Regulated Nuclear Trafficking of the Homeodomain Protein Otx1 in Cortical Neurons

doi:10.1006/mcne.2001.1076

Molecular and Cellular Neuroscience

Volume 19, Issue 3, March 2002, Pages 430-446

https://doi.org/10.1006/mcne.2001.1076 Get rights and content

Abstract

Otx1 is a homeodomain protein required for axon refinement by layer 5 neurons in developing cerebral cortex. Otx1 localizes to the cytoplasm of progenitor cells in the rat ventricular zone, and remains cytoplasmic as neurons migrate and begin to differentiate. Nuclear translocation occurs during the first week of postnatal life, when layer 5 neurons begin pruning their long-distance axonal projections. Deletion analysis reveals that Otx1 is imported actively into cell nuclei, that the N-terminus of Otx1 is necessary for nuclear import, and that a putative nuclear localization sequence within this domain is sufficient to direct nuclear import in a variety of cell lines. In contrast, GFP–Otx1 fusion proteins that contain the N-terminus are retained in the cytoplasm of cortical progenitor cells, mimicking the distribution of Otx1 in vivo. These results suggest that ventricular cells actively sequester Otx1 in the cytoplasm, either by preventing nuclear import or by promoting a balance of export over import signals.

References (54)

L.J. Briggs et al.
The cAMP-dependent protein kinase site (Ser312) enhances dorsal nuclear import through facilitating nuclear localization sequence/importin interaction
J. Biol. Chem.
(1998)
K.-Y. Chau et al.
Functional domains of the cone-rod homeobox (CRX) transcription factor
J. Biol. Chem.
(2000)
A. Chenn et al.
Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis
Cell
(1995)
S.J. Fashena et al.
LexA-based two-hybrid systems
Methods Enzymol.
(2000)
T. Furukawa et al.
Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation
Cell
(1997)
A. Komeili et al.
Nuclear transport and transcription
Curr. Opin. Cell Biol.
(2000)
X. Li et al.
Requirement of SpOtx in cell fate decisions in the sea urchin embryo and possible role as a mediator of beta-catenin signaling
Dev. Biol.
(1999)
H.C. Liou et al.
Regulation of the NF-kappa B/rel transcription factor and I kappa B inhibitor system
Curr. Opin. Cell Biol.
(1993)
D.C. Lo et al.
Neuronal transfection in brain slices using particle-mediated gene transfer
Neuron
(1994)
K.P. Mitton et al.
The leucine zipper of NRL interacts with the CRX homeodomain
J. Biol. Chem.
(2000)

D.D.M. O'Leary et al.

Occipital cortical neurons with transient pyramidal axons extend and maintain collaterals to subcortical but not intracortical targets

Brain Res.

(1985)

G.E. Parker et al.

The homeodomain coordinates nuclear entry of the Lhx3 neuroendocrine transcription factor and association with the nuclear matrix

J. Biol. Chem.

(2000)

G.E. Rieckhof et al.

Nuclear translocation of extradenticle requires homothorax, which encodes an extradenticle-related homeodomain protein

Cell

(1997)

E. Sock et al.

Identification of the nuclear localization signal of the POU domain protein Tst1/Oct6

J. Biol. Chem.

(1996)

P. Turpin et al.

Nuclear transport and transcriptional regulation

FEBS Lett.

(1999)

J.M. Weimann et al.

Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets

Neuron

(1999)

W. Wen et al.

Identification of a signal for rapid export of proteins from the nucleus

Cell

(1995)

A. Abu-Shaar et al.

Control of the nuclear localization of Extradenticle by competing nuclear import and export signals

Genes Dev.

(1999)

D. Acampora et al.

Epilepsy and brain abnormalities in mice lacking the Otx1 gene

Nature Genet.

(1996)

M. Affolter et al.

Balancing import and export in development

Genes Dev.

(1999)

S.E. Aspland et al.

Nucleocytoplasmic localisation of extradenticle protein is spatially regulated throughout development in Drosophila

Development

(1997)

Ausubel, F. M, Brent, R, Kingston, R. E, Moore, D. D, Seidman, J. G, Smith, J. A, and, Struhl, K. 1997, Current...

J. Berthelsen et al.

The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH

Genes Dev.

(1999)

H.P. Bogerd et al.

Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay

Mol. Cell Biol.

(1996)

J.T. Bryan et al.

