Endothelial and smooth muscle cells interact with each other to form new blood vessels. In this review, the cellular and molecular mechanisms underlying the formation of endothelium-lined channels (angiogenesis) and their maturation via recruitment of smooth muscle cells (arteriogenesis) during physiological and pathological conditions are summarized, alongside with possible therapeutic applications.

Mechanisms of angiogenesis and arteriogenesis.

Epithelial-mesenchymal transition is an indispensable mechanism during morphogenesis, as without mesenchymal cells, tissues and organs will never be formed. However, epithelial-cell plasticity, coupled to the transient or permanent formation of mesenchyme, goes far beyond the problem of cell-lineage segregation. Understanding how mesenchymal cells arise from an epithelial default status will also have a strong impact in unravelling the mechanisms that control fibrosis and cancer progression.

Complex networks orchestrate epithelial–mesenchymal transitions

Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of ~21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila , at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA

Charge, Sophie B. P., and Michael A. Rudnicki. Cellular and Molecular Regulation of Muscle Regeneration. Physiol Rev 84: 209–238, 2004; 10.1152/physrev.00019.2003.—Under normal circumstances, mamma...

Cellular and Molecular Regulation of Muscle Regeneration

Characterization of a promoter element required for transcription in myocardial cells

The chicken gene encoding cardiac troponin T (cTNT) is expressed in both cardiac and skeletal muscle during early embryonic development, but is specifically repressed in skeletal muscle during fetal development. To determine if the cis-acting sequences governing transcription of a single gene in these two related cell types are the same, we have transfected promoter/upstream segments of the cTNT gene coupled to the bacterial chloramphenicol acetyltransferase gene into primary cultures of early embryonic cardiac and skeletal muscle cells. Using this assay system, chloramphenicol acetyltransferase activity directed by the cTNT promoter/upstream region was between two and three orders of magnitude higher in cardiac or skeletal muscle cells than in fibroblast cells, indicating that cis elements responsible for cell-specific expression reside in this region of the cTNT gene. Deletion experiments showed that a 67-nucleotide DNA segment residing between 268 and 201 nucleotides upstream of the cTNT transcription initiation site is required for cTNT promoter activity in embryonic cardiac cells. This region is not required in embryonic skeletal muscle cells because a cTNT promoter construction containing only 129 upstream nucleotides is transcriptionally active in these cells. These results demonstrate that different cis-acting sequences are required for cTNT expression in early embryonic cardiac and skeletal muscle cells. Nonessential regions residing farther upstream, on the other hand, affected the level of expression of these minimum regions in a similar manner in both cell types. The data from these experiments indicate, therefore, that transcription of the cTNT promoter in early embryonic cardiac and skeletal muscle cells is governed both by common and divergent regulatory elements in cis and in trans.

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Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells.

The enzymatic mechanism of poly(ADP-ribose) polymerase (PARP-1) has been analyzed in two in vitro systems:  (a) in solution and (b) when the acceptor histones were attached to a solid surface. In system (a), it was established that the coenzymatic function of dsDNAs was sequence-independent. However, it is apparent from the calculated specificity constants that the AT homopolymer is by far the most effective coenzyme and randomly damaged DNA is the poorest. Rates of auto(poly-ADP-ribosylation) with dsDNAs as coenzymes were nearly linear for 20 min, in contrast to rates with dcDNA, which showed product [(ADPR)n] inhibition. An allosteric activation of auto(poly-ADP-ribosylation) by physiologic cellular components, Mg2+, Ca2+, and polyamines, was demonstrated, with spermine as the most powerful activator. On a molar basis, histones H1 and H3 were the most effective PARP-1 activators, and their action was abolished by acetylation of lysine end groups. It was shown in system (b) that oligo(ADP-ribosyl) transf...

Regulation of the enzymatic catalysis of poly(ADP-ribose) polymerase by dsDNA, polyamines, Mg2+, Ca2+, histones H1 and H3, and ATP.

We have studied the derivatives of the first somite using the quail-chick marking technique. After transplantation of the somite, the chick embryos were reincubated for periods ranging from 4 h to 11 days. Coronal and sagittal sections of the embryos were prepared for parallel staining with Feulgen-reaction, anti-quail antibody, anti-desmin antibody and QH-1 antibody. The first somite consists of an epithelial envelope surrounding somitocoele cells. Like other somites, it forms sclerotome, dermatome and myotome. Cells contribute to the occipital and parasphenoid bone, the meninges, the dermis in the occipital region and the pharyngeal connective tissue. The contribution of the first somite to bones, meninges, dermis and pharyngeal connective tissue is characterised by sharp anterior and posterior boundaries. In contrast, other derivatives such as connective tissue surrounding the vagus nerve, the carotid artery, and jugular vein exceed 10 to 18 segments. This is also true for myogenic cells participating in the formation of the cucullaris capitis muscle that extends from the temporal bone to the shoulder. In one third of the embryos, myocytes of the intrinsic laryngeal muscles are derived from the grafted first somite. Moreover, endothelial cells originate from this somite and migrate into the head (hindbrain, meninges, dermis), neck (pharynx, connective tissue surrounding the vagus nerve, carotid artery and jugular vein) and thorax. With respect to differentiation and derivatives the first somite is similar to other somites.

The fate of the first avian somite.

The cardiac troponin T (cTNT) pre-mRNA contains a single alternative exon (exon 5) which is either included or excluded from the processed mRNA. Using transient transfection of cTNT minigenes, we have previously localized pre-mRNA cis elements required for exon 5 alternative splicing to three small regions of the pre-mRNA which include exons 4, 5, and 6. In the present study, nucleotide substitutions were introduced into the region containing exon 5 to begin to define specific nucleotides required for exon 5 alternative splicing. A mutation within the 5' splice site flanking the cTNT alternative exon that increases its homology to the consensus sequence improves splicing efficiency and leads to increased levels of mRNAs that include the alternative exon. Surprisingly, substitution of as few as four nucleotides within the alternative exon disrupts cTNT pre-mRNA alternative splicing and prevents recognition of exon 5 as a bona fide exon. These results establish that the cTNT alternative exon contains information in cis that is required for its recognition by the splicing machinery.

Charles P. Ordahl

Papers

Characterization of a promoter element required for transcription in myocardial cells

Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells.

Regulation of the enzymatic catalysis of poly(ADP-ribose) polymerase by dsDNA, polyamines, Mg2+, Ca2+, histones H1 and H3, and ATP.

The fate of the first avian somite.

Nucleotide substitutions within the cardiac troponin T alternative exon disrupt pre-mRNA alternative splicing