Embryonic expression of three mouse genes with homology to the Drosophila melanogaster prickle gene

The Drosophila melanogaster gene prickle-spiny-legs (pk) functions in an intercellular feedback loop that is central to the establishment of planar cell polarity in the eye and epidermis of the fly, by modulating Frizzled-Disheveled signalling. Here we identify three mouse prickle-related genes (dyxin, testin and prickle) and describe their expression pattern during murine embryogenesis (E7.5-E15.5). We report that the three genes are expressed in restricted areas of the developing mouse brain: dyxin in the most ventral region of the neural tube and in some localized regions of the ventricular layer of the mesencephalon and rhombencephalon, prickle in the pons region, ventrolateral part of rhombencephalon and motoneurons in the spinal cord, and testin in differentiating neurons of the spinal cord and retina. At the stages analyzed, the main site of expression of testin is the migrating cranial neural crest, while the expression of dyxin is noticeable in myotomal cells and its derivatives, with prickle expression being reciprocally localized to some sclerotomal derivatives, like bone primordia. prickle is also expressed in the apical ectodermal ridge and the most distal mesenchyme of the forming limb buds.     

Retinoic acid-binding protein, rhombomeres and the neural crest.

We have investigated by immunocytochemistry the spatial and temporal distribution of cellular retinoic acid-binding protein (CRABP) in the developing nervous system of the chick embryo in order to answer two specific questions: do neural crest cells contain CRABP and where and when do CRABP-positive neuroblasts first arise in the neural tube? With regard to the neural crest, we have compared CRABP staining with HNK-1 staining (a marker of migrating neural crest) and found that they do indeed co-localise, but cephalic and trunk crest behave slightly differently. In the cephalic region in tissues such as the frontonasal mass and branchial arches, HNK-1 immunoreactivity is intense at early stages, but it disappears as CRABP immunoreactivity appears. Thus the two staining patterns do not overlap, but are complementary. In the trunk, HNK-1 and CRABP stain the same cell populations at the same time, such as those migrating through the anterior halves of the somites. In the neural tube, CRABP-positive neuroblasts first appear in the rhombencephalon just after the neural folds close and then a particular pattern of immunoreactivity appears within the rhombomeres of the hindbrain. Labelled cells are present in the future spinal cord, the posterior rhombencephalon up to rhombomere 6 and in rhombomere 4 thus producing a single stripe pattern. This pattern is dynamic and gradually changes as anterior rhombomeres begin to label. The similarity of this initial pattern to the arrangement of certain homeobox genes in the mouse stimulated us to examine the expression of the chicken Hox-2.9 gene. We show that at stage 15 the pattern of expression of this gene is closely related to that of CRABP. The relationship between retinoic acid, CRABP and homeobox genes is discussed.     

Expression patterns of Hox10 paralogous genes during lumbar spinal cord development

We have examined the expression of three paralogous Hox genes from E11.5 through E15.5 in the mouse spinal cord. These ages coincide with major phases of spinal cord neurogenesis, neuronal differentiation, cell migration, gliogenesis, and motor neuron cell death. The three genes, Hoxa10, Hoxc10, and Hoxd10, are all expressed in the lumbar spinal cord and have distinct expression patterns. Mutations in these three genes are known to affect motor neuron patterning. All three genes show lower levels of expression at the rostral limits of their domains, with selective regions of higher expression more caudally. Hoxa10 and Hoxd10 expression appears confined to postmitotic cell populations in the intermediate and ventral gray, while Hoxc10 is also expressed in proliferating cells in the dorsal ventricular zone. Hoxc10 and Hoxd10 expression is clearly excluded from the lateral motor columns at rostral lumbar levels but is present in this region more caudally. Double labeling demonstrates that Hoxc10 expression is correlated with ventrolateral LIM gene expression in the caudal part of the lumbar spinal cord.     

Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord

Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell‐adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M‐AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M‐AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH?/?) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M‐AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 494–504, 2015     

Retinoic acid induces changes in the localization of homeobox proteins in the antero-posterior axis of Xenopus laevis embryos.

