The Xiro-repressed gene CoREST is expressed in Xenopus neural territories.

The iroquois (iro) genes encode evolutionary conserved homeoproteins that participate in many developmental processes [reviewed in Development 128 (2001) 2847]. In Xenopus, the Iro protein Xiro1 is a repressor, required during gastrulation for neural plate formation, that downregulates Bmp4. During neurulation, Xiro1 participates in the pattering of the neuroectoderm. In this work, we report the cloning and pattern of expression of XCoREST, another gene repressed by Xiro1. During Xenopus development, XCoREST is expressed in territories in which neurogenesis takes place.     

The lipocalin Xlcpl1 expressed in the neural plate of Xenopus laevis embryos is a secreted retinaldehyde binding protein.

The cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate of Xenopus laevis embryos and the adult choroid plexus have been investigated. It was found that this lipocalin, termed Xlcpl1, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol. Furthermore, this protein also binds D/L thyroxine. The Xlcpl1 cDNA was expressed in cell culture using the vaccinia virus expression system. In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway. We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids. Therefore, cpl1-expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers. These retinoids may enter the cytosol by diffusion or receptor-controlled mechanisms, as has been shown for exogenously applied retinoids. Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development.     

A novel putative transmembrane protein, IZP6, is expressed in neural cells during embryogenesis

Gene trapping in mouse embryonic stem cells is an efficient method for identifying new genes and examining their functions. This method has been used in an effort to identify some novel genes involved in mouse development. In the present paper, one such gene named IZP6 is reported. Expression of the IZP6 gene, as monitored by beta-galactosidase expression in heterozygous mice, was detected in a developmentally regulated fashion: the expression pattern has two phases during the embryogenesis. In the first phase, from embryonic day 11.5 (E11.5) until E14.5, the reporter gene is mainly expressed in the forebrain. In the second phase, from E15.5 until birth, expression in the forebrain becomes weaker but is still observed in the olfactory bulb and the skin around the eyes, nose, limbs and tail. Thus, IZP6 gene expression changes from the central nervous system (the first phase) to the peripheral tissues (the second phase) during development. The IZP6 gene encodes a protein of 228 amino acids. Analysis of the secondary structure of the IZP6 protein revealed four hydrophobic regions, indicating that the IZP6 protein is a four transmembrane region protein. These results suggest that IZP6 is a transmembrane protein related to neurogenesis in the mouse.     

A homeobox-containing marker of posterior neural differentiation shows the importance of predetermination in neural induction

A homeobox sequence has been used to isolate a new Xenopus cDNA, named XIHbox6. A short probe from this gene serves as an early marker of posterior neural differentiation in the Xenopus nervous system. The gene recognized by this cDNA sequence is first transcribed at the late gastrula stage and solely in the posterior neural cells. The gene is expressed when ectodermal and mesodermal tissues of an early gastrula are placed in contact, but not by either tissue cultured on its own. However, gene expression is most easily inducible in ectoderm from the dorsal region, i.e., in ectoderm normally destined to form neural structures. This establishes the principle, in contrast to previous belief, that the induction of the embryonic nervous system involves a predisposition of the ectoderm and does not depend entirely on an interaction with inducing mesoderm.     

Darmin is a novel secreted protein expressed during endoderm development in Xenopus

Endoderm development is an area of intense interest in developmental biology, but progress has been hampered by the lack of specific markers for differentiated endodermal cells. In an unbiased secretion cloning screen of Xenopus gastrula embryos we isolated a novel gene, designated Darmin. Darmin encodes a secreted protein of 56 kDa containing a peptidase M20 domain characteristic of the glutamate carboxypeptidase group of zinc metalloproteases. We also identified homologous Darmin genes in other eukaryotes and in prokaryotes suggesting that Darmin is the founding member of a family of evolutionarily conserved proteins. Xenopus Darmin showed zygotic expression in the early endoderm and later became restricted to the midgut. By secretion cloning of Xenopus cleavage-stage embryos we isolated another novel protein, designated Darmin-related (Darmin-r) due to its sequence similarity with Darmin. Darmin-r was maternally expressed and showed at later stages expression in the lens and pronephric glomus. The endoderm-specific expression of Darmin makes this gene a useful marker for the study of endoderm development.     

Kremen is required for neural crest induction in Xenopus and promotes LRP6-mediated Wnt signaling

Kremen 1 and 2 (Krm1/2) are transmembrane receptors for Wnt antagonists of the Dickkopf (Dkk) family and function by inhibiting the Wnt co-receptors LRP5/6. Here we show that Krm2 functions independently from Dkks during neural crest (NC) induction in Xenopus. Krm2 is co-expressed with, and regulated by, canonical Wnts. Krm2 is differentially expressed in the NC, and morpholino-mediated Krm2 knockdown inhibits NC induction, which is mimicked by LRP6 depletion. Conversely, krm2 overexpression induces ectopic NC. Kremens bind to LRP6, promote its cell-surface localization and stimulate LRP6 signaling. Furthermore, Krm2 knockdown specifically reduces LRP6 protein levels in NC explants. The results indicate that in the absence of Dkks, Kremens activate Wnt/beta-catenin signaling through LRP6.     

The Xenopus laevis Hox 2.1 homeodomain protein is expressed in a narrow band of the hindbrain

The expression pattern of the Xenopus homeodomain protein Hox 2.1 during development was determined using an affinity-purified antibody directed against a carboxyterminal peptide. Nuclear staining was detected in a very narrow band of the hindbrain. This pattern was compared to that of the previously described Xenopus gene XIHbox 1 in serial sections and found to be more anterior than the XIHbox 1 long protein expression but overlapping with that of the short protein. Xenopus Hox 2.1 protein expression is restricted to a much narrower antero-posterior band than that reported for mouse Hox 2.1 RNA expression by in situ hybridization.     

