Loss of late primitive streak mesoderm and interruption of left-right morphogenesis in the Ednrb(s-1Acrg) mutant mouse

This study characterizes defects associated with abnormal mesoderm development in mouse embryos homozygous for the induced Ednrb(s-1Acrg) allele of the piebald deletion complex. The Ednrb(s-1Acrg) deletion results in recessive embryonic lethality and mutant embryos exhibit a truncated posterior body axis. The primitive streak and node become disfigured, consistent with evidence that cell migration is impaired in newly formed mesoderm. Additional defects related to mesoderm development include notochord degeneration, somite malformations, and abnormal vascular development. Arrested heart looping morphogenesis and a randomized direction of embryonic turning indicate that left-right development is also perturbed. The expression of nodal and leftb, Tgf-beta-related genes involved in a left-determinant signaling pathway, is variably lost in the left lateral plate mesoderm. Mutational analysis has demonstrated that Fgf8 and Brachyury (T) are required for normal mesoderm and left-right development and the asymmetric expression of nodal and leftb. Fgf8 expression in nascent mesoderm exiting the primitive streak is dramatically reduced in mutant embryos, and diminished T expression accompanies the progressive loss of paraxial, lateral, and primitive streak mesoderm. In contrast, axial mesoderm persists and T and nodal appear to be appropriately expressed in their specific domains in the node and notochord. We propose that this mutation disrupts a morphogenetic pathway, likely involving FGF signaling, important for the development of streak-derived posterior mesoderm and lateral morphogenesis.     

Molecular characterization of a deletion encompassing the splotch mutation on mouse chromosome 1

We have used a set of markers newly assigned to the proximal portion of mouse chromosome 1 to characterize the chromosomal segment deleted in the splotch-retarded (Spr) mouse mutant. Among nine markers tested in the heterozygote Spr/+mouse, we have identified four genes, Vil, Des, Inha, and Akp-3, which map within the Spr deletion. The closest distal marker to the deletion is the Acrg gene, with the distal deletion breakpoint mapping within the 0.8-cM segment separating Akp-3 and Acrg. The most proximal gene to the Spr deletion is Tp1. The proximal deletion breakpoint maps within the 0.8-cM segment separating Tp1 and Vil. The minimum size of the Spr deletion would therefore be limited to 14 cM, the genetic distance between Vil and Akp-3. The maximum size of the Spr deletion is estimated to be 16 cM, the genetic distance between Tp1 and Acrg.     

Two gene families clustered in a small region of the Drosophila genome

Three Drosophila genes that are clustered within 8 X 10(3) bases of DNA at the chromosomal region 44D have been identified and mapped, and the gene cluster entirely sequenced. The three genes are 55 to 60% homologous in DNA sequence. One gene contains an intron in its 5'-proximal protein coding sequence while the other two have none at this position; similarly, another gene has an intron in its 3'-proximal protein coding sequence which is not found in the other genes. All three genes are abundantly expressed together in Drosophila first, second, and early third instar larval stages and in adults, but they are not abundantly expressed in either embryonic, late third instar larval, or pupal stages. This gene family lies 11 X 10(3) bases away from another cluster containing four Drosophila larval cuticle protein genes plus a pseudogene. The cuticle genes are all abundantly expressed throughout third instar larval development. Thus, at least seven protein-coding genes and one pseudogene lie within 27 X 10(3) bases of DNA. Moreover, two small gene families can lie adjacent on a chromosome and exhibit different patterns of developmental regulation, even though individual genes within each clustered family are co-ordinately expressed.     

Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32----qter

The nicotinic acetylcholine receptor of skeletal muscle (CHRN in man, Acr in mouse) is a transmembrane protein composed of four different subunits (alpha, beta, gamma, and delta) assembled into the pentamer alpha 2 beta gamma delta. These subunits are encoded by separate genes which derive from a common ancestral gene by duplication. We have used a murine full-length 1,900-bp-long cDNA encoding the gamma subunit subcloned into M 13 (clone gamma 18) to prepare single-stranded probes for hybridization to EcoRI-digested DNA from a panel of human x rodent somatic cell hybrids. Using conditions of low stringency to favor cross-species hybridization, and prehybridization with rodent DNA to prevent rodent background, we detected a single major human band of 30-40 kb. The pattern of segregation of this 30-40 kb band correlated with the segregation of human chromosome 2 within the panel and the presence of a chromosomal translocation in the distal part of the long arm of this t(X;2)(p22;q32.1) chromosome allowing the localization of the gamma subunit gene (CHRNG) to 2q32----qter. The human genes encoding the gamma and delta subunits have been shown to be contained in an EcoRI restriction fragment of approximately 20 kb (Shibahara et al., 1985). Consequently, this study also maps the delta subunit gene (CHRND) to human chromosome 2q32.1----qter. In the mouse, the Acrd and Acrg genes have been shown to be linked to Idh-1, Mylf (IDH1 and MYL1 in humans, respectively) and to the gene encoding villin on chromosome 1. Interestingly, we have recently localized the human MYL1 gene to the same chromosomal fragment of human chromosome 2. These results clearly demonstrate a region of chromosomal homoeology between mouse chromosome 1 and human chromosome 2.     

Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo.

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5-7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.     

Identification and gene organization of three novel members of the IL-1 family on human chromosome 2

Members of the IL-1 family of cytokines are important in mediating inflammatory responses. The genes encoding IL-1alpha, IL-beta, and the IL-1 receptor antagonist (IL-1Ra) are clustered within 450 kb on human chromosome 2q. By searching the EST databases and sequencing this region of chromosome 2, we have identified three novel genes that show homology to the IL-1 family, which we have named IL-1-related protein 1, 2, and 3 (IL-1RP1, IL-1RP2, and IL-1RP3). All three genes contain a signature motif common to the IL-1 family and appear to be more closely related to IL-1Ra. Similar to the intracellular form of IL-1Ra, these genes lack conventional hydrophobic signal sequences. The expression of these genes appears to be highly restricted to various epithelial cell populations. Our results demonstrate the existence of additional IL-1 gene family members within the previously defined IL-1 cluster and point to this region of chromosome 2 as an evolutionary hotspot for IL-1 gene duplication. These genes may prove to have an important role in inflammatory responses.     

The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors

Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.     

Physical linkage of three CD3 genes on human chromosome 11.

T-cell antigen receptors are associated on T cell surfaces with a complex of proteins called CD3 (formerly T3). Human CD3 consists of at least four proteins, gamma, delta, epsilon and zeta, and all but the latter have been cloned as cDNA. Using standard cloning techniques, together with field inversion gel electrophoresis, we have demonstrated the physical linkage of three CD3 genes. The genes for CD3 gamma and CD3 delta are situated close together, about 1.6 kb apart, organized in a head-to-head orientation. The gene encoding CD3 gamma has been sequenced, and is split into seven exons spread over 9 kb of DNA. Like CD3 delta, CD3 gamma gene has an unusual promoter which lacks a TATA-box and potential Sp1 binding sites. The CD3 gamma-CD3 delta gene pair is within 300 kb of the CD3 epsilon gene, and therefore these genes form a tightly linked cluster in chromosome 11 band q23. The clustering of the CD3 genes may be significant in terms of their simultaneous activation during T-cell development.     

Genes and chromosomal breakpoints in the Langer-Giedion syndrome region on human chromosome 8

The tricho-rhino-phalangeal syndrome type II (TRPS II, or Langer-Giedion syndrome) is an example of contiguous gene syndromes, as it comprises the clinical features of two autosomal dominant diseases, TRPS I and a form of multiple cartilaginous exostoses caused by mutations in the EXT1 gene. We have constructed a contig of cosmid, lambda-phage, PAC, and YAC clones, which covers the entire TRPS I critical region. Using these clones we identified a novel submicroscopic deletion in a TRPS I patient and refined the proximal border of the minimal TRPS1 gene region by precisely mapping the inversion breakpoint of another patient. As a first step towards a complete inventory of genes in the Langer-Giedion syndrome chromosome region (LGCR) with the ultimate aim to identify the TRPS1 gene, we analyzed 23 human expressed sequence tags (ESTs) and four genes (EIF3S3, RAD21, OPG, CXIV) which had been assigned to human 8q24.1. Our analyses indicate that the LGCR is gene-poor, because none of the ESTs and genes map to the minimal TRPS1 gene region and only two of these genes, RAD21 and EIF3S3, are located within the shortest region of deletion overlap of TRPS II patients. Two genes, OPG and CXIV, which are deleted only in some patients with TRPS II may contribute to the clinical variability of this syndrome.     

Evolutionarily plastic regions at human 3p21.3 coincide with tumor breakpoints identified by the "elimination test"

We have previously found with the microcell hybrid-based "elimination test" that human chromosome 3 transferred into murine or human tumor cells regularly lost certain 3p regions during tumor growth in SCID mice. The most common eliminated region, CER1, is approximately 2.4 Mb at 3p21.3. CER1 breakpoints were clustered in approximately 200-kb regions at both telomeric and centromeric borders. We have also shown, earlier, that tumor-related deletions often coincide with human/mouse synteny breakpoints on 3p12-p22. Here we describe the results of a comparative genomic analysis on the CER1 region in Caenorhabditis elegans, Drosophila melanogaster, Fugu rubripes, Gallus gallus, Mus musculus, Rattus norvegicus, and Canis familiaris. First, four independent synteny breaks were found within the CER1 telomeric breakpoint cluster region, comparing human, dog, and chicken genomes, and two independent synteny breaks within the CER1 centromeric breakpoint cluster region, comparing human, mouse, and chicken genomes, suggesting a nonrandom involvement of tumor breakpoint regions in chromosome evolution. Second, both CER1 breakpoint cluster regions show recent tandem duplications (seven Zn finger protein family genes at the telomeric and eight chemokine receptor genes at the centromeric side). Finally, all genes from these regions underwent horizontal evolution in mammals, with formation of new genes and expansion of gene families, which were displayed in the human genome as tandem gene duplications and pseudogene insertions. In contrast the CER1 middle region contained evolutionarily well-conserved solitary genes and a minimal amount of retroposed genes. The coincidence of evolutionary plasticity with CER1 breakpoints may suggest that regional structural instability is expressed in both evolutionary and cancer-associated chromosome rearrangements.     

Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.     

Regulation of larval cuticle protein gene expression in Drosophila melanogaster

Genes that encode 3rd instar larval cuticle proteins (LCP's) of Drosophila melanogaster are located in at least two chromosomal sites. The genes encoding four of the five predominant LCP's are located in a cluster at the chromosomal region 44D. They are organized in pairs that are transcribed divergently, and expressed with different timing during the third larval instar. Towards understanding the basis of gene regulation within the 44D cluster, we have analyzed genetic variants, including the 2-3 variant, which has an insertion of a copia-like transposable element, H.M.S. Beagle, within the 44D cluster. The Beagle element appears to inactivate the LCP-3 gene by inserting into its TATA box, but also may cause the precocious expression of two other LCP genes, LCP-1 and LCP-f2, in the cluster. The long terminal repeat (LTR) of the Beagle element apparently contains a sequence, perhaps an enhancer-like element, which causes altered expression of these genes. We have also investigated the cis-regulatory elements involved in expression of the LCP-2 gene in wild-type larvae. We have identified two upstream regions that may contain separate cis-regulatory elements. The region between -252 bp and -515 bp may be essential for any expression of LCP-2. Additionally, the region between -515 bp and -795 bp appears to be required for the normal level of expression of the LCP-2 gene.     

Cytogenetic rearrangements involving the loss of the Sonic Hedgehog gene at 7q36 cause holoprosencephaly

Holoprosencephaly (HPE) is a genetically heterogeneous disorder that affects the midline development of the forebrain and midface in humans. As a step toward identifying one of the HPE genes, we have set out to refine the HPE3 critical region on human chromosome 7q36 by analyzing 34 cell lines from families with cytogenetic abnormalities involving 7q, 24 of which are associated with HPE. Genomic clones surrounding the DNA marker D7S104, which has previously been shown to be in the HPE3 critical region, have been examined by fluorescent in situ hybridization and microsatellite analysis of our panel of patient cell lines. We report the analysis of a cluster of four translocation breakpoints within a 300-kb region of 7q36 that serves to define the minimal critical region for HPE3 and that has directed the search for candidate genes. The human Sonic Hedgehog (hSHH) gene maps to this region and has been shown to be HPE3 on the basis of mutations within the coding region of the gene. We present evidence that cytogenetic deletions and/or rearrangements of this region of chromosome 7q containing Sonic Hedgehog, and translocations that may suppress Sonic Hedgehog gene expression through a position effect are common mechanisms leading to HPE. Received: 23 December 1996 / Accepted: 17 March 1997     

Concerted evolution within a trypsin gene cluster in Drosophila.

A cluster of four trypsin genes has previously been localized to cytological position 47D-F of the Drosophila melanogaster genome. One of these genes had been sequenced, and the presence of the other three genes was identified by cross-hybridization. Here, we present the DNA sequence of the entire genomic region encoding these four trypsin genes. In addition to the four previously inferred genes, we have identified a fifth trypsin-coding sequence located within this gene cluster. This new gene shows a high degree of sequence divergence (more than 30%) from the other four genes, although it retains all of the functional motifs that are characteristic of trypsin-coding sequences. In order to trace the molecular evolution of this gene cluster, we isolated and sequenced the homologous 7-kb region from the closely related species Drosophila erecta. A comparison of the DNA sequences between the two species provides strong evidence for the concerted evolution of some members of this gene family. Two genes within the cluster are evolving in concert, while a third gene appears to be evolving independently. The remaining two genes show an intermediate pattern of evolution. We propose a simple model, involving chromosome looping and gene conversion, to explain the relatively complex patterns of molecular evolution within this gene cluster.     

The human histone gene cluster at the D6S105 locus

The sequences and organization of the histone genes in the histone gene cluster at the chromosomal marker D6S105 have been determined by analyzing the Centre d’étude du Polymorphisme Humain yeast artificial chromosome (YAC) 964f1. The insert of the YAC was subcloned in cosmids. In the established contig of the histone-gene-containing cosmids, 16 histone genes and 2 pseudogenes were identified: one H1 gene (H1.5), five H2A genes, four H2B genes and one pseudogene of H2B, three H3 genes, and three H4 genes plus one H4 pseudogene. The cluster extends about 80 kb with a nonordered arrangement of the histone genes. The dinucleotide repeat polymorphic marker D6S105 was localized at the telomeric end of this histone gene cluster. Almost all human histone genes isolated until now have been localized within this histone gene cluster and within the previously described region of histone genes, about 2 Mb telomeric of the newly described cluster or in a small group of histone genes on chromosome 1. We therefore conclude that the data presented here complete the set of human histone genes. This now allows the general organization of the human histone gene complement to be outlined on the basis of a compilation of all known histone gene clusters and solitary histone genes. Received: 30 June 1997 / Accepted: 3 September 1997