Alternatively spliced variants of protocadherin 8 exhibit distinct patterns of expression during mouse development

Protocadherins, a subgroup of the cadherin superfamily of calcium-dependent cell adhesion molecules, are considered to play important roles in the developing embryo particularly in the central nervous system. The Protocadherin 8 (Pcdh8) gene comprises three coding exons in both human and mouse, and the exon junctions are precisely conserved between these two species. Alternative splicing of Pcdh8 RNA leads to the formation of two isoforms that differ in the length of the cytoplasmic domains. We have investigated the expression of these short and long variants of Pcdh8 during early mouse development by RT/PCR and in situ hybridization. We found that both isoforms were predominantly expressed in the nervous system, and that their expression patterns appeared to be developmentally regulated. However, the short variant had a broader pattern of expression than the long variant and was found in some non-neuronal tissues, such as paraxial mesoderm, developing somites, and in limb interdigital mesenchyme where massive programmed cell death occurs. The differential expression of two alternative cytoplasmic domain variants suggests that Pcdh8 may regulate cell adhesion in a variety of developmental processes, and that this may involve different intracellular interactions.     

Protocadherin-18a has a role in cell adhesion, behavior and migration in zebrafish development

Protocadherin-18a (Pcdh18a) belongs to the δ2-protocadherins, which constitute the largest subgroup within the cadherin superfamily. Here we present isolation of a full-length zebrafish cDNA that encodes a protein highly similar to human and mouse Pcdh18. Zebrafish pcdh18a is expressed in a complex and dynamic pattern in the nervous system from gastrula stages onward, with lesser expression in mesodermal derivatives. Pcdh18a-eGFP fusion protein is expressed in a punctate manner on the membranes between cells. Overexpression of pcdh18a in embryos caused cyclopia, mislocalization of hatching gland tissue, and duplication or splitting of the neural tube. Most neural markers tested were expressed in an approximately correct A-P pattern. By cell transplantation we showed that overexpression of pcdh18a causes diminished cell migration and reduced cell protrusions, resulting in a tendency of cells to stay more firmly aggregated, probably due to increased cell adhesion. In contrast, knockdown of pcdh18a by a morpholino oligonucleotide caused defects in epiboly, and led to reduced cell adhesion as shown by cell dissociation, sorting and transplantation experiments. These results suggest a role for Pcdh18a in cell adhesion, migration and behavior but not cell specification during gastrula and segmentation stages of development.     

Expression of multiple delta-protocadherins during feather bud formation

The expression of the chicken delta-protocadherin (Pcdh) subfamily was investigated in the developing feather buds of the chicken. The expression profiles of the eight investigated Pcdhs in the cells and tissues of the feather buds differ from each other. Pcdh1, Pcdh7, Pcdh8 and Pcdh10 are differentially expressed in the epidermis of the feather bud. Expression of Pcdh1 and Pcdh10 is restricted to the periderm and Pcdh17 expression to the epidermis of interbud region. Pcdh19 is mostly expressed at the anterior side of epidermis as well as in the blood vessels of the feather buds. Furthermore, Pcdh9 and Pcdh18 both are regionally expressed in the dermis of the feather bud. These results suggest that Pcdhs may play a variety of roles during avian feather bud formation.     

Developmentally regulated expression of the mouse homologues of the potassium channel encoding genes m-erg1, m-erg2 and m-erg3

Deciphering the expression pattern of K+ channel encoding genes during development can help in the understanding of the establishment of cellular excitability and unravel the molecular mechanisms of neuromuscular diseases. We focused our attention on genes belonging to the erg family, which is deeply involved in the control of neuromuscular excitability in Drosophila flies and possibly other organisms. Both in situ hybridisation and RNase Protection Assay experiments were used to study the expression pattern of mouse (m)erg1, m-erg2 and m-erg3 genes during mouse embryo development, to allow the pattern to be compared with their expression in the adult. M-erg1 is first expressed in the heart and in the central nervous system (CNS) of embryonic day 9.5 (E9.5) embryos; the gene appears in ganglia of the peripheral nervous system (PNS) (dorsal root (DRG) and sympathetic (SCG) ganglia, mioenteric plexus), in the neural layer of retina, skeletal muscles, gonads and gut at E13.5. In the adult m-erg1 is expressed in the heart, various structures of the CNS, DRG and retina. M-erg2 is first expressed at E9.5 in the CNS, thereafter (E13.5) in the neural layer of retina, DRG, SCG, and in the atrium. In the adult the gene is present in some restricted areas of the CNS, retina and DRG. M-erg3 displayed an expression pattern partially overlapping that of m-erg1, with a transitory expression in the developing heart as well. A detailed study of the mouse adult brain showed a peculiar expression pattern of the three genes, sometimes overlapping in different encephalic areas.     

Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation

The protocadherins comprise the largest subgroup within the cadherin superfamily, yet their cellular and developmental functions are not well understood. In this study, we demonstrate that pcdh 19 (protocadherin 19) acts synergistically with n-cadherin (ncad) during anterior neurulation in zebrafish. In addition, Pcdh 19 and Ncad interact directly, forming a protein-protein complex both in vitro and in vivo. Although both molecules are required for calcium-dependent adhesion in a zebrafish cell line, the extracellular domain of Pcdh 19 does not exhibit adhesive activity, suggesting that the involvement of Pcdh 19 in cell adhesion is indirect. Quantitative analysis of in vivo two-photon time-lapse image sequences reveals that loss of either pcdh 19 or ncad impairs cell movements during neurulation, disrupting both the directedness of cell movements and the coherence of movements among neighboring cells. Our results suggest that Pcdh 19 and Ncad function together to regulate cell adhesion and to mediate morphogenetic movements during brain development.     

Chicken protocadherin-1 functions to localize neural crest cells to the dorsal root ganglia during PNS formation

In vertebrate embryos, neural crest cells emerge from the dorsal neural tube and migrate along well defined pathways to form a wide diversity of tissues, including the majority of the peripheral nervous system (PNS). Members of the cadherin family of cell adhesion molecules play key roles during the initiation of migration, mediating the delamination of cells from the neural tube. However, a role for cadherins in the sorting and re-aggregation of the neural crest to form the PNS has not been established. We report the requirement for a protocadherin, chicken protocadherin-1 (Pcdh1), in neural crest cell sorting during the formation of the dorsal root ganglia (DRG). In embryos, cPcdh1 is highly expressed in the developing DRG, where it co-localizes with the undifferentiated and mitotically active cells along the perimeter. Pcdh1 can promote cell adhesion in vivo and disrupting Pcdh1 function in embryos results in fewer neural crest cells localizing to the DRG, with a concomitant increase in cells that migrate to the sympathetic ganglia. Furthermore, those cells that still localize to the DRG, when Pcdh1 is inhibited, are no longer found at the perimeter, but are instead dispersed throughout the DRG and are now more likely to differentiate along the sensory neuron pathway. These results demonstrate that Pcdh1-mediated cell adhesion plays an important role as neural crest cells coalesce to form the DRG, where it serves to sort cells to the mitotically active perimeter.     

Sox9 neural crest determinant gene controls patterning and closure of the posterior frontal cranial suture

Cranial suture development involves a complex interaction of genes and tissues derived from neural crest cells (NCC) and paraxial mesoderm. In mice, the posterior frontal (PF) suture closes during the first month of life while other sutures remain patent throughout the life of the animal. Given the unique NCC origin of PF suture complex (analogous to metopic suture in humans), we performed quantitative real-time PCR and immunohistochemistry to study the expression pattern of the NCC determinant gene Sox9 and select markers of extracellular matrix. Our results indicated a unique up-regulated expression of Sox9, a regulator of chondrogenesis, during initiation of PF suture closure, along with the expression of specific cartilage markers (Type II Collagen and Type X Collagen), as well as cartilage tissue formation in the PF suture. This process was followed by expression of bone markers (Type I Collagen and Osteocalcin), suggesting endochondral ossification. Moreover, we studied the effect of haploinsufficiency of the NCC determinant gene Sox9 in the NCC derived PF suture complex. A decrease in dosage of Sox9 by haploinsufficiency in NCC-derived tissues resulted in delayed PF suture closure. These results demonstrate a unique development of the PF suture complex and the role of Sox9 as an important contributor to timely and proper closure of the PF suture through endochondral ossification.     

