Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum.

We have raised an antiserum, designated alpha Enhb-1, to a portion of the mouse En-2 protein containing the homeodomain. The antiserum detects both the En-1 and En-2 proteins in mouse, chick and Xenopus embryos by Western blot analysis. Using whole-mount immunohistochemistry, combined in some cases with scanning electron microscopy, we have examined the distribution of the proteins in the early embryos of these species. The major features of expression were similar. The initial production of En protein occurred, just before or during the formation of the first somites, in a band of the anterior neural plate in the prospective mid/hindbrain region. Later in development En-1 protein accumulated in the ventral ectoderm of the developing mouse and chick limb buds, indicating that a dorsal-ventral polarity is present as soon as any limb bud swelling is apparent and that, at least in the mouse, this polarity is established independently of the apical ectodermal ridge. In all three species, alpha Enhb-1 bound to a subset of ventro-lateral differentiating neurons in the spinal cord and hindbrain and their pattern of birth in the mouse reflected the division of the hindbrain into rhombomeres. En-1 protein also accumulated in a lateral stripe of dermatome in the mouse and chick, indicating a dorsal-ventral subdivision of this tissue. The results show that En expression is a good marker for pattern formation in a variety of tissues and will be useful in experimental studies designed to characterize further these processes.     

N-cadherin function is required for differentiation-dependent cytoskeletal reorganization in lens cells in vitro

Members of the cadherin family of cell adhesion molecules participate in calcium-dependent cell-cell adhesions that are necessary for the cell sorting events that regulate early developmental processes. Although individual cadherin molecules have been shown to participate in tissue histogenesis, the regulation of function of these receptors in cell differentiation has been more difficult to identify. We have determined that N-cadherin linkage to the cytoskeleton is correlated with lens cell differentiation in vivo. Through the use of a chick embryo lens culture system that mimics differentiation in vivo, we have determined that N-cadherin linkage to the cytoskeleton is altered and lens differentiation is blocked by function-blocking antibodies to N-cadherin. In the presence of the N-cadherin function-blocking antibody, NCD-2, both N-cadherin and filamentous actin are prevented from organizing at the cortical membranes. This correlates with an inhibition of lens morphogenesis and differentiation. These results are paralleled by changes in the expression of the molecular components of the cadherin-catenin complex and their linkage to the actin cytoskeleton. In the presence of NCD-2, expression of N-cadherin, alpha-catenin, and beta-catenin is inhibited and their association with the cytoskeleton blocked. Overall cadherin expression, however, remains unchanged as demonstrated by studies with a pan-cadherin antibody. This is accompanied by an increase in expression of the cadherin cytoskeletal protein plakoglobin. Although the cells have tried to compensate for the loss of N-cadherin by up-regulation of another cadherin(s) and plakoglobin, this is unable to compensate for N-cadherin function. The data strongly suggest that N-cadherin and its associated cytoskeleton play an important role in the differentiation process that leads to the formation of the crystalline lens.     

Expression of calcium-dependent cell adhesion during ocular development: a biochemical, histochemical and functional analysis

Previous studies of the adhesive properties of embryonic chick neural retina cells indicate a gradual decrease in the expression of calcium-dependent adhesions during retinal histogenesis, a function which has been attributed in part to gp130/4.8, a retinal calcium-dependent adhesion-associated cell surface membrane glycoprotein with a molecular weight of approximately 130 kDa and an isoelectric point of 4.8 (G. B. Grunwald, R. Pratt, and J. Lilien, 1982, J. Cell Sci. 55, 69-83). The experiments described here were done to define the relationship of gp130/4.8 to N-cadherin, another calcium-dependent adhesion molecule found in chick retina, which has a reported molecular weight of 127 kDa and which is recognized by monoclonal antibody NCD-2 (K. Hatta and M. Takeichi, 1986, Nature (London) 320, 447-449). Using two-dimensional gel electrophoresis followed by Western blotting as well as quantitative solid-phase immunoassays, polyspecific antisera recognizing gp130/4.8 were compared with monoclonal antibody NCD-2 for reactivity with proteins of retina and other tissues. The data lead us to conclude that retinal calcium-dependent adhesion proteins gp130/4.8 and N-cadherin are likely to be the same molecule. In order to obtain evidence for a direct correlation of changes in expression of these adhesion proteins with changes in retinal cell adhesivity and related morphogenetic events, parallel studies were carried out with cells from various ocular tissues to examine the functional, biochemical, and immunohistochemical expression of N-cadherin during ocular development. Immunohistochemical mapping of N-cadherin in the developing chick eye reveals three modes of N-cadherin expression which occur simultaneously in different ocular tissues: (1) down-regulation, (2) up-regulation, and (3) steady-state expression. These patterns of expression correlate with changes in the adhesive behavior of cells as well as with discrete stages in the morphogenesis of several ocular tissues. The results suggest that N-cadherin is a versatile cell adhesion protein with a role in both the development of several ocular tissues and the maintenance of specialized structures in the mature eye.     