A Xenopus mRNA related to Drosophila twist is expressed in response to induction in the mesoderm and the neural crest

We have cloned a Xenopus cDNA related to the twist gene, which is required for mesodermal differentiation in Drosophila. Northern blots of dissected embryos and in situ hybridization show that the corresponding mRNA, called Xtwi, first appears in early gastrulae, and is present only in mesodermal cells. Within the mesoderm, Xtwi is expressed in the notochord and lateral plate, but not in the myotome; therefore there is a complementary pattern of Xtwi and muscle-specific gene expression in the mesoderm. Xtwi expression therefore marks the subdivision of the mesoderm. Xtwi is also activated a few hours later in the early development of the neural crest. This gene is thus expressed in response to two sequential early inductions in frog development.     

XCIRP (Xenopus homolog of cold-inducible RNA-binding protein) is expressed transiently in developing pronephros and neural tissue

The pronephros functions in the amphibian larval stage. It differentiates in certain presumptive regions of the amphibian embryo. The study of molecules functioning during pronephrogenesis is important for understanding the mechanism of kidney formation. Herein, we report a gene expressed during differentiation of the pronephros and neural tissues that we isolated by differential hybridization using our pronephros in-vitro induction system. The gene, XCIRP, is 887 bp in length, and encodes a putative protein composed of 163 amino acid residues. The deduced protein contains two CS-RBDs (consensus sequence RNA-binding domain) and a glycine-rich domain, and is 74% identical to homologs from other species (mouse, rat and human). The expression of XCIRP increased rapidly during gastrulation, and XCIRP localization was seen in the presumptive pronephros and neural tissues. These findings suggest that XCIRP may play important roles in pronephrogenesis and neurogenesis.     

The LIM domain protein Lmo4 is highly expressed in proliferating mouse epithelial tissues.

LMO4 belongs to the LIM-only family of zinc finger proteins that have been implicated in oncogenesis. The LMO4 gene is overexpressed in breast cancer and oral cavity carcinomas, and high levels of this protein inhibit mammary epithelial differentiation. Targeted deletion of Lmo4 in mice leads to complex phenotypic abnormalities and perinatal lethality. To further understand the role of LMO4, we have characterized Lmo4 expression in adult mouse tissues by immunohistochemical staining using monoclonal anti-Lmo4 antibodies. Lmo4 was highly expressed within specific cell types in diverse tissues. Expression was prevalent in epithelial-derived tissues, including the mammary gland, tongue, skin, small intestine, lung, and brain. High levels of Lmo4 were frequently observed in proliferating cells, such as the crypt cells of the small intestine and the basal cells of the skin and tongue. Lmo4 was highly expressed in the proliferative cap cell layer of the terminal end buds in the peripubertal mammary gland and in the lobuloalveolar units during pregnancy. The expression profile of Lmo4 suggests that this cofactor is an important regulator of epithelial proliferation and has implications for its role in the pathogenicity of cancer.     

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.     

Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

Gal-NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system

A new monoclonal antibody, 2E11, was produced by immunizing mice with the microsomal fraction of rat accessory olfactory bulb cells. This IgM recognizes a previously described complex alpha-galactosyl containing glycolipid, as well as N-linked glycoproteins at 170 and 210 kD. These proteins correspond to a new nerve cell adhesion molecule (NCAM) glycoform, Gal-NCAM, which contains a blood group B-like oligosaccharide. During embryonic development, the 2E11 epitope is expressed by a subset of mature olfactory sensory neurons randomly dispersed throughout the olfactory epithelium, whereas in the olfactory bulb, immunostaining is restricted to medial areas of the nerve layer. When compared to PSA-NCAM, another NCAM glycoform, Gal-NCAM has a mutually exclusive distribution pattern both in the olfactory epithelium and in the olfactory bulb. We propose a model for the hierarchy of neuronal maturation in the olfactory epithelium, including a switch from PSA-NCAM expression by immature neurons to the expression of Gal-NCAM by mature neurons.     

The B30 ganglioside is a cell surface marker for neural crest-derived neurons in the developing mouse

We have previously reported the isolation of a monoclonal antibody, mAb B30, that recognizes two minor gangliosides specifically expressed in a small subset of neurons in the developing mouse central nervous system (Stainier and Gilbert, 1989). B30 labels mesencephalic trigeminal neurons shortly after differentiation until about 2 weeks after birth. Postnatally, it also labels two specific monolayers of cerebellar neurons. In this study, we have characterized the B30 immunoreactivity in the developing peripheral nervous system of the mouse. We report that B30 is a marker for neural crest-derived neurons and have used it to follow the neuronal differentiation of neural crest cells in a serum-free chemically defined culture system. Within hours after plating, neural crest cells migrate away from the neural tube explant on a fibronectin or laminin substrate and by 24 hr, up to 15% of them have differentiated into morphologically identifable neurons. In vitro as in vivo, undifferentiated mouse neural crest cells express the GD3 ganglioside which is recognized by mAb B33, and neural crest-derived neurons can be labeled by mAbs B33, B30, and also E1.9, a specific neuronal cytoskeletal marker. We also show the unique biochemical specificity of mAb B30 and provide experimental evidence for the role of the B30 ganglioside in the cellular adhesion process.