The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1

J. Cell Sci.

(2000)

Y. Chagnac-Amitai et al.

Burst generating and regular spiking layer 5 pyramidal neurons of rat neocortex have different morphological features

J. Comp. Neurol.

(1990)

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OTX1 regulates cell cycle progression of neural progenitors in the developing cerebral cortex
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It was speculated that this locus may contain part of the Otx1 enhancer that contributes to the regulation of neocortical volume (9). OTX1 is a transcription factor expressed in cortical progenitor cells and emerging cortical plate during the embryonic development (10, 11). Mice bearing a targeted deletion of Otx1 exhibited a decrease in the number of cortical neurons and the thickness of the cortex (12, 13).
The progenitor cells in the cerebral cortex coordinate proliferation and mitotic exit to generate the correct number of neurons and glial cells during development. However, mechanisms for regulating the mitotic cycle of cortical progenitors are not fully understood. Otx1 is one of the homeobox-containing transcription factors frequently implicated in the development of the central nervous system. Mice bearing a targeted deletion of Otx1 exhibit brain hypoplasia and a decrease in the number of cortical neurons. We hypothesized that Otx1 might be crucial to the proliferation and differentiation of cortical progenitors. Otx1 knockdown by in utero electroporation in the mouse brain reduced the proportion of the G₁ phase while increasing the S and M phases of progenitor cells. The knockdown diminished Tbr1+ neurons but increased GFAP+ astrocytes in the early postnatal cortex as revealed by lineage tracing study. Tbr2+ basal progenitors lacking Otx1 were held at the transit-amplifying stage. In contrast, overexpression of wildtype Otx1 but not an astrocytoma-related mutant Y320C inhibited proliferation of the progenitor cells in embryonic cortex. This study demonstrates that Otx1 is one of the key elements regulating cortical neurogenesis, and a loss-of-function in Otx1 may contribute to the overproduction of astrocytes in vivo.
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Moreover, the basic amino acid clusters (BCs) at both ends of the homeodomain (Fig. 2) structurally resemble classical NLSs and can function as NLSs when coupled to an irrelevant cytoplasmic protein [61,62]. For the homeoproteins Chx10, Nkx2.5, Oct6, and Otx1, the N-terminal basic clusters (BC1) have been shown to function as a NLS [63–66]. For Pdx1, Pitx2 and Shox2, the C-terminal basic clusters (BC2) function as a NLS [62,67,68].
Homeodomain proteins are crucial transcription factors for cell differentiation, cell proliferation and organ development. Interestingly, their homeodomain signature structure is important for both their DNA-binding and their nucleocytoplasmic trafficking. The accurate nucleocytoplasmic distribution of these proteins is essential for their functions. We summarize information on (a) the roles of karyopherins for import and export of homeoproteins, (b) the regulation of their nuclear transport during development, and (c) the corresponding complexity of homeoprotein nucleocytoplasmic transport signals. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.
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Whether Arx has one or more functional nuclear export signals is also an open question. Some homeodomain-containing proteins (Vsx1, Chx10, Otx1, Otx2, Oct6, and Prospro) shuttle in and out of the nucleus (71–75). The localization of most of these proteins is sensitive to leptomycin B, suggesting a Crm1-mediated export pathway (76).
Nuclear import of proteins with nuclear localization signals (NLSs) is mediated by shuttling carriers, the importins. Some cargoes display more than a single NLS, and among these are homeodomain proteins such as Arx, which is critical for development of multiple tissues. Arx has two functional NLSs. The present studies show that several pathways can import Arx via its NLS2, which is within its DNA binding homeodomain. Using an in vitro nuclear import assay, we show that import of Arx via NLS2 can be mediated by importin β1, importin 9, or importin 13, with binding being strongest to importin β1. All binding is sensitive to RanGTP. Experiments based on precise domain deletions indicate that NLS2 binds impβ1, imp9, and imp13 and includes both an importin binding subdomain and a regulatory subdomain with arginine residues being important for function. Moreover, Arx can be co-precipitated with these importins when NLS2 is present. Although nuclear import of Arx can be mediated by these three importin βs, importin β1 seems to play the major role judging from in vivo small interfering RNA ablations and the in vitro import assay. This is the first evidence to show the role of importin β1 in nuclear import of paired-type homeodomain proteins. We propose a novel and possibly quite general mechanism for nuclear import of paired-type homeodomain proteins which is critical for development.
Molecular mechanisms of axon guidance in the developing corticospinal tract
2008, Progress in Neurobiology
The great repertoire of movements in higher order mammals comes courtesy of the corticospinal tract (CST) which is able to initiate precise movement of the entire musculature of the axial and limb muscle groups. It forms the longest axonal trajectory in the mammalian central nervous system and its axons must navigate the entire length of the central nervous system—from its origins in the deeper layers of the cerebral cortex down through the cerebral peduncles and brainstem and along the entire length of the spinal cord. This period of navigation is incredibly complex, and relies upon the coordinated regulation of a collection of molecular guidance cues – coming from all of the known major families of guidance cues – the ephrins, slits, Netrins and Semaphorins – that work together to steer the growing axonal tips through the brain and spinal cord. As such a long tract, the CST forms an excellent experimental model to investigate the nature of molecular cues that sequentially guide axons through the central nervous system. Using the rodent as a model system, this review discusses each step of axonal guidance through the major brain regions—starting from the decision to grow ventrally out of the cortical plate to the eventual activity-dependent refinement of circuitry in the spinal grey matter. In recent years, the identification of these guidance cues and their proposed mode of action is beginning to give us a picture at a molecular level of how the CST is guided so accurately over such a long distance.
Establishment of framework of the cortical area is influenced by Otx1
2008, Neuroscience Research
Subcortical projection from layer 5 neurons is the major cortical output. A transcription factor, Otx1, which is expressed in the layer 5 subcortical projection neurons in the visual cortex, was reported to be responsible for the establishment of visual area-specific layer 5 subcortical projections by inducing the sensorimotor cortex to adopt a visual cortex identity. However, we here demonstrate that the area of corticospinal neurons shifted caudo-medially in the Otx1-null mice of which cortex is 9 tenths in size compared with that of the wild-type littermates, while the whole visual cortex did not convert to the sensorimotor cortex in the absence of Otx1. This suggests that Otx1 is not crucial for the development of visual cortex identity but for the determination of the proportion of cortical areas to the whole neocortex.
Mechanisms of axon degeneration: From development to disease
2007, Progress in Neurobiology
Citation Excerpt :
Taken together, these results provide compelling experimental evidence that axonal degeneration induced during development and upon lesions is brought about by distinct mechanisms, and that WldS specifically inhibits Wallerian, but not developmental axon degeneration (Hoopfer et al., 2006; Luo and O’Leary, 2005). Interestingly, competence for developmental degeneration of cortical axon collaterals in vertebrates depends on the expression and nuclear translocation of the transcription factor Otx1 in these neurons (Weimann et al., 1999; Zhang et al., 2002) suggesting that induction of competence through transcriptional processes might be a general feature in developmental axon degeneration. One possibility could be that transcriptional switches during defined periods of development provide a prerequisite to induce competence for axon degeneration, and that additional signals, e.g. through axon guidance molecules, trophic factor deprivation, or signaling initiated in the absence of synchronized synaptic activity might confer competence for degeneration.
Axon degeneration is an active, tightly controlled and versatile process of axon segment self-destruction. Although not involving cell death, it resembles apoptosis in its logics. It involves three distinct steps: induction of competence in specific neurons, triggering of degeneration at defined axon segments of competent neurons, and rapid fragmentation and removal of the segments. The mechanisms that initiate degeneration are specific to individual settings, but the final pathway of pruning is shared; it involves microtubule disassembly, axon swellings, axon fragmentation, and removal of the remnants by locally recruited phagocytes. The tight regulatory properties of axon degeneration distinguish it from passive loss phenomena, and confer significance to processes that involve it. Axon degeneration has prominent roles in development, upon lesions and in disease. In development, it couples the progressive specification of neurons and circuits to the removal of defined axon branches. Competence might involve transcriptional switches, and local triggering can involve axon guidance molecules and synaptic activity patterns. Lesion-induced Wallerian degeneration is inhibited in the presence of Wld^S fusion protein in neurons; it involves early local, and later, distal degeneration. It has recently become clear that like in other settings, axon degeneration in disease is a rapid and specific process, which should not be confused with a variety of disease-related pathologies. Elucidating the specific mechanisms that initiate axon degeneration should open up new avenues to investigate principles of circuit assembly and plasticity, to uncover mechanisms of disease progression, and to identify ways of protecting synapses and axons in disease.