Design and application of a polyclonal peptide antiserum for the universal detection of leptin protein

An epitope-specific polyclonal antiserum was produced in rabbits immunized against a synthetic 15 amino acid peptide (QRVTGLDFIPGLHPV) derived from the coding sequence reported for the porcine leptin gene (GenBank Accession No. U59894). This peptide contains a core sequence comprised of eight amino acids (GLDFIPGL) that is totally conserved in all leptin proteins studied to date. Purified recombinant human, mouse, rat, pig, and chicken leptin proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotted onto PVDF membranes. Western blots were developed employing the leptin-specific peptide antiserum with an alkaline-phosphatase-conjugated anti-rabbit IgG second antibody chromogenic system. The peptide antiserum was found to be highly specific for leptin which exhibited an estimated molecular weight of about 16 kDa for all species analyzed. The sensitivity of the Western blot assay was not sufficient to permit the direct detection of leptin in chicken serum or plasma. However, with this assay we were able to detect native leptin protein in an enriched fraction prepared from chicken plasma using a combination of gel filtration and ion exchange column chromatography. Slot blots indicated a potential application of the immunostaining technique for quantitative analysis of leptin protein. Finally, the peptide antiserum was successfully employed to localize leptin protein by immunohistochemical staining of thin sections prepared from adipose (chicken and pig) and liver (chicken) tissue samples. This study is the first to report a polyclonal peptide antiserum that apparently recognizes intact leptin protein, both native and recombinant, regardless of the species of origin.     

N-Cadherin Gene Maps to Human Chromosome 18 and Is Not Linked to the E-Cadherin Gene

cDNA clones encoding the human N-cadherin cell adhesion molecule have been isolated from an embryonic muscle library by screening with an oligonucleotide probe complementary to the chick brain sequence and chick brain cDNA probe lambda N2. Comparison of the predicted protein sequences revealed greater than 91% homology between chick brain, mouse brain, and human muscle N-cadherin cDNAs over the 748 amino acids of the mature, processed protein. A single polyadenylation site in the chick clone was also present and duplicated in the human muscle sequence. Immediately 3' of the recognition site in chick a poly(A) tail ensued; however, in human an additional 800 bp of 3' untranslated sequence followed. Northern analysis identified a number of major N-cadherin mRNAs. These were of 5.2, 4.3, and 4.0 kb in C6 glioma, 4.3 and 4.0 kb in human foetal muscle cultures, and 4.3 kb in human embryonic brain and mouse brain with minor bands of 5.2 kb in human muscle and embryonic brain. Southern analysis of a panel of somatic cell hybrids allowed the human N-cadherin gene to be mapped to chromosome 18. This is distinct from the E-cadherin locus on chromosome 16. Therefore, it is likely that the cadherins have evolved from a common precursor gene that has undergone duplication and migration to other chromosomal locations.     

Specific immunolabeling of brain macrophages and microglial cells in the developing and mature chick central nervous system.

