Search

The kidney vasculature is critical for renal function, but its developmental assembly mechanisms remain poorly understood and models for studying its assembly dynamics are limited. Here, we tested whether the embryonic kidney contains endothelial cells (ECs) that are heterogeneous with respect to VEGFR2/Flk1/KDR, CD31/PECAM, and CD146/MCAM markers. Tie1Cre;R26RYFP-based fate mapping with a time-lapse in embryonic kidney organ culture successfully depicted the dynamics of kidney vasculature development and the correlation of the process with the CD31+EC network. Depletion of Tie1+or CD31+ECs from embryonic kidneys, with either Tie1Cre-induced diphtheria toxin susceptibility or cell surface marker–based sorting in a novel dissociation and reaggregation technology, illustrated substantial EC network regeneration. Depletion of the CD146+cells abolished this EC regeneration. Fate mapping of green fluorescent protein (GFP)-marked CD146+/CD31-cells indicated that they became CD31+cells, which took part in EC structures with CD31+wild-type ECs. EC network development depends on VEGF signaling, and VEGF and erythropoietin are expressed in the embryonic kidney even in the absence of any external hypoxic stimulus. Thus, the ex vivoembryonic kidney culture models adopted here provided novel ways for targeting renal EC development and demonstrated that CD146+cells are critical for kidney vasculature development.

ABSTRACTCiliated protists are model organisms for a number of molecular phenomena including telomerase function, self-splicing introns, and an RNA interference-related mechanism in programmed DNA elimination. Despite this relevance, our knowledge about promoters and transcriptional regulation in these organisms is very limited. The macronuclear genome of stichotrichous ciliates consists of minichromosomes which typically encode a single gene. The 5' nontranscribed spacers are usually no longer than 400 bp and highly suitable for promoter characterizations. We used microinjection of two artificial and differently tagged a1 tubulin minichromosomes into the macronucleus of Stylonychia lemnaeas a means to characterize in detail the corresponding promoter. Clonal cell lines that stably maintained both minichromosomes were generated, enabling comparative expression analysis by primer extension assays. Deletion and block substitution mutations of one of the minichromosomes revealed a TATA-like element, a putative initiator element, and two distinct upstream sequence elements (USEs). Determination of transcription initiation sites and a sequence alignment indicated that both TATA-like and initiator elements are conserved components of S. lemnaeminichromosomes, whereas the USEs appear to be specific for the a1 tubulin minichromosome. The a2 tubulin minichromosome promoter is very short, comprising the two proximal elements but not the USEs. Despite the latter finding, up-regulation of a-tubulin expression in cells treated with concanavalin A activated the a2 but not the a1 tubulin promoter. These results therefore show that gene expression regulation in S. lemnaeoccurs at the level of transcription initiation on the basis of structurally different promoters.

Ciliated protists are model organisms for a number of molecular phenomena including telomerase function, self-splicing introns, and an RNA interference-related mechanism in programmed DNA elimination. Despite this relevance, our knowledge about promoters and transcriptional regulation in these organisms is very limited. The macronuclear genome of stichotrichous ciliates consists of minichromosomes which typically encode a single gene. The 5' nontranscribed spacers are usually no longer than 400 bp and highly suitable for promoter characterizations. We used microinjection of two artificial and differently tagged 1 tubulin minichromosomes into the macronucleus of Stylonychia lemnae as a means to characterize in detail the corresponding promoter. Clonal cell lines that stably maintained both minichromosomes were generated, enabling comparative expression analysis by primer extension assays. Deletion and block substitution mutations of one of the minichromosomes revealed a TATA-like element, a putative initiator element, and two distinct upstream sequence elements (USEs). Determination of transcription initiation sites and a sequence alignment indicated that both TATA-like and initiator elements are conserved components of S. lemnae minichromosomes, whereas the USEs appear to be specific for the 1 tubulin minichromosome. The 2 tubulin minichromosome promoter is very short, comprising the two proximal elements but not the USEs. Despite the latter finding, up-regulation of -tubulin expression in cells treated with concanavalin A activated the 2 but not the 1 tubulin promoter. These results therefore show that gene expression regulation in S. lemnae occurs at the level of transcription initiation on the basis of structurally different promoters.