We have studied the localization of the proteins of Xeb1 and Xeb2, two homeobox (hbx)-containing genes that are expressed during the early development of Xenopus laevis. Both proteins are expressed in juxtaposed and partially overlapping domains along the antero-posterior axis of Xenopus laevis embryos, with clearly defined anterior boundaries. Xeb2 is predominantly expressed in the caudal region of the hindbrain, whereas the Xeb1 protein is located in the most rostral region of the spinal cord. Furthermore, both proteins are expressed in single cells dispersed in the lateral flanks of the embryo in positions that correlate with the expression domains in the neural tube. We suggest that these cells are migratory neural crest cells that have acquired positional information in the neural tube prior to migration. The Xeb2 protein was also detected in the most posterior branchial arches and the pronephros. In stage 45 embryos, nuclei of the IX-X cranial ganglia, the lung buds and cells spreading into the forelimb rudiment express the Xeb2 antigen. The Xeb1 protein was also detected in the lung buds and the forelimb rudiment. To examine the effect of retinoic acid on expression, gastrula embryos were treated with all-trans retinoic acid (RA). Increasing concentrations of RA caused progressive truncation of anterior structures. The most severely affected embryos lacked eyes, nasal pits, forebrain, midbrain and otic vesicles, and the anterior boundary of the hindbrain seemed to be displaced rostrally. This alteration correlates with a progressive displacement of the anterior boundary of the expression domain of Xeb2. On the other hand, 10(-6) M RA induces an ectopic site of Xeb1 expression at the anterior end of the central nervous system, located just anterior to the extended domain of Xeb2 whereas expression in the spinal cord remains unaffected.     

Region-specific and stage-dependent regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 homeodomain transcription factor

During early neural development, the Nkx6.1 homeodomain neural progenitor gene is specifically expressed in the ventral neural tube, and its activity is required for motoneuron generation in the spinal cord. We report that Nkx6.1 also controls oligodendrocyte development in the developing spinal cord, possibly by regulating Olig gene expression in the ventral neuroepithelium. In Nkx6.1 mutant spinal cords, expression of Olig2 in the motoneuron progenitor domain is diminished, and the generation and differentiation of oligodendrocytes are significantly delayed and reduced. The regulation of Olig gene expression by Nkx6.1 is stage dependent, as ectopic expression of Nkx6.1 in embryonic chicken spinal cord results in an induction of Olig2 expression at early stages, but an inhibition at later stages. Moreover, the regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 also appears to be region specific. In the hindbrain, unlike in the spinal cord, Olig1 and Olig2 can be expressed both inside and outside the Nkx6.1-expressing domains and oligodendrogenesis in this region is not dependent on Nkx6.1 activity.     

Transgenic Xenopus embryos reveal that anterior neural development requires continued suppression of BMP signaling after gastrulation.

In vertebrates, BMP signaling before gastrulation suppresses neural development. Later in development, BMP signaling specifies a dorsal and ventral fate in the forebrain and dorsal fate in the spinal cord. It is therefore possible that a change in the competence of the ectoderm to respond to BMP signaling occurs at some point in development. We report that exposure of the anterior neural plate to BMP4 before gastrulation causes suppression of all neural markers tested. To determine the effects of BMP4 after gastrulation, we misexpressed BMP4 using a Pax-6 promoter fragment in transgenic frog embryos and implanted beads soaked in BMP4 in the anterior neural plate. Suppression of most anterior neural markers was observed. We conclude that most neural genes continue to require suppression of BMP signaling into the neurula stages. Additionally, we report that BMP4 and BMP7 are abundantly expressed in the prechordal mesoderm of the neurula stage embryo. This poses the paradox of how the expression of most neural genes is maintained if they can be inhibited by BMP signaling. We show that at least one gene in the anterior neural plate suppresses the response of the ectoderm to BMP signaling. We propose that the suppressive effect of BMP signaling on the expression of neural genes coupled with localized suppressors of BMP signaling result in the fine-tuning of gene expression in the anterior neural plate.     

Spatial and temporal expression of cellular retinoic acid binding protein (CRABP) along the anteroposterior axis in the central nervous system of mouse embryos

In the central nervous system of 11.5-day mouse embryos, the expression of CRABP was spatially restricted to the anteroposterior axis. CRABP was most strongly expressed in the rhombencephalon and the anterior part of the neural tube. In 14-day mouse embryo, CRABP drastically decreased in the brain and the anterior part of the neural tube. The transient expression and spatial distribution of CRABP in the central nervous system strongly suggest that retinoic acid is involved in the neurogenesis during development.     