The present study showed that the HIS-C7 monoclonal antibody, which recognizes the chick form of CD45, is a specific marker for macrophages/microglial cells in the developing and mature chick central nervous system (CNS). HIS-C7-positive cells were characterized according to their morphological features and chronotopographical distribution patterns within developing and adult CNS, similar to those of macrophages/microglial cells in the quail CNS and confirmed by their histochemical labeling with Ricinus communis agglutinin I, a lectin that recognizes chick microglial cells. Therefore, the HIS-C7 antibody is a valuable tool to identify brain macrophage and microglial cells in studies of the function, development, and pathology of the chick brain. CD45 expression differed between chick microglia (as revealed with HIS-C7 antibody) and mouse microglial cells (as revealed with an antibody against mouse form of CD45). Thus, a discontinuous label was seen on mouse microglial cells with the anti-mouse CD45 immunostaining, whereas the entire surface of chick microglial cells was labeled with the anti-chick CD45 staining. The functional relevance of these differences between species has yet to be determined.     

Immunologically unique and common domains within a family of proteins related to the retina Ca2+-dependent cell adhesion molecule, NcalCAM

Rabbit polyclonal antibodies raised to gp90, a fragment of the embryonic chick neural retina Ca2+-dependent adhesive molecule, gp130, recognize gp130 and inhibit Ca2+-dependent cell-cell adhesion. When tested against a panel of 10-day embryonic tissues, one of these antisera recognizes a component with a molecular weight identical to that of gp130 in embryonic chick cerebrum, optic lobe, hind brain, spinal cord and neural retina only; the second antiserum recognizes a similar component in all of the embryonic chick tissues tested. These data imply the existence of an extended family of closely related cell surface components with immunologically distinct subgroups each of which may mediate Ca2+-dependent cell-cell adhesion. As the term CAM, or cell adhesion molecule, has become common usage we propose to refer to these molecules as calCAMs, reflecting their calcium dependence. Analysis of fragments and endoglycosidase digests of NcalCAM have allowed a comparison of its structure with similar molecules from different tissues and species that have been implicated in Ca2+-dependent cell-cell adhesion.     

Characterisation of the genomic structure of chick Fgf8.

The Fgf8 gene encodes a series of secreted signalling molecules important in the normal development of the face, brain and limbs. The genomic structure of the chick Fgf8 gene has been analysed and compared to the human and mouse sequences. Divergence between the chick, human and mouse genomic structure was observed. Data indicates that the long alternatively spliced form of exon 1b observed in mouse and exon 1c observed in human and mouse do not exist in the chick Fgf8 gene. RT-PCR analysis indicates that chick Fgf8, like its mouse and human counterpart is alternatively spliced. This data along with the genomic structure data indicates that in the chick there are only two isoforms of Fgf8. This is in contrast to the human and mouse, where evidence suggests that there are 4 and 8 isoforms, respectively. Approximately 400 bp of intron 1d is highly conserved between chick, human and mouse genomic sequences. Using TRANSFAC possible conserved regulatory element binding sites within this domain were identified.     

Cloning and expression of hbves, a novel and highly conserved mRNA expressed in the developing and adult heart and skeletal muscle in the human

bves is a novel mRNA expressed in the developing heart in chick and mouse. Here we describe hbves, the human homolog of chick and mouse bves. Northern and dot blot analyses reveal restricted expression in the heart and skeletal muscle in the embryo and adult. BLAST searches of the NCBI databases confirm that hbves is novel. Portions of the sequence are an exact match with genomic PAC 52202, which localizes to Chromosome (Chr) 6q21. Presumably, these matches and intervening sequences match the intron-exon borders of the gene. Computer conformation analysis of the derived amino acid predicts three transmembrane helices with an extracellular C-terminus that is conserved in chick, mouse, and human. bves is highly conserved among all three species at the amino acid level with 75% identity and 92% similarity. Received: 15 February 1999 / Accepted: 29 April 1999     

Conservation and diversity in the cis-regulatory networks that integrate information controlling expression of Hoxa2 in hindbrain and cranial neural crest cells in vertebrates

