An amphioxus winged helix/forkhead gene, AmphiFoxD: insights into vertebrate neural crest evolution.

During amphioxus development, the neural plate is bordered by cells expressing many genes with homologs involved in vertebrate neural crest induction. However, these amphioxus cells evidently lack additional genetic programs for the cell delaminations, migrations, and differentiations characterizing definitive vertebrate neural crest. We characterize an amphioxus winged helix/forkhead gene (AmphiFoxD) closely related to vertebrate FoxD genes. Phylogenetic analysis indicates that the AmphiFoxD is basal to vertebrate FoxD1, FoxD2, FoxD3, FoxD4, and FoxD5. One of these vertebrate genes (FoxD3) consistently marks neural crest during development. Early in amphioxus development, AmphiFoxD is expressed medially in the anterior neural plate as well as in axial (notochordal) and paraxial mesoderm; later, the gene is expressed in the somites, notochord, cerebral vesicle (diencephalon), and hindgut endoderm. However, there is never any expression in cells bordering the neural plate. We speculate that an AmphiFoxD homolog in the common ancestor of amphioxus and vertebrates was involved in histogenic processes in the mesoderm (evagination and delamination of the somites and notochord); then, in the early vertebrates, descendant paralogs of this gene began functioning in the presumptive neural crest bordering the neural plate to help make possible the delaminations and cell migrations that characterize definitive vertebrate neural crest.     

Ncx, a Hox11 related gene, is expressed in a variety of tissues derived from neural crest cells

We have isolated the murine homeobox gene (Ncx) that belong to a Hox11 gene family. Expression of the Ncx gene was analyzed in total RNAs from embryos by reverse transcribed polymerase chain reaction (RT-PCR). The mRNA was detected in embryos after 9.5 days of embryogenesis (E9.5) and was maximal at E12.5. The RT-PCR also detected the message in total RNAs from adrenal glands and intestine in adult mice. The expression was further examined in various tissues from embryos by in situ hybridization. It was detected in dorsal root ganglia, cranial nerve ganglia (V, IX, X), enteric nerve ganglia and adrenal glands from embryos between E9.5 and E13.5. Since its expression is restricted to tissues derived from neural crest cells, Ncx may play a role in differentiation and proliferation of neural crest lineage cells.     

Endogenous bone morphogenetic protein antagonists regulate mammalian neural crest generation and survival.

We demonstrate here that Chordin and Noggin function as bone morphogenetic protein (BMP) antagonists in vivo to promote mammalian neural crest development. Using Chrd and Nog single and compound mutants, we find that Noggin has a major role in promoting neural crest formation, in which Chordin is partially redundant. BMP signaling is increased in dorsal tissues lacking Noggin and is further increased when Chordin is also absent. The early neural crest domain is expanded with decreased BMP antagonism in vivo. Noggin and Chordin also regulate subsequent neural crest cell emigration from the neural tube. However, reduced levels of these BMP antagonists ultimately result in perturbation of neural crest cell derived peripheral nervous system and craniofacial skeletal elements. Such defects reflect, at least in part, a function to limit apoptosis in neural crest cells. Noggin and Chordin, therefore, function together to regulate both the generation and survival of neural crest cells in mammalian development.     

Differential expression of beta3 integrin gene in chick and mouse cranial neural crest cells.

RNA in situ hybridization on early chicken embryos revealed that the beta3 integrin gene started to be expressed after Hamburger and Hamilton (HH) stage 6 in the presumptive epidermis adjacent to the neural plate, before closure of the neural tube. The beta3 integrin gene was also strongly expressed in cephalic neural crest cells at the same stage in which they begin their migration but disappeared progressively in these cells along the route they take to the branchial arches. The gene was weakly expressed in the differentiating cranial neural crest cells. The alphaVbeta3 integrin protein complex was also mainly detected in the migratory cephalic neural crest cells. However, during early mouse embryogenesis and in contrast to the chick, the beta3 integrin gene was expressed in the foregut diverticulum and in the heart and not in the cephalic neural crest cells. Therefore, the difference in the beta3 integrin expression suggests that mouse and chicken cranial neural crest cells may have distinct integrin requirements during their ontogenesis.     

Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells.

We report the cloning and characterization of a novel epidermal growth factor (EGF) domain gene that was identified in a retroviral gene entrapment screen and is expressed in endothelial cells. This gene encodes a protein of 278 amino acids with an amino-terminal signal peptide and two centrally located EGF-like domains. We have named this novel gene in accordance with the guidelines of the Mouse Genome Informatics group Egfl7, for EGF-like domain 7. Egfl7 mRNA is expressed in highly vascularized adult tissues such as the lung, heart, uterus, and ovary. In addition, Egfl7 is expressed early during mouse embryogenesis and in undifferentiated murine embryonic stem cells. The analysis of Egfl7 expression in embryonic day 9.5 embryos by in situ hybridization indicates that Egfl7 is expressed in vascular structures in both the embryo proper and the yolk sac and at sites of mesodermal precursors of angioblasts. Within the cell, EGFL7 protein is localized to the endoplasmic reticulum and Golgi apparatus, suggesting that the protein is targeted for secretion. Indeed, recombinant EGFL7 is readily detectable in the supernatant media of transiently transfected HEK293 cells. We also report the identification of an Egfl7 paralog, Egfl8, and show that EGFL8 protein shares similar domains and molecular weight with EGFL7.     

