MC3R links nutritional state to childhood growth and the timing of puberty.

The state of somatic energy stores in metazoans is communicated to the brain, which regulates key aspects of behaviour, growth, nutrient partitioning and development1. The central melanocortin system acts through melanocortin 4 receptor (MC4R) to control appetite, food intake and energy expenditure2. Here we present evidence that MC3R regulates the timing of sexual maturation, the rate of linear growth and the accrual of lean mass, which are all energy-sensitive processes. We found that humans who carry loss-of-function mutations in MC3R, including a rare homozygote individual, have a later onset of puberty. Consistent with previous findings in mice, they also had reduced linear growth, lean mass and circulating levels of IGF1. Mice lacking Mc3r had delayed sexual maturation and an insensitivity of reproductive cycle length to nutritional perturbation. The expression of Mc3r is enriched in hypothalamic neurons that control reproduction and growth, and expression increases during postnatal development in a manner that is consistent with a role in the regulation of sexual maturation. These findings suggest a bifurcating model of nutrient sensing by the central melanocortin pathway with signalling through MC4R controlling the acquisition and retention of calories, whereas signalling through MC3R primarily regulates the disposition of calories into growth, lean mass and the timing of sexual maturation.

Sonic hedgehog--regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system.

During development, basic helix-loop-helix (bHLH) proteins regulate formation of neurons from multipotent progenitor cells. However, bHLH factors linked to gliogenesis have not been described. We have isolated a pair of oligodendrocyte lineage genes (Olg-1 and Olg-2) that encode bHLH proteins and are tightly associated with development of oligodendrocytes in the vertebrate central nervous system (CNS). Ectopic expression of Olg-1 in rat cortical progenitor cell cultures promotes formation of oligodendrocyte precursors. In developing mouse embryos, Olg gene expression overlaps but precedes the earliest known markers of the oligodendrocyte lineage. Olg genes are expressed at the telencephalon-diencephalon border and adjacent to the floor plate, a source of the secreted signaling molecule Sonic hedgehog (Shh). Gain- and loss-of-function analyses in transgenic mice demonstrate that Shh is both necessary and sufficient for Olg gene expression in vivo.

Olig bHLH proteins interact with homeodomain proteins to regulate cell fate acquisition in progenitors of the ventral neural tube.

BACKGROUND: Organizing signals such as Sonic hedgehog are thought to specify neuronal subtype identity by regulating the expression of homeodomain proteins in progenitors of the embryonic neural tube. One of these, Nkx2.2, is necessary and sufficient for the development of V3 interneurons. RESULTS: We report that Olig genes, encoding basic helix-loop-helix (bHLH) proteins, are expressed in a subset of Nkx2.2 progenitors before the establishment of interneurons and oligodendroglial precursors. Gain-of-function analysis in transgenic mouse embryos indicates that Olig genes specifically inhibit the establishment of Sim1-expressing V3 interneurons. Moreover, coexpression of Olig2 with Nkx2.2 in the chick neural tube generated cells expressing Sox10, a marker of oligodendroglial precursors. Colocalization of Olig and Nkx2.2 proteins at the dorsal extent of the Nkx2.2 expression domain is consistent with regulatory interactions that define the potential of progenitor cells in the border region. CONCLUSIONS: Interactions between homeodomain and Olig bHLH proteins evidently regulate neural cell fate acquisition and diversification in the ventral neural tube. In particular, interactions between Olig and Nkx2.2 proteins inhibit V3 interneuron development and promote the formation of alternate cell types, including those expressing Sox10.

Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.