Spatially and temporally restricted expression of Pax2 during murine neurogenesis

The expression of the murine paired-box-containing gene, Pax2, is examined in the developing central nervous system by in situ hybridization. Pax2 expression is detected along the boundaries of primary divisions of the neural tube. Initially, Pax2 is expressed in the ventricular zone in two compartments of cells on either side of the sulcus limitans and along the entire rhombencephalon and spinal cord. At later times, Pax2 is restricted to progeny cells that have migrated to specific regions of the intermediate zone. In the eye, Pax2 expression is restricted to the ventral half of the optic cup and stalk and later to the optic disc and nerve. In the ear, expression is restricted to regions of the otic vesicle that form neuronal components. The transient and restricted nature of Pax2 expression suggests that this murine segmentation gene homologue may also establish compartmental boundaries and contribute to the specification of neuronal identity, as do certain Drosophila segmentation genes.     

小鼠ERb 基因片段的克隆及其在胚胎发育中的表达

为探讨ERb在小鼠胚胎发育过程中的表达, 首先设计ERb引物并扩增ERb基因片段, 构建ERb/pGEM-3Z重组质粒进行克隆, 分别用EcoRⅠ和HindⅢ进行酶切得到线性化DNA片段, 以Sp6和T7聚合酶合成地高辛标记的(dig )正、反义RNA探针。然后通过胚胎整体原位杂交技术分析ERb 在小鼠胚胎中的表达。运用该探针检测到ERb 基因在10.5 dpc胚胎的脑、脊神经管、生殖脊、心包、肢芽及颌弓部位表达, 在13.5 dpc胚胎的端脑、中脑、延髓、脊髓、肢芽中表达。推测ERb基因可能在小鼠胚胎性别分化过程中起调节作用; 可能在小鼠胚胎神经管的早期区域化过程中起作用并在3个原始脑泡进一步分化及脊髓的分化过程中起作用; 可能在小鼠胚胎肢芽中的骨与软骨的形成与分化中起调控作用; 可能在小鼠胚胎心脏发育过程中起作用, 可能在小鼠胚胎颌弓的表面分化过程中起调控作用。     

Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice

Retinoid control of vertebrate development depends upon tissue-specific metabolism of retinol to retinoic acid (RA). The RA biosynthetic enzyme RALDH2 catalyzes much, but not all, RA production in mouse embryos, as revealed here with Raldh2 null mutants carrying an RA-responsive transgene. Targeted disruption of Raldh2 arrests development at midgestation and eliminates all RA synthesis except that associated with Raldh3 expression in the surface ectoderm of the eye field. Conditional rescue of Raldh2(-/-) embryos by limited maternal RA administration allows development to proceed and results in the establishment of additional sites of RA synthesis linked to Raldh1 expression in the dorsal retina and to Raldh3 expression in the ventral retina, olfactory pit and urinary tract. Unexpectedly, conditionally rescued Raldh2(-/-) embryos also possess novel sites of RA synthesis in the neural tube and heart that do not correspond to expression of Raldh1-3. RA synthesis in the mutant neural tube was localized in the spinal cord, posterior hindbrain and portions of the midbrain and forebrain, whereas activity in the mutant heart was localized in the conotruncus and sinus venosa. In the posterior hindbrain, this novel RA-generating activity was expressed during establishment of rhombomeric boundaries. In the spinal cord, the novel activity was localized in the floorplate plus in the intermediate region where retinoid-dependent interneurons develop. These novel RA-generating activities in the neural tube and heart fill gaps in our knowledge of how RA is generated spatiotemporally and may, along with Raldh1 and Raldh3, contribute to rescue of Raldh2(-/-) embryos by producing RA locally.     

