RESUMO
The embryonic development of the pineal organ, a neuroendocrine gland on top of the diencephalon, remains enigmatic. Classic fate-mapping studies suggested that pineal progenitors originate from the lateral border of the anterior neural plate. We show here, using gene expression and fate mapping/lineage tracing in zebrafish, that pineal progenitors originate, at least in part, from the non-neural ectoderm. Gene expression in chick indicates that this non-neural origin of pineal progenitors is conserved in amniotes. Genetic repression of placodal, but not neural crest, cell fate results in pineal hypoplasia in zebrafish, while mis-expression of transcription factors known to specify placodal identity during gastrulation promotes the formation of ectopic pineal progenitors. We also demonstrate that fibroblast growth factors (FGFs) position the pineal progenitor domain within the non-neural border by repressing pineal fate and that the Otx transcription factors promote pinealogenesis by inhibiting this FGF activity. The non-neural origin of the pineal organ reveals an underlying similarity in the formation of the pineal and pituitary glands, and suggests that all CNS neuroendocrine organs may require a non-neural contribution to form neurosecretory cells.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Glândula Pineal/citologia , Glândula Pineal/embriologia , Transdução de Sinais , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Linhagem da Célula , Embrião de Galinha , Ectoderma/citologia , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Crista Neural/citologia , Placa Neural/citologia , Neuroglia/citologia , Neurônios/citologia , Sistemas Neurossecretores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Neurogenesis in the post-embryonic vertebrate brain varies in extent and efficiency between species and brain territories. Distinct neurogenesis modes may account for this diversity, and several neural progenitor subtypes, radial glial cells (RG) and neuroepithelial progenitors (NE), have been identified in the adult zebrafish brain. The neurogenic sequences issued from these progenitors, and their contribution to brain construction, remain incompletely understood. Here we use genetic tracing techniques based on conditional Cre recombination and Tet-On neuronal birthdating to unravel the neurogenic sequence operating from NE progenitors in the zebrafish post-embryonic optic tectum. We reveal that a subpopulation of her5-positive NE cells of the posterior midbrain layer stands at the top of a neurogenic hierarchy involving, in order, the amplification pool of the tectal proliferation zone (TPZ), followed by her4-positive RG cells with transient neurogenic activity. We further demonstrate that the adult her5-positive NE pool is issued in lineage from an identically located NE pool expressing the same gene in the embryonic neural tube. Finally, we show that these features are reminiscent of the neurogenic sequence and embryonic origin of the her9-positive progenitor NE pool involved in the construction of the lateral pallium at post-embryonic stages. Together, our results highlight the shared recruitment of an identical neurogenic strategy by two remote brain territories, where long-lasting NE pools serve both as a growth zone and as the life-long source of young neurogenic RG cells.
Assuntos
Envelhecimento/fisiologia , Linhagem da Célula , Mesencéfalo/embriologia , Células-Tronco Neurais/citologia , Peixe-Zebra/embriologia , Animais , Linhagem da Célula/efeitos dos fármacos , Doxiciclina/farmacologia , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/efeitos dos fármacos , Células Neuroepiteliais/metabolismo , Neurogênese/efeitos dos fármacos , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Recombinação Genética/genética , Colículos Superiores/citologia , Colículos Superiores/efeitos dos fármacos , Colículos Superiores/embriologia , Colículos Superiores/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
A detrimental perceptive consequence of damaged auditory sensory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1(-/-) mice displayed OHC hair-bundle shape anomalies in the mid and basal cochlea, normally tuned to mid- and high-frequency tones, and mild (22-35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the cochlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1(-/-) mice to high-frequency (20-40 kHz) test tones were not masked by tones of neighboring frequencies. Instead, efficient maskers were characterized by their frequencies up to two octaves below the probe-tone frequency, unusually low intensities up to 25 dB below probe-tone level, and growth-of-masker slope (2.2 dB/dB) reflecting their compressive amplification. Together, these properties do not fit the current acknowledged features of a hypersensitivity of the basal cochlea to lower frequencies, but rather suggest a previously unidentified mechanism. Low-frequency maskers, we propose, may interact within the unaffected cochlear apical region with midhigh frequency sounds propagated there via a mode possibly using the persistent contact of misshaped OHC hair bundles with the tectorial membrane. Our findings thus reveal a source of misleading interpretations of hearing thresholds and of hypervulnerability to low-frequency sound interference.
