resetting of the clock. At the molecular level the clock genes mPer1 and mPer2 are believed to be target for the light signaling to the clock. In this study, we investigated the possible role of PACAP-type 1 receptor signaling in light-induced resetting of the behavioral rhythm and light-induced clock gene expression in the SCN. Light stimulation at early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice (PAC1−/−) compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and c-fos gene expression. Light stimulation at late night induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type andPAC1−/− mice. However, in contrast to the phase advance seen in wild-type mice,PAC1−/− mice responded with phase delays after photic stimulation. These data indicate that PAC1 receptor signaling participates in the gating control of photic sensitivity of the clock and suggest thatmPer1, mPer2, and c-fosare of less importance for light-induced phase shifts at night.

Evidence for neural stem cells in the medaka optic tectum proliferation zones

Few adult neural stem cells have been characterized in vertebrates. Although teleosts continually generate new neurons in many regions of the brain after embryogenesis, only two types of neural stem cells (NSCs) have been reported in zebrafish: glial cells in the forebrain resembling mammalian NSCs, and neuroepithelial cells in the cerebellum. Here, following our previous studies on dividing progenitors (Nguyen et al. [ 1999 ]: J Comp Neurol 413:385–404.), we further evidenced NSCs in the optic tectum (OT) of juvenile and adult in the medaka, Oryzias latipes. To detect very slowly cycling progenitors, we did not use the commonly used BrdU/PCNA protocol, in which PCNA may not be present during a transiently quiescent state. Instead, we report the optimizations of several protocols involving long subsequent incubations with two thymidine analogs (IdU and CldU) interspaced with long chase times between incubations. These protocols allowed us to discriminate and localize fast and slow cycling cells in OT of juvenile and adult in the medaka. Furthermore, we showed that adult OT progenitors are not glia, as they express neither brain lipid‐binding protein (BLBP) nor glial fibrillary acidic protein (GFAP). We also showed that expression of pluripotency‐associated markers (Sox2, Musashi1 and Bmi1) colocalized with OT progenitors. Finally, we described the spatio‐temporally ordered population of NSCs and progenitors in the medaka OT. Hence, the medaka appears as an invaluable model for studying neural progenitors that will open the way to further exciting comparative studies of neural stem cells in vertebrates.

Up-regulation of the PACAP type-1 receptor (PAC1) promoter by neurotrophins in rat PC12 cells and mouse cerebellar granule cells via the Ras/mitogen-activated protein kinase cascade.

The pituitary adenylate cyclase‐activating polypeptide type‐1 receptor (PAC1) has been involved in the survival and differentiation of neuroblasts during development. This study examined the effects of various neurotrophins on the activity of the mouse PAC1 promoter/luciferase reporter constructs in rat PC12 cells and in 8‐day‐old mouse cerebellar granule cells. In PC12 cells, both differentiating factors such as nerve growth factor (NGF) and mitogens such as epidermal growth factor (EGF) and insulin growth factor‐1 (IGF‐1) up‐regulated PAC1 promoter activity by 2–4‐fold in a concentration‐dependent manner. Although PACAP differentiated the PC12 cells, it had no effect on the PAC1 promoter and antagonized the stimulatory effect of NGF. In cerebellar granule cells, IGF‐1 and brain‐derived neurotrophic factor (BDNF) also stimulated the activity of the PAC1 promoter. NGF and IGF‐1 increased endogenous PAC1 mRNA levels, and the NGF‐induced up‐regulation is the result of an increase in transcription from PAC1 promoter instead of an increase in mRNA stability. The mitogen‐activated protein kinase (MAPK) kinase inhibitor, PD98059, prevented the transcriptional effects both in PC12 and cerebellar granule cells. Moreover, expression of dominant‐negative Ras protein in PC12 cells also prevented the NGF effect. Our results show that the PAC1 promoter can be up‐regulated by diverse neurotrophins via an MAPK‐dependent pathway and suggest a role for the Ras protein.

Transcription of the mouse PAC1 receptor gene: cell-specific expression and regulation by Zac1

Regulations of the PACAP type 1 (PAC1) receptor expression have been described in the brain and the anterior pituitary. To understand the molecular mechanisms underlying mouse PAC1 gene regulation, we first mapped its transcription start sites (tss). PAC1 receptor RNA initiates from two major sites in embryos and adult tissues. Functional analysis revealed a basal promoter within the first 180 bp upstream of transcription start. Negative regulatory sequences upstream of this minimal promoter control the cell type-specific transcription of a luciferase reporter gene. Zac1, a zinc finger protein mainly expressed in the brain and the pituitary gland, binds to a GC-rich motif of the promoter regulatory elements. The Zac1 DNA binding site is required to positive and negative regulations of the promoter. Our findings provide bases for future studies on the regulatory elements controlling PAC1 gene transcription and demonstrate the PAC1 receptor promoter as a target of Zac1.