The ability to generate specific genetic modifications in mice provides a powerful approach to assess gene function. When genetic modifications have been generated in the germ line, however, the resulting phenotype often only reflects the first time a gene has an influence on - or is necessary for - a particular biological process. Therefore, systems allowing conditional genetic modification have been developed (for a review, see [1]); for example, inducible forms of the Cre recombinase from P1 phage have been generated that can catalyse intramolecular recombination between target recognition sequences (loxP sites) in response to ligand [2] [3] [4] [5]. Here, we assessed whether a tamoxifen-inducible form of Cre recombinase (Cre-ERTM) could be used to modify gene activity in the mouse embryo in utero. Using the enhancer of the Wnt1 gene to restrict the expression of Cre-ERTM to the embryonic neural tube, we found that a single injection of tamoxifen into pregnant mice induced Cre-mediated recombination within the embryonic central nervous system, thereby activating expression of a reporter gene. Induction was ligand dependent, rapid and efficient. The results demonstrate that tamoxifen-inducible recombination can be used to effectively modify gene function in the mouse embryo.

Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors.

Sonic hedgehog (Shh) signal transduction via the G-protein-coupled receptor, Smoothened, is required for proliferation of cerebellar granule neuron precursors (CGNPs) during development. Activating mutations in the Hedgehog pathway are also implicated in basal cell carcinoma and medulloblastoma, a tumor of the cerebellum in humans. However, Shh signaling interactions with cell cycle regulatory components in neural precursors are poorly understood, in part because appropriate immortalized cell lines are not available. We have utilized primary cultures from neonatal mouse cerebella in order to determine (i) whether Shh initiates or maintains cell cycle progression in CGNPs, (ii) if G(1) regulation by Shh resembles that of classical mitogens, and (iii) whether individual D-type cyclins are essential components of Shh proliferative signaling in CGNPs. Our results indicate that Shh can drive continued cycling in immature, proliferating CGNPs. Shh treatment resulted in sustained activity of the G(1) cyclin-Rb axis by regulating levels of cyclinD1, cyclinD2, and cyclinE mRNA transcripts and proteins. Analysis of CGNPs from cyclinD1(-/-) or cyclinD2(-/-) mice demonstrates that the Shh proliferative pathway does not require unique functions of cyclinD1 or cyclinD2 and that D-type cyclins overlap functionally in this regard. In contrast to many known mitogenic pathways, we show that Shh proliferative signaling is mitogen-activated protein kinase independent. Furthermore, protein synthesis is required for early effects on cyclin gene expression. Together, our results suggest that Shh proliferative signaling promotes synthesis of regulatory factor intermediates that upregulate or maintain cyclin gene expression and activity of the G(1) cyclin-Rb axis in proliferating granule neuron precursors.

Sonic hedgehog regulates proliferation and inhibits differentiation of CNS precursor cells.

Activation of the Sonic hedgehog (Shh) signal transduction pathway is essential for normal pattern formation and cellular differentiation in the developing CNS. However, it is also thought to be etiological in primitive neuroectodermal tumors. We adapted GAL4/UAS methodology to ectopically express full-length Shh in the dorsal neural tube of transgenic mouse embryos commencing at 10 d postcoitum (dpc), beyond the period of primary dorsal-ventral pattern formation and floorplate induction. Expression of Shh was maintained until birth, permitting us to investigate effects of ongoing exposure to Shh on CNS precursors in vivo. Proliferative rates of spinal cord precursors were twice that of wild-type littermates at 12.5 dpc. In contrast, at late fetal stages (18.5 dpc), cells that were Shh-responsive but postmitotic were present in persistent structures reminiscent of the ventricular zone germinal matrix. This tissue remained blocked in an undifferentiated state. These results indicate that cellular competence restricts the proliferative response to Shh in vivo and provide evidence that proliferation and differentiation can be regulated separately in precursor cells of the spinal cord. Thus, Hedgehog signaling may contribute to CNS tumorigenesis by directly enhancing proliferation and preventing neural differentiation in selected precursor cells.

Cloning and expression of the filamentous bacteriophage Pf1 major coat protein gene in Escherichia coli. Membrane protein processing and virus assembly.