The Hoxa2 and Hoxb2 genes are members of paralogy group II and display segmental patterns of expression in the developing vertebrate hindbrain and cranial neural crest cells. Functional analyses have demonstrated that these genes play critical roles in regulating morphogenetic pathways that direct the regional identity and anteroposterior character of hindbrain rhombomeres and neural crest-derived structures. Transgenic regulatory studies have also begun to characterize enhancers and cis-elements for those mouse and chicken genes that direct restricted patterns of expression in the hindbrain and neural crest. In light of the conserved role of Hoxa2 in neural crest patterning in vertebrates and the similarities between paralogs, it is important to understand the extent to which common regulatory networks and elements have been preserved between species and between paralogs. To investigate this problem, we have cloned and sequenced the intergenic region between Hoxa2 and Hoxa3 in the chick HoxA complex and used it for making comparative analyses with the respective human, mouse, and horn shark regions. We have also used transgenic assays in mouse and chick embryos to test the functional activity of Hoxa2 enhancers in heterologous species. Our analysis reveals that three of the critical individual components of the Hoxa2 enhancer region from mouse necessary for hindbrain expression (Krox20, BoxA, and TCT motifs) have been partially conserved. However, their number and organization are highly varied for the same gene in different species and between paralogs within a species. Other essential mouse elements appear to have diverged or are absent in chick and shark. We find the mouse r3/r5 enhancer fails to work in chick embryos and the chick enhancer works poorly in mice. This implies that new motifs have been recruited or utilized to mediate restricted activity of the enhancer in other species. With respect to neural crest regulation, cis-components are embedded among the hindbrain control elements and are highly diverged between species. Hence, there has been no widespread conservation of sequence identity over the entire enhancer domain from shark to humans, despite the common function of these genes in head patterning. This provides insight into how apparently equivalent regulatory regions from the same gene in different species have evolved different components to potentiate their activity in combination with a selection of core components.     

Antibodies to the cytoplasmic domain of the MUC1 mucin show conservation throughout mammals.

An antiserum against the carboxy-terminal seventeen amino acids of the human MUC1 mucin has been raised and extensively characterized. This antiserum, CT1, immunoprecipitates two high molecular weight polymorphic bands (greater than 200 kDa) from a metabolically labelled breast cancer cell line corresponding to the two alleles which have previously been shown to contain different numbers of a twenty amino acid repeat. The CT1 antiserum reacted with tissues from many mammalian species and immunoprecipitated large polymorphic proteins, suggesting that the cytoplasmic portion of the molecule is well conserved. The cell and tissue distribution of Muc-1 mucin in the mouse has been studied by immunocytochemistry. This protein is abundant at the apical surfaces of epithelial tissues and is found expressed in the stomach, kidney, mammary gland, pancreas, salivary gland, lung, trachea, uterus, cervix and vagina.     

A cadherin-like protein in eggs and cleaving embryos of Xenopus laevis is expressed in oocytes in response to progesterone

A new cadherin-like protein (CLP) was identified in oocytes, eggs, and cleavage stage embryos of Xenopus laevis. As a probe for detecting new cadherin proteins, an antiserum was raised to a 17 amino acid peptide derived from a highly conserved region in the cytoplasmic domain of all cadherins which have been sequenced to date. This antipeptide antibody recognized Xenopus E-cadherin and a polypeptide in Xenopus brain extracts similar to N-cadherin, which were independently identified by specific mAbs. In extracts of eggs and midblastula stage embryos the antipeptide antibody recognized specifically a 120-kD glycoprotein that migrated faster on SDS gels than the 140-kD E- and N-cadherin polypeptides. This 120-kD polypeptide was not recognized by the mAbs specific to E- and N-cadherin. In fact, E- and N-cadherin were not detectable in eggs or midblastula stage embryos. The possible relationship of CLP to P-cadherin, which has been identified in mouse tissues, has not yet been determined. CLP was synthesized by large, late stage oocytes. When oocytes were induced to mature in vitro with progesterone it accumulated to the same level found in normally laid eggs. It did not accumulate further to any significant extent during the early cleavage stages. CLP was detected on the surface of stage 8 blastomeres by cell surface biotinylation, but only after the tight junctions of the blastula epithelium were opened by removal of Ca2+. We conclude that CLP is a maternally encoded protein that is the major, if not only, cadherin-related protein present in the earliest stages of Xenopus development, and we propose that it may play a role in the Ca2(+)-dependent adhesion and junction formation between cleavage stage blastomeres.     