Assuntos
Percepção Auditiva/fisiologia , Células Ciliadas Auditivas Externas/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Som , Animais , Células Ciliadas Auditivas Externas/citologia , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
The mechanotransducer channels of auditory hair cells are gated by tip-links, oblique filaments that interconnect the stereocilia of the hair bundle. Tip-links stretch from the tips of stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. They are made of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes USH1D and USH1F, respectively. In this study we address the role of sans, a putative scaffold protein and product of the USH1G gene. In Ush1g(-/-) mice, the cohesion of stereocilia is disrupted, and both the amplitude and the sensitivity of the transduction currents are reduced. In Ush1g(fl/fl)Myo15-cre(+/-) mice, the loss of sans occurs postnatally and the stereocilia remain cohesive. In these mice, there is a decrease in the amplitude of the total transducer current with no loss in sensitivity, and the tips of the stereocilia in the short and middle rows lose their prolate shape, features that can be attributed to the loss of tip-links. Furthermore, stereocilia from these rows undergo a dramatic reduction in length, suggesting that the mechanotransduction machinery has a positive effect on F-actin polymerization. Sans interacts with the cytoplasmic domains of cadherin-23 and protocadherin-15 in vitro and is absent from the hair bundle in mice defective for either of the two cadherins. Because sans localizes mainly to the tips of short- and middle-row stereocilia in vivo, we conclude that it belongs to a molecular complex at the lower end of the tip-link and plays a critical role in the maintenance of this link.
Assuntos
Actinas/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Proteínas Relacionadas a Caderinas , Caderinas/metabolismo , Cílios/metabolismo , Eletrofisiologia , Imunofluorescência , Vetores Genéticos/genética , Células Ciliadas Auditivas/ultraestrutura , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Proteínas do Tecido Nervoso/genética , Polimerização , Precursores de Proteínas/metabolismo , Transdução de Sinais/genéticaRESUMO
Macroglia fulfill essential functions in the adult vertebrate brain, producing and maintaining neurons and regulating neuronal communication. However, we still know little about their emergence and diversification. We used the zebrafish D. rerio as a distant vertebrate model with moderate glial diversity as anchor to reanalyze datasets covering over 600 million years of evolution. We identify core features of adult neurogenesis and innovations in the mammalian lineage with a potential link to the rarity of radial glia-like cells in adult humans. Our results also suggest that functions associated with astrocytes originated in a multifunctional cell type fulfilling both neural stem cell and astrocytic functions before these diverged. Finally, we identify conserved elements of macroglial cell identity and function and their time of emergence during evolution.
Assuntos
Astrócitos , Peixe-Zebra , Animais , Humanos , Neurogênese/fisiologia , Neuroglia/fisiologia , Perfilação da Expressão Gênica , MamíferosRESUMO
Epithelial cells acquire diverse shapes relating to their different functions. This is particularly relevant for the cochlear outer hair cells (OHCs), whose apical and basolateral shapes accommodate the functioning of these cells as mechano-electrical and electromechanical transducers, respectively. We uncovered a circumferential shape transition of the apical junctional complex (AJC) of OHCs, which occurs during the early postnatal period in the mouse, prior to hearing onset. Geometric analysis of the OHC apical circumference using immunostaining of the AJC protein ZO1 and Fourier-interpolated contour detection characterizes this transition as a switch from a rounded-hexagon to a non-convex circumference delineating two lateral lobes at the neural side of the cell, with a negative curvature in between. This shape tightly correlates with the 'V'-configuration of the OHC hair bundle, the apical mechanosensitive organelle that converts sound-evoked vibrations into variations in cell membrane potential. The OHC apical circumference remodeling failed or was incomplete in all the mouse mutants affected in hair bundle morphogenesis that we tested. During the normal shape transition, myosin VIIa and myosin II (A and B isoforms) displayed polarized redistributions into and out of the developing lobes, respectively, while Shroom2 and F-actin transiently accumulated in the lobes. Defects in these redistributions were observed in the mutants, paralleling their apical circumference abnormalities. Our results point to a pivotal role for actomyosin cytoskeleton tensions in the reshaping of the OHC apical circumference. We propose that this remodeling contributes to optimize the mechanical coupling between the basal and apical poles of mature OHCs.