A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the replicative form (RF) DNA of the class II filamentous bacteriophage Pf1, which infects Pseudomonas aeruginosa. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter. In transformed Escherichia coli JM101 cells, in the presence of the inducer isopropyl-beta-D-thiogalactoside, the bacteriophage Pf1 gene was strongly expressed. The bacteriophage Pf1 coat protein displays the same pattern of negatively charged N-terminal region, hydrophobic middle region and positively charged C-terminal region as that of its counterpart in the class I bacteriophage fd, which infects E. coli, but otherwise the two proteins have no sequence homology. However, the Pf1 procoat protein was found to undergo processing and insertion into the E. coli cell inner membrane, like its fd counterpart, demonstrating that this part of the assembly process is the same for these different bacteriophages. The complete transcriptional unit, incorporating the tac promoter and rrnB transcription terminators flanking the Pf1 coat protein gene, was excised from the expression plasmid and cloned into the intergenic space of bacteriophage R252, an fd bacteriophage that carries an amber mutation in its own major coat protein gene. The Pf1 coat protein gene was again well expressed in infected E. coli cells but the chimeric bacteriophage had growth properties identical to those of the parent bacteriophage R252 on suppressor and non-suppressor strains of E. coli. The class I bacteriophage Pf1 coat protein evidently cannot be recognized by the class I bacteriophage assembly complex at or in the E. coli cell inner membrane, either at the point of initiation of assembly or during the elongation process.

Variable electrostatic interaction between DNA and coat protein in filamentous bacteriophage assembly.

A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the DNA of the class I filamentous bacteriophage fd, which infects Escherichia coli. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter, generating plasmid pKf8P. Bacteriophage fd gene VIII was similarly cloned into the plasmid pEMBL9+, enabling it to be subjected to site-directed mutagenesis. By this means the positively charged lysine residue at position 48, one of four positively charged residues near the C terminus of the protein, was turned into a negatively charged glutamic acid residue. The mutated fd gene VIII was cloned back from the pEMBL plasmid into the expression plasmid pKK223-3, creating plasmid pKE48. In the presence of the inducer isopropyl-beta-D-thiogalactoside, the wild-type and mutated coat protein genes were strongly expressed in E. coli TG1 cells transformed with plasmids pKf8P and pKE48, respectively, and the product procoat proteins underwent processing and insertion into the E. coli cell inner membrane. A net positive charge of only 2 on the side-chains in the C-terminal region is evidently sufficient for this initial stage of the virus assembly process. However, the mutated coat protein could not encapsidate the DNA of bacteriophage R252, an fd bacteriophage carrying an amber mutation in its own gene VIII, when tested on non-suppressor strains of E. coli. On the other hand, elongated hybrid bacteriophage particles could be generated whose capsids contained mixtures of wild-type (K48) and mutant (E48) subunits. This suggests that the defect in assembly may occur at the initiation rather than the elongation step(s) in virus assembly. Other mutations of lysine-48 that removed or reversed the positive charge at this position in the C-terminal region of the coat protein were also found to lead to the production of commensurately longer bacteriophage particles. Taken together, these results indicate direct electrostatic interaction between the DNA and the coat protein in the capsid and support a model of non-specific binding between DNA and coat protein subunits with a stoicheiometry that can be varied during assembly.

Pax-2 expression in the murine neural plate precedes and encompasses the expression domains of Wnt-1 and En-1.

In the Drosophila embryo, activation of wingless and engrailed in the parasegment requires paired, a member of the Pax family of transcription factors. We have explored the possible conservation of this regulatory hierarchy in the developing mouse brain. We find that Pax-2 is expressed prior to somite formation in the presumptive mid/hindbrain region. Shortly thereafter, Wnt-1 (the wingless orthologue) and Engrailed-1 are expressed in overlapping regions within the Pax-2 domain. Pax-5 expression commences later, at the 3-somite stage. Thus, the spatial and temporal expression of Pax-2 is consistent with a possible regulatory role in the activation of Wnt-1 and En-1.

A single homeodomain binding site restricts spatial expression of Wnt-1 in the developing brain.

