Krox20 inactivation in the PNS leads to CNS/PNS boundary transgression by central glia.

CNS/PNS interfaces constitute cell boundaries, since they delimit territories with different neuronal and glial contents. Despite their potential interest in regenerative medicine, the mechanisms restricting oligodendrocytes and astrocytes to the CNS, and Schwann cells to the PNS in mammals are not known. To investigate the involvement of peripheral glia and myelin in the maintenance of the CNS/PNS boundary, we have first made use of different mouse mutants. We show that inactivation of Krox20/Egr2, a master regulatory gene for myelination in Schwann cells, results in transgression of the CNS/PNS boundary by astrocytes and oligodendrocytes and in myelination of nerve root axons by oligodendrocytes. In contrast, such migration does not occur with the Trembler(J) mutation, which prevents PNS myelination without affecting Krox20 expression. Altogether these data suggest that maintenance of the CNS/PNS boundary requires a new Krox20 function separable from myelination control. Finally, we have analyzed a human patient affected by a congenital amyelinating neuropathy, associated with the absence of the KROX20 protein in Schwann cells. In this case, the nerve roots were also invaded by oligodendrocytes and astrocytes. This indicates that transgression of the CNS/PNS boundary by central glia can occur in pathological situations in humans and suggests that the underlying mechanisms are common with the mouse.

Boundary cap cells are highly competitive for CNS remyelination: fast migration and efficient differentiation in PNS and CNS myelin-forming cells.

During development, boundary cap cells (BC) and neural crest cell (NCC) derivatives generate Schwann cells (SC) of the spinal roots and a subpopulation of neurons and satellite cells in the dorsal root ganglia. Despite their stem-like properties, their therapeutic potential in the diseased central nervous system (CNS) was never explored. The aim of this work was to explore BC therapeutic potential for CNS remyelination. We derived BC from Krox20(Cre) x R26R(Yfp) embryos at E12.5, when Krox20 is exclusively expressed by BC. Combining microdissection and cell fate mapping, we show that acutely isolated BC are a unique population closely related but distinct from NCC and SC precursors. Moreover, when grafted in the demyelinated spinal cord, BC progeny expands in the lesion through a combination of time-regulated processes including proliferation and differentiation. Furthermore, when grafted away from the lesion, BC progeny, in contrast to committed SC, show a high migratory potential mediated through enhanced interactions with astrocytes and white matter, and possibly with polysialylated neural cell adhesion molecule expression. In response to demyelinated axons of the CNS, BC progeny generates essentially myelin-forming SC. However, in contact with axons and astrocytes, some of them generate also myelin-forming oligodendrocytes. There are two primary outcomes of this study. First, the high motility of BC and their progeny, in addition to their capacity to remyelinate CNS axons, supports the view that BC are a reservoir of interest to promote CNS remyelination. Second, from a developmental point of view, BC behavior in the demyelinated CNS raises the question of the boundary between central and peripheral myelinating cells.

Effects of cannabinoids in Krox-24 targeted mice.

Krox-24 is an immediate early gene encoding a zinc-finger transcription factor implicated in several adaptive responses, and its induction by cannabinoids has been reported. We used mice targeted in the Krox-24 gene to specifically dissect the role of this protein in the acute and chronic central actions of cannabinoids. We report here on the ability of cannabinoids to activate G-proteins and to inhibit adenylyl cyclase, and to elicit behavioral responses in wild-type and mutant mice. The behavioral parameters and the biochemical correlates of abstinence after delta9-THC withdrawal were evaluated. We show that Krox-24 is not involved in the acute analgesic effects of delta9-THC and in the SR precipitated delta9-THC withdrawal syndrome.

Targeting of the EphA4 tyrosine kinase receptor affects dorsal/ventral pathfinding of limb motor axons.

The Eph family of tyrosine kinase receptors has recently been implicated in various processes involving the detection of environmental cues such as axonal guidance, targeted cell migration and boundary formation. We have inactivated the mouse EphA4 gene to investigate its functions during development. Homozygous EphA4 mutant animals show peroneal muscular atrophy correlating with the absence of the peroneal nerve, the main dorsal nerve of the hindlimb. This phenotype is also observed, although with a lower penetrance, in heterozygotes. During normal hindlimb innervation, motor axons converge towards the sciatic plexus region at the base of the limb bud, where they must choose between dorsal and ventral trajectories within the limb. Among the axons emerging from the sciatic plexus, dorsal projections show higher levels of EphA4 protein than ventral axons. In EphA4 mutant mice, presumptive dorsal motor axons fail to enter the dorsal compartment of the limb and join the ventral nerve. Our data therefore suggest that the level of EphA4 protein in growing limb motor axons is involved in the selection of dorsal versus ventral trajectories, thus contributing to the topographic organisation of motor projections.

A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool.