Assuntos
Cóclea/fisiologia , Células Ciliadas Auditivas Externas/fisiologia , Animais , Cílios/fisiologia , Cílios/ultraestrutura , Cóclea/anatomia & histologia , Cóclea/inervação , Cóclea/ultraestrutura , Orelha Interna/citologia , Cabras , Células Ciliadas Auditivas Externas/citologia , Células Ciliadas Auditivas Externas/ultraestrutura , Camundongos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Neurônios/citologia , Neurônios/fisiologia , Órgão Espiral/fisiologia , Órgão Espiral/ultraestruturaRESUMO
Spatiotemporal variations of neurogenesis are thought to account for the evolution of brain shape. In the dorsal telencephalon (pallium) of vertebrates, it remains unresolved which ancestral neurogenesis mode prefigures the highly divergent cytoarchitectures that are seen in extant species. To gain insight into this question, we developed genetic tools to generate here the first 4-dimensional (3D + birthdating time) map of pallium construction in the adult teleost zebrafish. Using a Tet-On-based genetic birthdating strategy, we identify a "sequential stacking" construction mode where neurons derived from the zebrafish pallial germinal zone arrange in outside-in, age-related layers from a central core generated during embryogenesis. We obtained no evidence for overt radial or tangential neuronal migrations. Cre-lox-mediated tracing, which included following Brainbow clones, further demonstrates that this process is sustained by the persistent neurogenic activity of individual pallial neural stem cells (NSCs) from embryo to adult. Together, these data demonstrate that the spatiotemporal control of NSC activity is an important driver of the macroarchitecture of the zebrafish adult pallium. This simple mode of pallium construction shares distinct traits with pallial genesis in mammals and non-mammalian amniotes such as birds or reptiles, suggesting that it may exemplify the basal layout from which vertebrate pallial architectures were elaborated.
Assuntos
Neocórtex/embriologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Telencéfalo/citologia , Peixe-Zebra/embriologia , Animais , Biomarcadores/metabolismo , Telencéfalo/anatomia & histologia , Peixe-Zebra/anatomia & histologiaRESUMO
Little is known on the embryonic origin and related heterogeneity of adult neural stem cells (aNSCs). We use conditional genetic tracing, activated in a global or mosaic fashion by cell type-specific promoters or focal laser uncaging, coupled with gene expression analyses and Notch invalidations, to address this issue in the zebrafish adult telencephalon. We report that the germinal zone of the adult pallium originates from two distinct subtypes of embryonic progenitors and integrates two modes of aNSC formation. Dorsomedial aNSCs derive from the amplification of actively neurogenic radial glia of the embryonic telencephalon. On the contrary, the lateral aNSC population is formed by stepwise addition at the pallial edge from a discrete neuroepithelial progenitor pool of the posterior telencephalic roof, activated at postembryonic stages and persisting lifelong. This dual origin of the pallial germinal zone allows the temporally organized building of pallial territories as a patchwork of juxtaposed compartments.
Assuntos
Células-Tronco Adultas/citologia , Linhagem da Célula , Globo Pálido/citologia , Células-Tronco Neurais/citologia , Células-Tronco Adultas/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Globo Pálido/embriologia , Globo Pálido/crescimento & desenvolvimento , Células-Tronco Neurais/metabolismo , Neurogênese , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Especificidade de Órgãos , Regiões Promotoras Genéticas , Transcrição Gênica , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Planar cell polarity (PCP) refers to coordinated polarization of cells within the plane of a cell sheet. A conserved signaling pathway is required for the establishment of PCP in epithelial tissues and for polarized cellular rearrangements known as convergent extension. During PCP signaling, core PCP proteins are sorted asymmetrically along the polarization axis; this sorting is thought to direct coordinated downstream morphogenetic changes across the entire tissue. Here, we show that a gene encoding a ciliary protein (a 'ciliary gene'), Ift88, also known as Polaris, is required for establishing epithelial PCP and for convergent extension of the cochlear duct of Mus musculus. We also show that the proper positioning of ciliary basal bodies and the formation of polarized cellular structures are disrupted in mice with mutant ciliary proteins ('ciliary mutants'), whereas core PCP proteins are partitioned normally along the polarization axis. Thus, our data uncover a distinct requirement for ciliary genes in basal body positioning and morphological polarization during PCP regulation.
Assuntos
Polaridade Celular , Órgão Espiral/citologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Cílios , Cóclea , Células Epiteliais/citologia , Cinesinas/genética , Camundongos , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
The thalamic complex is the major sensory relay station in the vertebrate brain and comprises three developmental subregions: the prethalamus, the thalamus and an intervening boundary region - the zona limitans intrathalamica (ZLI). Shh signalling from the ZLI confers regional identity of the flanking subregions of the ZLI, making it an important local signalling centre for regional differentiation of the diencephalon. However, our understanding of the mechanisms responsible for positioning the ZLI along the neural axis is poor. Here we show that, before ZLI formation, both Otx1l and Otx2 (collectively referred to as Otx1l/2) are expressed in spatially restricted domains. Formation of both the ZLI and the Irx1b-positive thalamus require Otx1l/2; embryos impaired in Otx1l/2 function fail to form these areas, and, instead, the adjacent pretectum and, to a lesser extent, the prethalamus expand into the mis-specified area. Conditional expression of Otx2 in these morphant embryos cell-autonomously rescues the formation of the ZLI at its correct location. Furthermore, absence of thalamic Irx1b expression, in the presence of normal Otx1l/2 function, leads to a substantial caudal broadening of the ZLI by transformation of thalamic precursors. We therefore propose that the ZLI is induced within the competence area established by Otx1l/2, and is posteriorly restricted by Irx1b.