In this study we investigate the molecular mechanisms that are responsible for the restricted expression of Wnt-1 during embryogenesis. We report that a single homeodomain binding site, HBS1, within the Wnt-1 enhancer contributes to appropriate spatial expression of Wnt-1 in the developing nervous system. This HBS1 site may be required for repressing Wnt-1 expression in the developing forebrain since specific mutations of this site result in an extension of the rostral boundary of Wnt-1/lacZ staining in transgenic embryos. We further demonstrate that a subset of homeodomain proteins expressed in the forebrain (i.e., Dix2, Emx2) interact specifically with HBS1. These findings suggest that these (or related) homeodomain proteins may regulate expression of Wnt-1 during normal brain development by interacting with the HBS1 site in the Wnt-1 enhancer.

Expression of the homeobox-containing genes EN1 and EN2 in human fetal midgestational medulla and cerebellum.

Homeobox-containing genes En-1 and En-2 have been implicated in the control of pattern formation during development of the central nervous system in experimental animals. In order to determine whether the expression of homologous human EN genes can be used as a developmental genetic marker of the arcuate nucleus of the medulla (a putative precerebellar nucleus that shows developmental deficiency in a subset of sudden infant death syndrome [SIDS]), we performed in situ hybridization with human EN1 and EN2 RNA probes in human fetal midgestational medulla and cerebellum (18-21 weeks gestational age, n=4). Expression of EN genes was demonstrated in all neuronal groups of the medulla and throughout the cerebellum. The RNA signal for both EN1 and EN2 was strongest in the cerebellar granule cell layers, white matter of the vermis and flocculus, inferior olive, arcuate nucleus, caudal raphe nuclei, corpus pontobulbare and nucleus ambiguus. Most of the structures that showed the strongest EN signal originate in the rhombic lip. Some of these structures are functionally interconnected, and show pathologic changes in the syndrome of infantile olivopontocerebellar hypoplasia/atrophy. Strong expression of EN signal in the arcuate nucleus could be used as a genetic marker of this nucleus in further developmental studies of the arcuate nucleus in SIDS. Although EN expression is not specific to the arcuate nucleus or to the rhombic lip derivatives, our results suggest that rhombic lip derivatives have the highest levels of EN RNA message among the medullary structures at midgestation.

Topical iodine and neonatal hypothyroidism.

OBJECTIVES: To determine whether skin care practices with iodine-containing disinfectants are putting patients in the neonatal intensive care unit at risk for primary hypothyroidism. Cutaneous exposure to povidone-iodine antiseptic solutions may be a cause of primary hypothyroidism in neonates. DESIGN: Prospective pilot study. SETTING: Level III neonatal intensive care unit of a university-affiliated hospital. PARTICIPANTS: Sequential sample of 47 medial and surgical patients admitted to the neonatal intensive care unit who received cutaneous povidone-iodine applications in preparation for invasive or surgical procedures. METHODS: Seven to 10 days after iodine exposure, capillary blood samples were obtained on filter paper blots for thyroid function testing and urine samples were collected to determine quantitative iodine concentrations. A plasma creatinine level was determined for each subject. RESULTS: A total of 47 patients were enrolled. The gestational ages of subjects ranged from 26 to 41 weeks (mean, 33.6 weeks); the male-to-female ratio was 28:19; and the birth weights ranged from 0.7 to 5.1 kg(mean, 2.42 kg). The thyroxine level ranged from 20 to 187 nmol/L (1.6 to 14.6 micrograms/dL) (mean, 102 nmol/L [7.9 micrograms/dL];reference, > or = 90 nmol/L [> or = 7 micrograms/dL]; and the thyrotropin level ranged from 0.1 to 16.5 mU/L (mean, 6.4 mU/L; reference, < 20 mU/L). The mean uridine iodine concentration was 2798.0 micrograms/dL (reference, < 40 micrograms/dL), and the mean plasma creatinine level was 60 mumol/L (0.69 mg/dL) (reference, < or = 50 mumol/L [ < or = 0.6 mg/dL] for males and < or = 40 mumol/L [ < or = 0.5 mg/dL] for females). CONCLUSIONS: There was no documentation of primary hypothyroidism in our subjects despite elevated urine iodine levels. While it is still possible that patients who receive long-term iodine exposure in other settings (eg, cardiac catheterization) are at risk for primary hypothyroidism, our study suggests that the amount of iodine absorbed through routine neonatal intensive care unit procedures does not substantially alter thyroid function during the first 10 days of life. An important confounding variable is that seven patients were receiving dopamine hydrochloride infusions and four were receiving dexamethasone phosphate at the time of sample collection. We therefore cannot rule out the possibility that these medications masked a thyrotropin level elevation that would have occurred in a primary hypothyroid state. We discuss implications for the interpretation of the results of neonatal thyroid function tests.