Altered gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA) placentae

Imprinted genes are known to be crucial for placental development and fetal growth in mammals, but no primary epigenetic abnormality in placenta has been documented to compromise human fetal growth. Imprinted genes demonstrate parent-of-origin-specific allelic expression that is epigenetically regulated i.e. extrinsic to the primary DNA sequence. To undertake an epigenetic analysis of poor fetal growth in placentae and cord blood tissues, we first established the tissue-specific patterns of methylation and imprinted gene expression for two imprinting clusters (KvDMR and H19 DMR) on chromosome 11p15 in placentae and neonatal blood for 20 control cases and 24 Small for Gestational Age (SGA) cases. We confirmed that, in normal human placenta, the H19 promoter is unmethylated. In contrast, most other human tissues show paternal methylation. In addition, we showed that the IGF2 DMR2, also paternally methylated in most human tissues, exhibits hypomethylation in placentae. However, in neonatal blood DNA, these two regions maintain the differential methylation status seen in most other tissues. Significantly, we have been able to demonstrate that placenta does maintain differential methylation at the imprinting control regions H19 DMR and KvDMR. Of note, in one SGA placenta, we found a methylation alteration at the H19 DMR and concomitant biallelic expression of the H19 gene, suggesting that loss of imprinting at H19 is one cause of poor fetal growth in humans. Of particular interest, we demonstrated also a decrease in IGF2 mRNA levels in all SGA placentae and showed that the decrease is, in most cases, independent of H19 regulation.     

A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion

During embryonic morphogenesis, adhesion molecules are required for selective cell-cell interactions. The classical cadherins mediate homophilic calcium-dependent cell adhesion and are founding members of the large and diverse cadherin superfamily. The protocadherins are the largest subgroup within this superfamily, yet their participation in calcium-dependent cell adhesion is uncertain. In this paper, we demonstrate a novel mechanism of adhesion, mediated by a complex of Protocadherin-19 (Pcdh19) and N-cadherin (Ncad). Although Pcdh19 alone is only weakly adhesive, the Pcdh19-Ncad complex exhibited robust adhesion in bead aggregation assays, and Pcdh19 appeared to play the dominant role. Adhesion by the Pcdh19-Ncad complex was unaffected by mutations that disrupt Ncad homophilic binding but was inhibited by a mutation in Pcdh19. In addition, the complex exhibited homophilic specificity, as beads coated with Pcdh19-Ncad did not intermix with Ncad- or Pcdh17-Ncad-coated beads. We propose a model in which association of a protocadherin with Ncad acts as a switch, converting between distinct binding specificities.     

Expression of Dll4 during mouse embryogenesis suggests multiple developmental roles

Delta-Notch signalling regulates cell-fate decisions in a variety of tissues in diverse organisms, through cell-to-cell interactions. Here, we report the expression pattern of a Delta gene family member, Delta-like 4 (Dll4). Dll4 expression was analyzed in mouse embryos and selected adult organs by monitoring beta-galactosidase (beta-gal) expression from a lacZ reporter cassette inserted downstream of the Dll4 promoter, which allowed for high sensitivity and single cell resolution. Expression was detected in several tissues where Notch signalling is known to control cell-fate decisions, like the vascular system, the nervous system, the gastrointestinal system, and the thymus. Throughout embryonic cardiovascular development, Dll4 expression was seen only on endocardial cells and endothelial cells of the arteries, arterioles, and capillaries, being absent from vascular smooth muscle cells and veins. In the nervous system, expression was detected in the brain, neural tube, retina, and, for the first time, in the olfactory epithelium, vomeronasal organs and para-aortic bodies. Extensive Dll4 expression was also observed in the gut. This detailed expression analysis reveals new clues for both endothelial and non-endothelial Dll4 function in different organs.     

Ezrin gene, coding for a membrane-cytoskeleton linker protein, is regionally expressed in the developing mouse neuroepithelium

Ezrin is a member of the Ezrin, Radixin, Moesin (ERM) proteins family that are proposed to act as linkers between the cytoskeleton and plasma membrane. Ezrin regulates cell-cell and cell-matrix interactions playing a role in the regulation of cellular adhesion, movement and morphology in epithelia. Alterations in the expression of Ezrin and other members of ERM family have also been observed in brain tumours. Here we report the expression pattern of Ezrin during mouse neural development, from early stages to postnatal stages. In young and middle gestation embryos, Ezrin is expressed in the roof plate of the neural tube, in the presumptive domain of the choroidal plexus, and in some precise domains of ventricular epithelium. These domains are distributed in basal and alar neuroepithelial regions, some of them in relation to the expression of cadherins. At later gestation and postnatal stages, Ezrin expression is maintained on the mature choroidal plexus and is weakly detected in the proliferative regions of the mature brain.     