Neuregulin-1 provides an important axonally derived signal for the survival and growth of developing Schwann cells, which is transmitted by the ErbB2/ErbB3 receptor tyrosine kinases. Null mutations of the neuregulin-1, erbB2, or erbB3 mouse genes cause severe deficits in early Schwann cell development. Here, we employ Cre-loxP technology to introduce erbB2 mutations late in Schwann cell development, using a Krox20-cre allele. Cre-mediated erbB2 ablation occurs perinatally in peripheral nerves, but already at E11 within spinal roots. The mutant mice exhibit a widespread peripheral neuropathy characterized by abnormally thin myelin sheaths, containing fewer myelin wraps. In addition, in spinal roots the Schwann cell precursor pool is not correctly established. Thus, the Neuregulin signaling system functions during multiple stages of Schwann cell development and is essential for correct myelination. The thickness of the myelin sheath is determined by the axon diameter, and we suggest that trophic signals provided by the nerve determine the number of times a Schwann cell wraps an axon.

Thyroid hormone effects on Krox-24 transcription in the post-natal mouse brain are developmentally regulated but are not correlated with mitosis.

Krox-24 (NGFI-A, Egr-1) is an immediate-early gene encoding a zinc finger transcription factor. As Krox-24 is expressed in brain areas showing post-natal neurogenesis during a thyroid hormone (T3)-sensitive period, we followed T3 effects on Krox-24 expression in newborn mice. We analysed whether regulation was associated with changes in mitotic activity in the subventricular zone and the cerebellum. In vivo T3-dependent Krox-24 transcription was studied by polyethylenimine-based gene transfer. T3 increased transcription from the Krox-24 promoter in both areas studied at post-natal day 2, but was without effect at day 6. An intact thyroid hormone response element (TRE) in the Krox-24 promoter was necessary for these inductions. These stage-dependent effects were also seen in endogenous Krox-24 mRNA levels: activation at day 2 and no effect at day 6. Moreover, similar results were obtained by examining beta-galactosidase expression in heterozygous mice in which one allele of the Krox-24 gene was disrupted with an inframe Lac-Z insertion. However, bromodeoxyuridine incorporation showed mitosis to continue through to day 6. We conclude first, that T3 activates Krox-24 transcription during early post-natal mitosis but that this effect is extinguished as development proceeds and second, loss of T3-dependent Krox-24 expression is not correlated with loss of mitotic activity.

Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice.

The zinc finger transcription factor Krox-24 (NGFI-A, Egr-1) is encoded by an immediate-early serum response gene expressed in various physiological situations and tissues. To investigate its function, we have created a null allele. Mice homozygous for the mutation have a reduced body size, and both males and females are sterile. These phenotypes were related to defects in the anterior pituitary of both sexes and in the ovary. In the pituitary, two cell lineages expressing Krox-24 are differentially affected by the mutation: somatotropes present abnormal cytological features and are reduced in number, consistent with the decreased GH content observed in these animals; in contrast gonadotropes are normal in number, but specifically fail to synthesize the beta-subunit of LH. In the ovary, LH receptor expression is prevented, indicating an involvement of Krox-24 at two levels at least of the pituitary-gonadal axis. Our data, together with the results of a previous report describing another Krox-24 mutant allele, suggest that Krox-24 may have two distinct molecular functions in the anterior pituitary: transcriptional activation of the LHbeta gene in gonadotropes and control of cell proliferation and/or survival in somatotropes by unknown mechanisms.

[Role of the Krox-20 gene in the development of rhombencephalon]. / Rôle du gène Krox-20 dans le développement du rhombencéphale.

In the hindbrain region of the developing CNS, anteroposterior patterning involves a transient segmentation process which leads to the formation of morphological bulges called rhombomeres. The rhombomeres constitute cell lineage restriction units and participate in the establishment of a metameric pattern which is responsible for the segmental organisation of motor and reticular neurons. Like Drosophila compartments, rhombomeres also constitute domains of specific gene expression. Genes expressed in a rhombomere-specific manner so far identified encode various types of putative regulatory molecules, including transcription factors, like Hox proteins, the zinc finger protein Krox-20 and the basic domain leucine-zipper protein kreisler, and receptor type molecules, like Sek-1, a member of the EPH family of tyrosine kinase receptors. Such genes are thought to play a role either in the definition of segmental territories or in the specification of the identity of the rhombomeres. Initial analysis of the function of some of these genes have indeed supported this hypothesis. This is the case for the Krox-20 gene. It is expressed within the developing hindbrain in two transverse domains which prefigure and then coincide with r3 and r5. We have inactivated Krox-20 by homologous recombination in ES cells and demonstrated that the mutation leads to the deletion of r3 and r5. The mutation introduced into the Krox-20 gene involved the in-frame insertion of the lacZ coding sequence. This allowed us to follow the late expression pattern of the gene and to identify two additional phenotypes, affecting myelination of the peripheral nervous system and endochondral ossification. The lacZ reporter also permitted a detailed analysis of the expression of Krox-20 in peripheral glial cells, revealing important steps in the control of their development. Recently we have performed a detailed analysis of specific neuronal populations affected by the mutation which shed new light on the role of Krox-20 in the segmentation and on the physiological consequences of its inactivation. We have also identified several new members of the Sek-1 family of receptor tyrosine kinases, which are also expressed in a rhombomere-specific manner. Finally, we have provided evidence that Krox-20 is as a key regulator of r3/r5-specific transcription, controlling the expression of at least five other regulator genes in these rhombomeres. In three cases, Hoxb-2, Hoxa-2 and Sek-1, we could demonstrate that Krox-20 was directly involved in the transcriptional activation of these genes.