Oligodendrocyte development in the spinal cord and telencephalon: common themes and new perspectives.

There are clear parallels between oligodendrocyte development in the spinal cord and forebrain. However, there is new evidence that in both of these regions oligodendrocyte lineage development may be more complex than we earlier thought. This stems from the recent identification of three new transcription factor genes, Olig1, Olig2 and Sox10, that are expressed from the early stages of oligodendrocyte lineage development. In this article, we highlight the common themes underlying specification and early development of oligodendrocytes in the spinal cord and telencephalon. Then, we discuss recent studies of Sox10 and the Olig genes and their implications for oligodendrocyte specification. We conclude that although the mechanisms of oligodendrogenesis appear to be fundamentally similar at different rostro-caudal levels of the neuraxis, there are still many unanswered questions about the details of oligodendrocyte specification.

Pax-2 regulatory sequences that direct transgene expression in the developing neural plate and external granule cell layer of the cerebellum.

Expression of Pax-2 in the mouse gastrula is the first marker of the midbrain-hindbrain region. To address roles played by transcription factors in the process of neural plate pattern formation and to facilitate gain-of-function approaches in the study of midbrain-hindbrain and cerebellar development, we characterized regulatory sequences at the Pax-2 locus using an in vivo transgenic mouse reporter assay. An 8.5 kb fragment of genomic DNA located upstream of Pax-2 directed lacZ expression prior to neurulation (7.5 days post-coitum, dpc) in a region fated to become midbrain and hindbrain, and subsequently in developing neuroepithelium. While similar to the pattern of Pax-2 expression, reporter gene activity extended beyond the boundaries of Pax-2 expression, most probably reflecting purdurance of beta-galactosidase activity and an absence of DNA sequences that restrict Pax-2 expression to rhombomere 1 by 9. 5 dpc. In the fetal and neonatal brain, Pax-2-lacZ activity was confined largely to Purkinje cells and the external granule cell layer (EGL) of the cerebellum. A 4 kb regulatory element, in contrast, initiated neural expression at 8.25 dpc in the anterior hindbrain, but recapitulated all later aspects of Pax-2-lacZ activity observed with the larger transgene. These results indicate the presence of regulatory sequences upstream of the Pax-2 locus capable of directing gene expression in the developing midbrain, first rhombomere of the hindbrain, and its principal derivative, the cerebellum. Successful misexpression of Sonic hedgehog demonstrates that Pax-2 regulatory sequences should prove generally useful for transgenic gain-of-function approaches in mice.

Pro-neural miR-128 is a glioma tumor suppressor that targets mitogenic kinases.

MicroRNAs (miRNAs) carry out post-transcriptional control of a multitude of cellular processes. Aberrant expression of miRNA can lead to diseases, including cancer. Gliomas are aggressive brain tumors that are thought to arise from transformed glioma-initiating neural stem cells (giNSCs). With the use of giNSCs and human glioblastoma cells, we investigated the function of miRNAs in gliomas. We identified pro-neuronal miR-128 as a candidate glioma tumor suppressor miRNA. Decreased expression of miR-128 correlates with aggressive human glioma subtypes. With a combination of molecular, cellular and in vivo approaches, we characterize miR-128's tumor suppressive role. miR-128 represses giNSC growth by enhancing neuronal differentiation. miR-128 represses growth and mediates differentiation by targeting oncogenic receptor tyrosine kinases (RTKs) epithelial growth factor receptor and platelet-derived growth factor receptor-α. Using an autochthonous glioma mouse model, we demonstrated that miR-128 repressed gliomagenesis. We identified miR-128 as a glioma tumor suppressor that targets RTK signaling to repress giNSC self-renewal and enhance differentiation.

