ABSTRACT
Vitamin A and its derivatives such as retinoic acid (RA) are important signaling molecules for morphogenesis of vertebrate embryos. Little is known, however, about morphogenetic factors controlling the development of the gastrointestinal tract and RA is likely to be involved. In the mouse, teratogenic doses of RA cause truncation of the embryonic caudal body axis that parallel the caudal regression syndrome as described in humans. These changes are often associated with anomalies of the lower digestive tract. Overlapping spatiotemporal expression of retinoic acid receptor-beta (RAR beta) and cellular retinol-binding protein I, CRBPI, with Hoxb5 and c-ret in the gut mesoderm imply possible cooperation required for proper neuromuscular development. To determine susceptibility and responsiveness of the developing gut and its neuromusculature to exogenous retinoids we used a mouse model of RA-induced caudal regression syndrome. The results showed that stage-specific RA treatment both in vivo and in vitro affected gut looping/rotation morphogenesis and growth of asymmetrical structures such as the cecum together with delayed differentiation of the gut mesoderm and colonization of the postcecal gut by neural crest-derived enteric neuronal precursors. These observations demonstrate that RA has a direct effect on gut morphogenesis and innervation.
Subject(s)
Intestines/drug effects , Lumbar Vertebrae/drug effects , Sacrum/drug effects , Tretinoin/administration & dosage , Animals , Disease Models, Animal , Embryo, Mammalian/abnormalities , Embryo, Mammalian/drug effects , Female , Intestines/embryology , Intestines/pathology , Lumbar Vertebrae/embryology , Lumbar Vertebrae/pathology , Mice , Muscles/drug effects , Muscles/embryology , Muscles/pathology , Nervous System/drug effects , Nervous System/embryology , Nervous System/pathology , Organ Culture Techniques , Pregnancy , Sacrum/embryology , Sacrum/pathology , Syndrome , Time FactorsABSTRACT
We describe the cloning of HOXD1 in human unfertilised oocytes and detailed expression analyses during mouse oogenesis and embryogenesis. The cDNA of 1991bp has an open reading frame of 987bp encoding a protein of 329 amino acids. A comparison of the amino acid sequence with the mouse homologue revealed an overall homology of 85.5% with 99% identity within the homeodomain. Expression was detected in unfertilised human oocytes and 2-, 4-, 8-cell and blastocyst stage embryos. Expression analyses in mature mouse ovaries, early embryos and isolated gut revealed expression in the oocytes of the primary and secondary ovarian follicles, and in embryonal mesodermal derivatives such as dermatomes, urogenital tubercle, tail bud, kidney, ovaries, testes and enteric mesoderm adjacent to the caecum where expression was up-regulated in vitro in response to increasing doses of retinoic acid. Our observations indicate a possible role for HOXD1/Hoxd1 in the ovarian oocytes and the establishment of mesodermal derivatives during embryogenesis.
Subject(s)
DNA-Binding Proteins/genetics , Embryo, Mammalian/metabolism , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Oocytes/metabolism , Transcription Factors/genetics , Amino Acid Sequence , Animals , Basic Helix-Loop-Helix Transcription Factors , Cloning, Molecular , DNA, Complementary/metabolism , DNA-Binding Proteins/biosynthesis , Humans , In Situ Hybridization , Mice , Molecular Sequence Data , Oogenesis , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Time Factors , Tissue Distribution , Transcription Factors/biosynthesisABSTRACT
BACKGROUND & AIMS: Hox genes are highly conserved developmental control genes that may be organized and expressed in the form of a code required for correct morphogenesis. Little is known about their control of the embryonal gut. However, Hox paralogues 4 and 5, which are expressed at the sites of origin of vagal neural crest cells and splanchnic mesoderm, are likely to be important. We have studied the expression domains of these genes in the gut both spatially and temporally. METHODS: CD1 mice embryos of embryonic days E8.5-E17.5 were studied. The spatial and temporal expression patterns of messenger RNA of Hoxa4, b4, c4, d4, a5, c5, and b5 homeoprotein were determined by in situ hybridization and immunohistochemistry in whole embryos, whole gastrointestinal tracts, and vibratome sections. RESULTS: There were different spatial, temporal, and combinatorial expression patterns in different morphological regions: foregut, prececal gut, cecum, and postcecal gut. Two dynamic gradients, rostral and caudal, were coordinated with nested expression domains along the gut primordium. Region-specific domains were present in the stomach and cecum. CONCLUSIONS: The expression patterns of genes in paralogous groups 4 and 5 suggest that they are organized to form a specific enteric Hox code required for correct enteric development.
Subject(s)
Gene Expression , Genes, Homeobox , Intestines/embryology , Phosphoproteins , Animals , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Embryonic and Fetal Development , Female , Gene Expression Profiling , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Intestinal Mucosa/metabolism , Mice , Muscles/metabolism , Neurons/metabolism , Pregnancy , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
An evaluation of nonisotopic in situ hybridization (NISH) for mRNA in archival lymphoid tissue was carried out and an analysis of factors affecting the final outcome was performed. A modification of the in situ reverse transcription method for RNA preservation assessment has been used and described. We have shown that, for frozen samples mRNA detection is optimal within 3 months of the biopsy being taken, while preservation declines after 1 year of storage.
Subject(s)
In Situ Hybridization/methods , Lymphoma/genetics , RNA, Messenger/analysis , Biopsy , Evaluation Studies as Topic , Frozen Sections , Humans , Lymph Nodes/chemistry , Preservation, Biological , Time Factors , Transcription, GeneticABSTRACT
DNA from 47 patients with non-Hodgkin's lymphoma (NHL) was studied for immunoglobulin and T-cell receptor (TCR) gene rearrangements with Southern blot hybridization. In 83% of the cases the genotypic changes were consistent with immunophenotypic and morphologic examination. Two cases showed mixed genotype and 9 cases of B-cell NHL (67% of centroblastic, 36% of follicular and 33% of large cell anaplastic) showed a population of cells with TCR gamma rearrangements in addition to immunoglobulin rearranged bands. We compared the TCR gamma variable region usage in these rearrangements in B-cell NHL with T-cell NHL and reactive hyperplasia. In T-cell NHL TCR gamma variable regions located at the 3' part of the variable locus were used more often, whilst in B-cell NHL regions of the 5' portion of the locus were preferentially used. Our results confirm the genotypic heterogeneity of histologically defined subtypes of NHL.
Subject(s)
Lymphoma, B-Cell/genetics , Lymphoma, Non-Hodgkin/genetics , Lymphoma, T-Cell/genetics , Adult , Aged , Aged, 80 and over , DNA Probes , DNA Restriction Enzymes , Female , Gene Rearrangement, B-Lymphocyte/genetics , Gene Rearrangement, T-Lymphocyte , Genotype , Humans , Male , Middle Aged , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, gamma-delta/analysisABSTRACT
The standard proteinase K/phenol DNA isolation method was found to produce unsatisfactory yields of DNA from small tissue biopsies (less than 50 mg). The influences of the volume of cell lysis buffer and the amount of proteinase K on the final DNA yield and quality were studied, and an improved method was devised and compared with both the standard procedure and a phenol-free protocol. The optimal volume of cell lysis buffer was found to be 200 microliters per mg of tissue while the optimal amount of proteinase K was 60 micrograms per mg of tissue. A mean yield of 12 mu/mg tissue of pure, high molecular weight DNA was achieved from 50 frozen samples prepared by crushing. Yields from 20 microns thick cryostat sections reached 30 micrograms/mg.