Inhibition of protocadherin-alpha function results in neuronal death in the developing zebrafish

The pcdhα/CNR gene comprises a diverse array of neuronal cell-surface proteins of the cadherin superfamily, although very little is known about their role in neural development. Here we provide the first in-depth characterization of pcdh1α in zebrafish. Whole-mount immunocytochemistry demonstrates that a large proportion of endogenous cytoplasmic domain immunoreactivity is present in the nucleus, suggesting that endoproteolytic cleavage and nuclear translocation of the intracellular domain are important aspects of pcdh1α activity in vivo. Using whole-mount immunocytochemistry and BAC-based expression of Pcdh1α-GFP fusion proteins, we find that Pcdh1α does not appear to form stable, synaptic puncta at early stages of synaptogenesis. We also demonstrate that the presence of the Pcdh1α cytoplasmic domain is essential for normal function. Truncation of Pcdh1α proteins, using splice-blocking antisense morpholinos to prevent the addition of the common intracellular domain to the entire pcdh1α cluster, results in neuronal apoptosis throughout the developing brain and spinal cord, demonstrating an essential role for pcdh1α in early neural development. This cell death phenotype can be attenuated by the expression of a soluble Pcdh1α cytoplasmic domain.     

Identification of novel oocyte and granulosa cell markers

Here we present novel gene expression patterns in the ovary as part of an ongoing assessment of published micro-array data from mouse oocytes and embryos. We present the expression patterns of 13 genes that had been determined by micro-array to be expressed in the mature egg, but not during subsequent preimplantation development. In-situ hybridization of sectioned ovaries revealed that these genes were expressed in one of two distinct patterns: (1) oocyte-specific or (2) expressed in both the oocyte and surrounding granulosa cells. Despite the fact that micro-array data demonstrated expression in the egg, several of these genes are expressed at low levels in the oocyte, but strongly expressed in granulosa cells. Eleven of these genes have no reported function or expression during oogenesis, indicating that this approach is a necessary step towards functional annotation of the genome. Also of note is that while some of these gene products have been well characterized in other tissues and cell types, others are relatively unstudied in the literature. Our results provide novel gene expression information that may provide insights into the molecular mechanisms of follicular recruitment, oocyte maturation and ovulation and will direct further experimentation into the role these genes play during oogenesis.     

Grainyhead-like 2 regulates neural tube closure and adhesion molecule expression during neural fold fusion

Defects in closure of embryonic tissues such as the neural tube, body wall, face and eye lead to severe birth defects. Cell adhesion is hypothesized to contribute to closure of the neural tube and body wall; however, potential molecular regulators of this process have not been identified. Here we identify an ENU-induced mutation in mice that reveals a molecular pathway of embryonic closure. Line2F homozygous mutant embryos fail to close the neural tube, body wall, face, and optic fissure, and they also display defects in lung and heart development. Using a new technology of genomic sequence capture and high-throughput sequencing of a 2.5 Mb region of the mouse genome, we discovered a mutation in the grainyhead-like 2 gene (Grhl2). Microarray analysis revealed Grhl2 affects the expression of a battery of genes involved in cell adhesion and E-cadherin protein is drastically reduced in tissues that require Grhl2 function. The tissue closure defects in Grhl2 mutants are similar to that of AP-2α null mutants and AP-2α has been shown to bind to the promoter of E-cadherin. Therefore, we tested for a possible interaction between these genes. However, we find that Grhl2 and AP-2α do not regulate each other's expression, E-cadherin expression is normal in AP-2α mutants during neural tube closure, and Grhl2;AP-2α trans-heterozygous embryos are morphologically normal. Taken together, our studies point to a complex regulation of neural tube fusion and highlight the importance of comparisons between these two models to understand more fully the molecular pathways of embryonic tissue closure.     

Teashirt 3 expression in the chick embryo reveals a remarkable association with tendon development

Drosophila teashirt (tsh) is involved in the patterning of the trunk identity together with the Hox genes. In addition, it is also a player in the Wingless and the Hedgehog pathways. In birds and mammals, three Tshz genes are identified and the expression patterns for mouse Tshz1 and Tshz2 have been reported during embryogenesis. Recently, we showed that all three mouse Tshz genes can rescue the Drosophila tsh loss-of-function phenotype, indicating that the function of the teashirt genes has been conserved during evolution. Here we describe the expression pattern of chick TSHZ3 during embryogenesis. Chick TSHZ3 is expressed in several tissues including mesodermal derivatives, the central and peripheral nervous systems. Emphasis is laid on the dynamic expression occurring in regions of the somites and limbs where tendons develop. We show that TSHZ3 is activated in the somites by FGF8, a known inducer of the tendon marker SCX.