Differential Expression of Micro-Heterogeneous LewisX-Type Glycans in the Stem Cell Compartment of the Developing Mouse Spinal Cord

Complex glycan structures and their respective carrier molecules are often expressed in a cell type specific manner. Thus, glycans can be used for the enrichment of specific cell types such as neural precursor cells (NPCs). We have recently shown that the monoclonal antibodies 487^LeX and 5750^LeX differentially detect the LewisX (LeX) glycan on NPCs in the developing mouse forebrain. Here, we analysed the staining pattern of both antibodies during late embryonic mouse spinal cord development. At E13.5 both antibodies strongly label the central canal region. Along these lines they detect the LeX glycan primarily on Nestin-positive NPCs at that age. Moreover, we show that spinal cord NPCs cultured as free floating neurospheres display a high immunoreactivity to both antibodies. In that context, we also demonstrate that the 487^LeX antibody can be used to deplete a subpopulation of neurosphere forming NPCs from a mixed E13.5 spinal cord cell suspension. Towards the end of embryogenesis the overall immunoreactivity to both antibodies increases and the staining appears very diffuse. However, the 5750^LeX antibody still labels the central canal region. The increase in immunoreactivity correlates with an expression increase of the extracellular matrix molecules Tenascin C and Receptor Protein Tyrosine Phosphatase β/ζ, two potential LeX carrier proteins. In line with this, immunoprecipitation analyses confirmed Tenascin C as a LeX carrier protein in the embryonic mouse spinal cord. However, the immunoreactivity to both antibodies appears only to be marginally affected in the absence of Tenascin C, arguing against Tenascin C being the major LeX carrier. In conclusion our study gives some novel insights into the complex expression of LeX glycans and potential carrier proteins during the development of the mouse spinal cord.     

Xenopus Polycomblike 2 (XPcl2) controls anterior to posterior patterning of the neural tissue

A novel gene, Xenopus Polycomblike 2 (XPcl2), which encodes a protein similar to Drosophila Polycomblike was cloned and characterized. Polycomblike belongs to the Polycomb group proteins, which maintain stable expression patterns for the clustered homeotic genes in the Drosophila embryo by forming multimeric complexes on chromatin. XPcl2 shows greater amino acid sequence homology to human and mouse M96 (hPcl2, mPcl2) than Xenopus Pcl1 (XPcl1), mouse Tctex3 (mPcl1) and human PHF1 (hPcl1), indicating that at least two types of Polycomblike genes are conserved between amphibians and mammals. XPcl2 mRNA is present both maternally and zygotically, and the temporal expression profile is distinct from XPcl1, another member of the Polycomblike family in Xenopus. XPcl2 is highly expressed in the anterior-dorsal region of Xenopus following the neurula stage in a manner similar to XPcl1. Overexpression of XPcl2 disturbs the development of the anterior central nervous system, eye and cement gland. In the XPcl2-overexpressing embryo, a hindbrain marker, Krox20, and a spinal cord marker, HoxB9, are expressed more posteriorly, suggesting an alteration in the anterior-posterior patterning of the neural tissue. In addition, XPcl2 represses Zic3- and noggin-induced anterior neural markers, but not neural crest markers in animal cap explants. These results indicate that XPcl2 regulates anterior neural tissue development and the anterior-posterior patterning of the neural tissue.     

Regionalisation of the mouse embryonic ectoderm: allocation of prospective ectodermal tissues during gastrulation

The regionalisation of cell fate in the embryonic ectoderm was studied by analyzing the distribution of graft-derived cells in the chimaeric embryo following grafting of wheat germ agglutinin--gold-labelled cells and culturing primitive-streak-stage mouse embryos. Embryonic ectoderm in the anterior region of the egg cylinder contributes to the neuroectoderm of the prosencephalon and mesencephalon. Cells in the distal lateral region give rise to the neuroectoderm of the rhombencephalon and the spinal cord. Embryonic ectoderm at the archenteron and adjacent to the middle region of the primitive streak contributes to the neuroepithelium of the spinal cord. The proximal-lateral ectoderm and the ectodermal cells adjacent to the posterior region of the primitive streak produce the surface ectoderm, the epidermal placodes and the cranial neural crest cells. Some labelled cells grafted to the anterior midline are found in the oral ectodermal lining, whereas cells from the archenteron are found in the notochord. With respect to mesodermal tissues, ectoderm at the archenteron and the distal-lateral region of the egg cylinder gives rise to rhombencephalic somitomeres, and the embryonic ectoderm adjacent to the primitive streak contributes to the somitic mesoderm and the lateral mesoderm. Based upon results of this and other grafting studies, a map of prospective ectodermal tissues in the embryonic ectoderm of the full-streak-stage mouse embryo is constructed.