Interactions between DNA and coat protein in the structure and assembly of filamentous bacteriophage fd.

Bacteriophage fd is a class I filamentous virus (others are M13 and f1) that comprises a circular, single-stranded DNA molecule enclosed in a cylindrical protein sheath to form a flexible particle approximately 890 nm long and 7 nm in diameter. The viral DNA contains 6,408 nucleotides incorporating 10 genes, and the protein sheath is composed of about 2,700 major coat protein subunits in a shingled helical array, the symmetry of which is defined by a fivefold rotational axis combined with a twofold screw axis of pitch 3.2 nm. The DNA extends throughout the length of the particle but is not base-paired and has a symmetry different from that of the protein helix. How the DNA is packed remains unclear but the number (2.4) of nucleotides packaged per major coat protein subunit is certainly not integral, in contrast with, say, the packaging of RNA in tobacco mosaic virus. The coat protein subunit is 50 amino-acid residues in length and, in the virus particle, adopts a largely alpha-helical conformation, with the long axis of the helix aligned close to the long axis of the filament. This protein is arranged with its negatively charged N-terminal region on the outside of the filament and its positively charged C-terminal region on the inside abutting the DNA. We report here that positive charge on one of the four lysine side chains in the latter region has a direct effect on DNA packaging, because when this charge is absent, elongated particles are produced with lengths that can be correlated with the residual positive charge in the C-terminal region of the coat protein subunit.

Fate of the mammalian cardiac neural crest.

A subpopulation of neural crest termed the cardiac neural crest is required in avian embryos to initiate reorganization of the outflow tract of the developing cardiovascular system. In mammalian embryos, it has not been previously experimentally possible to study the long-term fate of this population, although there is strong inference that a similar population exists and is perturbed in a number of genetic and teratogenic contexts. We have employed a two-component genetic system based on Cre/lox recombination to label indelibly the entire mouse neural crest population at the time of its formation, and to detect it at any time thereafter. Labeled cells are detected throughout gestation and in postnatal stages in major tissues that are known or predicted to be derived from neural crest. Labeling is highly specific and highly efficient. In the region of the heart, neural-crest-derived cells surround the pharyngeal arch arteries from the time of their formation and undergo an altered distribution coincident with the reorganization of these vessels. Labeled cells populate the aorticopulmonary septum and conotruncal cushions prior to and during overt septation of the outflow tract, and surround the thymus and thyroid as these organs form. Neural-crest-derived mesenchymal cells are abundantly distributed in midgestation (E9.5-12.5), and adult derivatives of the third, fourth and sixth pharyngeal arch arteries retain a substantial contribution of labeled cells. However, the population of neural-crest-derived cells that infiltrates the conotruncus and which surrounds the noncardiac pharyngeal organs is either overgrown or selectively eliminated as development proceeds, resulting for these tissues in a modest to marginal contribution in late fetal and postnatal life.

Cystic malformation of the posterior cerebellar vermis in transgenic mice that ectopically express Engrailed-1, a homeodomain transcription factor.

In WEXPZ-En-1 transgenic mice, Engrailed-1, a homeodomain-containing transcription factor, is ectopically expressed in the developing brain under control of the Wnt-1 enhancer. En-1 is a developmental regulatory control gene which has an essential role in the formation of the midbrain and cerebellum. Approximately 28% of WEXPZ-En-1 + mice develop cystic malformations of the posterior lobe of the cerebellar vermis, fourth ventricular dilatation, and postnatal hydrocephalus. These anatomic features are also found among the spectrum of posterior fossa malformations in humans. Expression characteristics of the WEXP transgene suggest that the neuropathology observed in WEXPZ-En-1+ mice stems from overexpression of En-1 during fetal and neonatal phases of cerebellar development. These observations raise the possibility that abnormal regulation of Engrailed genes, or targets of Engrailed, may be involved in the pathogenesis of cystic central nervous system malformations of the posterior fossa in humans.