Differential regulation of the zinc finger genes Krox-20 and Krox-24 (Egr-1) suggests antagonistic roles in Schwann cells.

Krox-20 and Krox-24 (Egr-1) encode closely related zinc finger transcription factors, which interact with the same DNA target sequences. Krox-20 is required for myelination in the peripheral nervous system. Using lacZ knock-in mutant mouse lines as well as immunohistochemical analyses, we have studied the expression of Krox-20 and Krox-24 in the Schwann cell lineage during normal development and following nerve lesion in the mouse and in human neuropathies. During embryogenesis, the two genes are expressed in a successive and mutually exclusive manner, Krox-24 being restricted to Schwann cell precursors and Krox-20 to mature Schwann cells. At birth, Krox-24 is reactivated and the two genes are coexpressed. In the adult, Krox-20 is expressed in myelinating cells, while Krox-24 is restricted to nonmyelinating cells. Following nerve lesion, Krox-24 is strongly induced in Schwann cells, reinforcing the link between its expression and the nonmyelinating and/or proliferative state, whereas Krox-20 is downregulated. These data are consistent with Krox-20 and Krox-24 playing antagonistic roles during the development of the Schwann cell lineage. In particular, their balance of expression might participate in the choice between myelinating and nonmyelinating pathways.

Reorganization of pontine rhythmogenic neuronal networks in Krox-20 knockout mice.

We have shown previously that the inactivation of the zinc finger gene Krox-20 affects hindbrain segmentation, resulting in the elimination of rhombomeres 3 and 5. We demonstrate here that Krox-20 homozygous mutant mice exhibit abnormally slow respiratory and jaw opening rhythms, indicating that a modification of hindbrain segmentation influences the function of neuronal networks after birth. Central neuronal networks that control respiratory frequency are made predominantly depressant by the elimination of a previously undescribed rhythm-promoting system. Recordings of rhythmic activity from the isolated hindbrain following progressive tissue transections indicate that the reorganization takes place in the caudal pontine reticular formation. The newborn (PO) Krox-20-/- mice, in which apneas are ten times longer than in wild-type animals, may be a valuable model for the study of life-threatening apneas during early infancy.

The regulation of Krox-20 expression reveals important steps in the control of peripheral glial cell development.

The zinc finger transcription factor gene Krox-20 is expressed in Schwann cells and is required for the myelination of peripheral nerves. We show that the regulation of Krox-20 expression in peripheral glial cells reveals three important steps in the development and differentiation of these cells. (i) Expression of Krox-20 in Schwann cells requires continuous neuronal signalling via direct axonal contact. Therefore Krox-20 appears to be a key component of the transduction cascade linking axonal signalling to myelination. (ii) Krox-20 inducibility is acquired by Schwann cells at the time that they are formed from their precursors. Diffusible factor(s) synthesised by the neural tube can mediate this transition and can be mimicked by NDFbeta or a combination of CNTF and bFGF. Furthermore, the neural tube activity is blocked by a hybrid protein containing the NDF-binding domain of the ErbB4 receptor, strongly implicating NDF in the physiological transition. (iii) In sensory ganglia, the microenvironment is capable of negatively regulating Krox-20, presumably by preventing the conversion of satellite glial cells toward a Schwann cell-like phenotype.

Embryonic Development of Schwann Cells: Multiple Roles for Neuregulins along the Pathway

During the past few years important advances have been realized in the understanding of the molecular and cellular mechanisms operating in the differentiation of Schwann cells from neural crest cells. In particular the development of in vitro culture systems has allowed the analysis of commitment to the Schwann cell lineage, identification of an intermediate between the neural crest cell and the Schwann cell, and initial analysis of the factors controlling determination and differentiation processes. These studies point in particular to major roles for neuregulins which appear to be involved in the control of cell survival, proliferation, and differentiation, as well as of gene expression at different levels of the pathway.

Embryonic development of Schwann cells: multiple roles for neuregulins along the pathway.