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¹: These authors contributed equally to the work.

²: Current address: Cell Therapy Center, Beijing Geriatric Institute, Xuanwu Hospital, Capital University of Medical Sciences, 45 Changchun St., Beijing 100053, China.

³: To whom correspondence and reprint requests should be addressed. Fax: (650) 725-9832. E-mail: [email protected].

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Regular ArticleRegulated Nuclear Trafficking of the Homeodomain Protein Otx1 in Cortical Neurons

Abstract

J. Biol. Chem.

J. Biol. Chem.

Cell

Methods Enzymol.

Cell

Curr. Opin. Cell Biol.

Dev. Biol.

Curr. Opin. Cell Biol.

Neuron

J. Biol. Chem.

Brain Res.

J. Biol. Chem.

Cell

J. Biol. Chem.

FEBS Lett.

Neuron

Cell

Control of the nuclear localization of Extradenticle by competing nuclear import and export signals

Genes Dev.

Epilepsy and brain abnormalities in mice lacking the Otx1 gene

Nature Genet.

Balancing import and export in development

Genes Dev.

Nucleocytoplasmic localisation of extradenticle protein is spatially regulated throughout development in Drosophila

Development

The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH

Genes Dev.

Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay

Mol. Cell Biol.

The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1

J. Cell Sci.

Burst generating and regular spiking layer 5 pyramidal neurons of rat neocortex have different morphological features

J. Comp. Neurol.

Regular Article
Regulated Nuclear Trafficking of the Homeodomain Protein Otx1 in Cortical Neurons