Lineage specification in neural crest cell pathfinding.

There are two principal models to explain neural crest patterning. One assumes that neural crest cells are multipotent precursors that migrate throughout the embryo and differentiate according to cues present in the local environment. A second proposes that the neural crest is a population of cells that becomes restricted to particular fates early in its existence and migrates along particular pathways dependent on unique cell-autonomous properties. Although it is now evident that the neural crest cell population, as a whole, is actually heterogenous (composed of both multipotent and restricted progenitors), evidence supporting the model of prespecification has increased over the past few years. This review will begin by telling the story of melanoblasts: a neural crest subpopulation that is biased toward a single fate and subsequently acquires intrinsic properties that guide cells of this lineage to their final destination. The remainder of this review will explore whether this model is exclusive to melanoblasts or if it can also be used to explain the patterning of other neural crest cells like those of the sensory, sympathoadrenal, and enteric lineages.     

M17: a novel gene expressed in germinal centers

Germinal centers are histologically distinct structures thatform within the draining lymphoid tissues following immunizationwith T cell-dependent antigens. Here, antigen-specific B cellstransform the lymphoid follicle into a site of intense B cellproliferation, differentiation and selection. To understandthe molecular basis for these cellular events, we sought toisolate germinal center B cell-specific genes using subtractivecDNA libraries derived from FACS-sorted (CD45R/B220⁺, lgD^–,Thy1.2^–) lymph node B cells of immunized mice. A novelgene isolated from this library, designated M17, was found tobe transcribed in spleen and, to a lesser extent, bone marrow.Strikingly, only PNA⁺ (germinal center) but not PNA^– splenicB cells express M17. Germinal center-specific expression ofM17 was confirmed by staining of histoiogical sections of spleenwith an antiserum raised against a glutathione-S-transferase-M17fusion protein. The M17 gene comprises four exons spanning 13.2kb, and encodes a 25 kDa cytoplasmic protein of 159 amlno acids.Analysis of the amino acid sequence revealed the presence ofa possible llpid binding domain and multiple potential phosphorylationsites, including a tyrosine-based activation motif. We speculatethat M17 may be a signaling molecule involved in the transductionof signals from the cell surface to the cytoplasm.     

PX-RICS, a novel splicing variant of RICS, is a main isoform expressed during neural development

In our previous study, we identified RICS, a novel beta-catenin-interacting protein with the GAP activity toward Cdc42 and Rac1, and found that RICS plays an important role in the regulation of neural functions, including postsynaptic NMDA signaling and neurite outgrowth. Here we report the characterization of an N-terminal splicing variant of RICS, termed PX-RICS, which has additional phox homology (PX) and src homology 3 (SH3) domains in its N-terminal region. The PX domain of PX-RICS interacted specifically with phosphatidylinositol 3-phosphate [PtdIns(3)P], PtdIns(4)P and PtdIns(5)P. Consistent with this binding affinity, PX-RICS was found to be localized at the endoplasmic reticulum (ER), Golgi and endosomes. We also found that wild-type PX-RICS possessed much lower GAP activity than RICS, whereas a mutant form of PX-RICS whose PX domain lacks the binding ability to phosphoinositides (PIs) exhibited the GAP activity comparable to that of RICS. However, PX-RICS and RICS exhibited similar inhibitory effects on neurite elongation of Neuro-2a cells. Furthermore, we demonstrate that PX-RICS is a main isoform expressed during neural development. Our results suggest that PX-RICS is involved in early brain development including extension of axons and dendrites, and postnatal remodeling and fine-tuning of neural circuits.     

Generation and characterization of a novel neural crest marker allele,Inka1‐LacZ,reveals a role for Inka1 in mouse neural tube closure

Previous studies identified Inka1 as a gene regulated by AP‐2α in the neural crest required for craniofacial morphogenesis in fish and frog. Here, we extend the analysis of Inka1 function and regulation to the mouse by generating a LacZ knock‐in allele. Inka1‐LacZ allele expression occurs in the cephalic mesenchyme, heart, and paraxial mesoderm prior to E8.5. Subsequently, expression is observed in the migratory neural crest cells and their derivatives. Consistent with expression of Inka1 in tissues of the developing head during neurulation, a low percentage of Inka1^?/? mice show exencephaly while the remainder are viable and fertile. Further studies indicate that AP‐2α is not required for Inka1 expression in the mouse, and suggest that there is no significant genetic interaction between these two factors during embryogenesis. Together, these data demonstrate that while the expression domain of Inka1 is conserved among vertebrates, its function and regulation are not. Developmental Dynamics 239:1188–1196, 2010. © 2010 Wiley‐Liss, Inc.