Assuntos
Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição Otx/fisiologia , Subtálamo/embriologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Modelos Biológicos , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Tálamo/embriologia , Tálamo/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.
Assuntos
Sequência Conservada , Regulação da Expressão Gênica/fisiologia , Sintenia , Peixe-Zebra/genética , Animais , Galinhas/genética , Evolução Molecular , Duplicação Gênica , Ligação Genética , Genoma Humano , Humanos , Tetraodontiformes/genéticaRESUMO
Although the secreted molecule Fgf8 is a key player of the isthmic organiser function, the mechanisms by which it acts remain unclear. Here, we present evidence indicating that Fgf8 is not instructive in establishing zebrafish cerebellar cell identities, although it is required for proliferation and morphogenesis of this territory. We first show that, as in mouse, lack of Otx function in zebrafish leads to transformation of the presumptive mesencephalon into an extended rhombomere 1 (r1). Expanded Fgf8 expression was proposed to be the cause of this fate transformation. However, this report demonstrates that zebrafish embryos lacking both Otx and fgf8 functions retain an extended r1 and display differentiation of at least two cerebellar cell fates. We show that this is not caused by presence of other Fgfs, which implies that in absence of Otx, Fgf function is not necessary for the differentiation of cerebellar cell types. Otx proteins are therefore potent repressors of cerebellar fates, kept out of r1 progeny by Fgf8. Because Otx transcripts are not present in presumptive r1 territory prior to fgf8 expression, Fgf8 is required to maintain, rather than induce, the posterior boundary of Otx expression. This maintenance is enough to allow cerebellar differentiation.
Assuntos
Diferenciação Celular , Cerebelo/citologia , Fatores de Transcrição Otx/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proliferação de Células , Cerebelo/embriologia , Embrião não Mamífero , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Mesencéfalo/embriologia , Microinjeções , Mutação , Oligonucleotídeos Antissenso/farmacologia , Pirróis/farmacologia , Rombencéfalo/embriologia , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismoRESUMO
The MAP1B (Mtap1b) promoter presents two evolutionary conserved overlapping homeoproteins and Hepatocyte nuclear factor 3beta (HNF3beta/Foxa2) cognate binding sites (defining putative homeoprotein/Fox sites, HF1 and HF2). Accordingly, the promoter domain containing HF1 and HF2 is recognized by cerebellum nuclear extracts containing Engrailed and Foxa2 and has regulatory functions in primary cultures of embryonic mesmetencephalic nerve cells. Transfection experiments further demonstrate that Engrailed and Foxa2 interact physiologically in a dose-dependent manner: Foxa2 antagonizes the Engrailed-driven regulation of the MAP1B promoter, and vice versa. This led us to investigate if Engrailed and Foxa2 interact directly. Direct interaction was confirmed by pull-down experiments, and the regions participating in this interaction were identified. In Foxa2 the interacting domain is the Forkhead box DNA-binding domain. In Engrailed, two independent interacting domains exist: the homeodomain and a region that includes the Pbx-binding domain. Finally, Foxa2 not only binds Engrailed but also Lim1, Gsc and Hoxa5 homeoproteins and in the four cases Foxa2 binds at least the homeodomain. Based on the involvement of conserved domains in both classes of proteins, it is proposed that the interaction between Forkhead box transcription factors and homeoproteins is a general phenomenon.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/embriologia , Fator 3-beta Nuclear de Hepatócito , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismoRESUMO
We report that Emx2 homeogene is expressed at the mRNA and protein levels in the adult mouse olfactory neuroepithelium. As expected for a transcription factor, Emx2 is present in the nucleus of immature and mature olfactory sensory neurons. However, the protein is also detected in the axonal compartment of these neurons, both in the olfactory mucosa axon bundles and in axon terminals within the olfactory bulb. Emx2 axonal staining is heterogeneous, suggesting an association with particles. Subcellular fractionations of olfactory bulb synaptosomes, combined with chemical lesions of olfactory neurons, confirm the presence of Emx2 in axon terminals. Significant amounts of Emx2 protein cosediment with high density synaptosomal subfractions containing eukaryotic translation initiation factor 4E (eIF4E). Nonionic detergents and RNase treatments failed to detach eIF4E and Emx2 from these high-density fractions enriched in vesicles and granular structures. In addition, Emx2 and eIF4E can be coimmunoprecipitated from olfactory mucosa and bulb extracts and interact directly, as demonstrated in pull-down experiments. Emx2 axonal localization, association with high-density particles and interaction with eIF4E strongly suggest that this transcription factor has new nonnuclear functions most probably related to the local control of protein translation in the olfactory sensory neuron axons. Finally, we show that two other brain-expressed homeoproteins, Otx2 and Engrailed 2, also bind eIF4E, indicating that several homeoproteins may modulate eIF4E functions in the developing and adult nervous system.