Cloning and expression analysis of Fgf5, 6 and 7 during early chick development

FGFs with similar sequences can play different roles depending on the model organisms examined. Determining these roles requires knowledge of spatio-temporal Fgf gene expression patterns. In this study, we report the cloning of chick Fgf5, 6 and 7, and examine their gene expression patterns by whole mount in situ hybridization. We show that Fgf5's spatio-temporally restricted expression pattern indicates a potentially novel role during inner ear development. Fgf6 and Fgf7, although belonging to different subfamilies with diverged sequences, are expressed in similar patterns within the mesoderm. Alignment of protein sequences and phylogenetic analysis demonstrate that FGF5 and FGF6 are highly conserved between chick, human, mouse and zebrafish. FGF7 is similarly conserved except for the zebrafish, which has considerably diverged.     

Molecular structure and functional testing of human L1CAM: an interspecies comparison.

The rodent, avian, and insect L1-like cell adhesion molecules are members of the immunoglobulin superfamily that have been implicated in axon growth. We have isolated an L1-like molecule from human brain and found that it also supports neurite growth in vitro. We have also cloned and sequenced the entire coding region of human L1CAM and found that it shows a very high degree of homology to mouse L1cam, with 92% identity at the amino acid level. This similarity suggests that L1CAM is an important molecule in normal human nervous system development and nerve regeneration. Overall, there is substantially less homology to chick Ng-CAM; they are 40% identical at the amino acid level but many regions are highly conserved. Comparison of the sequences from human, mouse, chick, and Drosophila indicates that the L1 immunoglobulin domain 2 and fibronectin type III domain 2 are strongly conserved and thus are likely functionally important.     

Conservation of molecular structure of DNA polymerase beta in vertebrates probed by tryptic peptide mapping

DNA polymerase beta's from mouse myeloma, chick embryo, and cherry salmon testis were all composed of a single polypeptide of about 40K daltons as judged by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified enzyme preparations. Although the enzyme from bullfrog ovary was not fully purified, its molecular weight was estimated to be the same as that of the chick enzyme by immunological detection after electrophoresis. All the enzymes tested cross-reacted immunologically with the antibody against chick DNA polymerase beta, indicating that they have a common molecular structure, at least in part. Two-dimensional maps of radioiodinated tryptic peptides directly showed the presence of highly conserved amino acid sequences among mouse, chick, and cherry salmon enzymes. This conserved structure is thought to be essential for the enzyme activity, which is very similar among all these vertebrates.     

A cross-reactive idiotype on anti-collagen antibodies in collagen-induced arthritis: identification and relevance to disease

Immunization of mice with type II collagen (CII) leads to the production of anti-CII antibodies and, in susceptible strains, to the induction of arthritis. Specifically purified anti-CII antibodies from arthritic DBA/1 mice were used to prepare a rabbit anti-idiotypic antiserum. This antiserum recognizes a cross-reactive idiotype (CRI) present on 20-25% of anti-CII antibodies from DBA/1 mice immunized with bovine CII. The CRI is not present on DBA/1 anti-trinitrophenyl, undetectable in normal Ig and not Igh allotype linked. The presence of this CRI was examined after antigen specific suppression of the anti-CII antibody response by intravenous administration of chick or bovine CII. While intravenous injection of bovine CII, prior to immunization with chick CII, greatly reduces both the incidence of arthritis and the anti-CII response, the fraction of anti-bovine CII which expresses the CRI is increased by this treatment. These findings suggest that the CRI characterizes a disease-unrelated fraction of anti-CII which recognizes bovine and chick CII, but probably not mouse CII. In addition, attempts at idiotypic regulation of arthritis incidence and antibody response by in vivo administration of anti-idiotypic serum also indicate that the CRI-bearing antibody is not important for the induction of arthritis.