Zebrafish midbrain slow-amplifying progenitors exhibit high levels of transcripts for nucleotide and ribosome biogenesis

Investigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs.

Nerve Growth Factor Upregulates the PAC1 Promoter by Activating the MAP Kinase Pathway in Rat PC12 Cells

We report that: (1) An increase in the transcription activity is a mechanism by which trophic peptides may regulate the expression of PAC1. (2) An activation of the PAC1 promoter does not necessarily correlate with the neurotrophin-promoted neuritogenesis. (3) Activation of the PAC1 promoter is probably an early event since the EGF response is rather weak and transient in PC12 cells. (4) MAP kinase pathway activation is necessary for the NGF effect. The mechanism of the antagonism between PACAP and NGF observed on the PAC1 promoter activity and already described in regulating chromaffin cell proliferation, remains to be explained.

Genome-wide analysis of the POU genes in medaka, focusing on expression in the optic tectum.

The highly conserved POU genes encode homeodomain transcription factors involved in various developmental events, with some, the Brn genes, playing key roles in neurogenesis. We investigated the evolutionary relationships between these genes, by studying the POU gene complement of a model teleost, the medaka (Oryzias latipes). We identified 17 POU genes and carried out a comprehensive in situ hybridization analysis focusing on the optic tectum, a cortical structure of the mesencephalon, in which cell positions and their differentiation states are spatially and temporally correlated. Six POU genes displayed patterned expression in the optic tectum: two genes were expressed in the center of the organ (a zone with differentiated neurons), two in an intermediate zone in which cells exit the cell cycle and two in the peripheral proliferation zone. These results suggest that POU genes may play key roles in both late neurogenesis and in multipotent neural progenitors. Developmental Dynamics 240:2354–2363, 2011.

PACAP Receptor Knockout and Transgenics: What Have We Learnt?

Since the discovery of PACAP, originally isolated from hypothalamus in 1989 (Miyata et al, 1989), numerous studies devoted to the expression of this peptide have clearly demonstrated that it is not restricted to hypothalamic level but it is also expressed in various other brain areas and peripheral tissues. Functional studies have also established that the role of PACAP is not restricted to activation of the hypothalamic-pituitary complex, but that PACAP serves additional functions in the brain and peripheral organs. Most of these studies, herein reported in the precedent chapters, have been conducted both in vitro by incubation of PACAP, VIP and related peptides on either tumoral cell lines, primary cell cultures, or isolated perfused organs; and in vivo by injection of PACAP, or by administration of antagonists or PACAP antibodies to block the peptide activity. These approaches had the advantage of being technically simple, reproducible and/or relatively fast but were limited to a cellular or tissular context and to application of exogenous drugs. Recently, an alternative more appropriate approach to studying the physiology of PACAP has been the generation of mouse strains deficient in PACAP type 1 receptor and overexpressing the VIP (vasoactive intestinal peptide)/PACAP type 2 receptor (VPAC2).

17-P005 Molecular characterization of neural stem cells in medaka

While adult neurogenesis in mammals is mainly restricted to two telencephalic zones, in teleost fishes, neurogenesis has been observed in many brain areas. Hence, the medaka (Oryzias latipes) represents an excellent model to study neural stem cells (NSCs). In order to detect NSCs, we have relied on the fact that adult stem cells divide more slowly than quickly amplifying progenitor cells. By using two different thymidine analogs and performing long pulses separated by a long period of time, we have managed to identify cells that are double-labelled in the medaka brain. These slow-proliferating cells are likely to be NSCs. We have looked more precisely at neurogenesis in a cortical structure of the dorsal midbrain, the optic tectum. Cell proliferation occurs at its margin throughout life. With the thymidine analog labeling, we have identified some slow-dividing cells, in the tectum, forming a narrow line at the very periphery of the proliferative zone. This topographic organization allowed us to perform an insitu hybridization screen on medaka embryos and find a dozen of genes whose expression corresponds to this thin line of putative NSCs. By analyzing the functions of these genes, we hope to identify essential genes for adult NSC and embryonic NSC biology. We should be able to bring cues on the conserved features between fish and mammal stem cells and examine whether neurogenesis in adult teleosts is merely the continuation of an embryonic phase or a distinct process involving adult specific mechanisms for stemness maintenance.