Assuntos
Axônios/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas de Homeodomínio/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células COS , Feminino , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurônios Receptores Olfatórios/citologia , Fatores de Transcrição Otx , Ligação Proteica , Transporte Proteico , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Sinaptossomos/química , Sinaptossomos/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
Transgenic mice expressing the homeobox gene Hoxa5 under the control of Hoxb2 regulatory elements present a growth arrest during weeks two and three of postnatal development, resulting in proportionate dwarfism. These mice present a liver phenotype illustrated by a 12-fold increase in liver insulin-like growth factor binding protein 1 (IGFBP1) mRNA and a 50% decrease in liver insulin-like growth factor 1 (IGF1) mRNA correlated with a 50% decrease in circulating IGF1. We show that the Hoxa5 transgene is expressed in the liver of these mice, leading to an overexpression of total (endogenous plus transgene) Hoxa5 mRNA in this tissue. We have used several cell lines to investigate a possible physiological interaction of Hoxa5 with the main regulator of IGFBP1 promoter activity, the Forkhead box transcription factor FKHR. In HepG2 cells, Hoxa5 has little effect by itself but inhibits the FKHR-dependent activation of the IGFBP1 promoter. In HuF cells, Hoxa5 cooperates with FKHR to dramatically enhance IGFBP1 promoter activity. This context-dependent physiological interaction probably corresponds to the existence of a direct interaction between Hoxa5 and FKHR and FoxA2/HNF3beta, as demonstrated by pull-down experiments achieved either in vitro or after cellular co-expression. In conclusion, we propose that the impaired growth observed in this transgenic line relates to a liver phenotype best explained by a direct interaction between Hoxa5 and liver-specific Forkhead box transcription factors, in particular FKHR but also Foxa2/HNF3beta. Because Hoxa5 and homeogenes of the same paralog group are normally expressed in the liver, the present results raise the possibility that homeoproteins, in addition to their established role during early development, regulate systemic physiological functions.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Nanismo/genética , Proteínas de Homeodomínio/fisiologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Sistema Nervoso/embriologia , Fosfoproteínas/fisiologia , Fatores de Transcrição/metabolismo , Animais , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Fator 3-beta Nuclear de Hepatócito , Proteínas de Homeodomínio/genética , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Insulin-like growth factor binding protein-1 (IGFBP-1) is abundantly expressed in the liver and decidualized endometrium. FKHR, a FOXO forkhead transcription factor, stimulates IGFBP-1 promoter activity in liver cells through the insulin response sequences (IRSs). HOXA10, a homeobox transcription factor, is important in the decidualization process. Here we show that FKHR and HOXA10 are expressed in baboon endometrium during the menstrual cycle and pregnancy. Levels are lowest during the follicular phase and highest in pregnancy. Reporter gene studies reveal that FKHR stimulates both baboon and human IGFBP-1 promoter activity, whereas HOXA10 alone has a relatively weak effect. When FKHR and HOXA10 are expressed together, promoter activity is markedly up-regulated, which is indicative of cooperativity. A DNA binding-deficient FKHR mutant fails to stimulate promoter activity, even in the presence of HOXA10, and deletion or mutation of IRSs also disrupts the effect of FKHR and cooperativity with HOXA10. Conversely, the IRS region placed upstream of the 31 base pair IGFBP-1 minimal promoter is sufficient to mediate effects of FKHR and cooperativity with HOXA10. Pull-down studies reveal physical association between GST-FKHR and (35)S-HOXA10. These studies show that FKHR and HOXA10 interact directly and can function cooperatively to stimulate IGFBP-1 promoter activity in endometrial cells and perhaps in other settings.