During the past few years important advances have been realized in the understanding of the molecular and cellular mechanisms operating in the differentiation of Schwann cells from neural crest cells. In particular the development of in vitro culture systems has allowed the analysis of commitment to the Schwann cell lineage, identification of an intermediate between the neural crest cell and the Schwann cell, and initial analysis of the factors controlling determination and differentiation processes. These studies point in particular to major roles for neuregulins which appear to be involved in the control of cell survival, proliferation, and differentiation, as well as of gene expression at different levels of the pathway.

Defective bone formation in Krox-20 mutant mice.

Endochondral ossification is the prevalent mode of vertebrate skeleton formation; it starts during embryogenesis when cartilage models of long bones develop central regions of hypertrophy which are replaced by bony trabeculae and bone marrow. Although several transcription factors have been implicated in pattern formation in the limbs and axial skeleton, little is known about the transcriptional regulations involved in bone formation. We have created a null allele in the mouse Krox-20 gene, which encodes a zinc finger transcription factor, by in frame insertion of the E. coli lacZ gene and shown that hindbrain segmentation and peripheral nerve myelination are affected in Krox-20-/- embryos. We report here that Krox-20 is also activated in a subpopulation of growth plate hypertrophic chondrocytes and in differentiating osteoblasts and that its disruption severely affects endochondral ossification. Krox-20-/- mice develop skeletal abnormalities including a reduced length and thickness of newly formed bones, a drastic reduction of calcified trabeculae and severe porosity. The periosteal component to bone formation and calcification does not appear to be affected in the homozygous mutant suggesting that the major role for Krox-20 is to be found in the control of the hypertrophic chondrocyte-osteoblast interactions leading to endosteal bone formation.

Krox-20 controls myelination in the peripheral nervous system.

The molecular mechanisms controlling the process of myelination by Schwann cells remain elusive, despite recent progress in the identification and characterization of genes encoding myelin components (reviewed in ref. 1). We have created a null allele in the mouse Krox-20 gene, which encodes a zinc-finger transcription factor, by in-frame insertion of the Escherichia coli lacZ gene, and have shown that hindbrain segmentation is affected in Krox-20-/- embryos. We demonstrate here that Krox-20 is also activated in Schwann cells before the onset of myelination and that its disruption blocks Schwann cells at an early stage in their differentiation, thus preventing myelination in the peripheral nervous system. In Krox-20-/- mice, Schwann cells wrap their cytoplasmic processes only one and a half turns around the axon, and although they express the early myelin marker, myelin-associated glycoprotein, late myelin gene products are absent, including those for protein zero and myelin basic protein. Therefore Krox-20 is likely to control a set of genes required for completion of myelination in the peripheral nervous system.

Several receptor tyrosine kinase genes of the Eph family are segmentally expressed in the developing hindbrain.

Pattern formation in the hindbrain involves a segmentation process leading to the formation of metameric units, manifested as successive swellings known as rhombomeres (r). In search for genes involved in cell-cell interactions during hindbrain segmentation, we have screened for protein kinase genes with restricted expression patterns in this region of the CNS. We present the cloning of three novel mouse genes, Sek-2, Sek-3 and Sek-4 (members of the Eph subfamily of putative transmembrane receptor protein tyrosine kinases (RTKs)), the identification of their chromosomal locations, and the analysis of their expression between 7.5 and 10.5 days of development. Before morphological segmentation, Sek-2 is transcribed in a transverse stripe corresponding to prospective r4 and the adjacent mesoderm, suggesting possible roles both in hindbrain segmentation and signalling between neuroepithelium and mesoderm. Sek-3 and Sek-4 have common domains of expression, including r3, r5 and part of the midbrain, as well as specific domains in the diencephalon, telencephalon, spinal cord and in mesodermal and neural crest derivatives. Together with our previous finding that Sek (Sek-1) is expressed in r3 and r5 (Gilardi-Hebenstreit et al., 1992; Nieto et al., 1992), these data indicate that members of the Eph family of RTKs may co-operate in the segmental patterning of the hindbrain.

Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain.

The zinc finger gene Krox-20 is transcribed in two alternate segments (rhombomeres) of the developing hindbrain. To investigate its function, we have used homologous recombination to generate mice carrying an in-frame insertion of the E. coli lacZ gene within Krox-20. Analysis of the beta-galactosidase pattern in heterozygous embryos confirmed the known profile with expression restricted to rhombomeres (r) 3 and 5. Mice homozygous for the mutation die during the first two weeks after birth. Anatomical analysis of the hindbrain and of the cranial nerves during embryogenesis, combined with the determination of the expression patterns of rhombomere-specific genes, demonstrated that Krox-20 inactivation results in a marked reduction or elimination of r3 and r5. We conclude that Krox-20, although not required for the initial delimitation of r3 and r5, plays an important role in the process of segmentation governing hindbrain development.