Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division.

The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from preexisting vasculature) during embryonic development, tissue regeneration and tumor progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis during development. The role of CCBE1 orthologs is highly conserved in zebrafish, mice and humans with mutations in CCBE1 causing generalized lymphatic dysplasia and lymphedema (Hennekam syndrome). To date, the mechanism by which CCBE1 acts remains unknown. Here, we find that ccbe1 genetically interacts with both vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by increased Vegfc are suppressed in the absence of Ccbe1, and Vegfcdriven sprouting is enhanced by local Ccbe1 overexpression. Moreover, Vegfc-and Vegfr3-dependent Erk signaling is impaired in the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the levels of fully processed, mature VEGFC in vitro and the overexpression of mature VEGFC rescues ccbe1 loss-of-function phenotypes in zebrafish. Taken together, these data identify Ccbe1 as a crucial component of the Vegfc/Vegfr3 pathway in the embryo.

SummaryThe lymphatic vascular system develops from the pre-existing blood vasculature of the vertebrate embryo. New insights into lymphatic vascular development have recently been achieved with the use of alternative model systems, new molecular tools, novel imaging technologies and growing interest in the role of lymphatic vessels in human disorders. The signals and cellular mechanisms that facilitate the emergence of lymphatic endothelial cells from veins, guide migration through the embryonic environment, mediate interactions with neighbouring tissues and control vessel maturation are beginning to emerge. Here, we review the most recent advances in lymphatic vascular development, with a major focus on mouse and zebrafish model systems.

Maternally and paternally derived alleles can utilize different promoters, but allele-specific differences in cotranscriptional processes have not been reported. We show that alternative polyadenylation sites at a novel murine imprinted gene (H13) are utilized in an allele-specific manner. A differentially methylated CpG island separates polyA sites utilized on maternal and paternal alleles, and contains an internal promoter. Two genetic systems show that alleles lacking methylation generate truncated H13 transcripts that undergo internal polyadenylation. On methylated alleles, the internal promoter is inactive and elongation proceeds to downstream polyadenylation sites. This demonstrates that epigenetic modifications can influence utilization of alternative polyadenylation sites.Supplemental material is available at http://www.genesdev.org.Received January 28, 2008; revised version accepted March 10, 2008. Transcription by RNA polymerase II (Pol II) requires multiple linked steps, including the assembly of an initiation complex, promoter release, elongation followed by splicing, polyadenylation (polyA), and dissociation of the polymerase complex from the template DNA. Each step provides an opportunity for the cell to regulate gene expression, either by changing the abundance or availability of mRNAs or by generating variant gene products. Epigenetic modifications to DNA and histones can influence transcription at the initiation stage by altering the accessibility of promoter sequences to initiation complex components (Kass et al. 1997a,b). Following initiation, regions of heterochromatin can subtly impede the progress of an elongating polymerase complex, reducing transcriptional output (Lorincz et al. 2004). However, it is not clear whether epigenetic modifications downstream from a promoter can lead to variant gene products through alternative polyadenylation or splicing.Polyadenylation, the addition of multiple adenyl residues to the 3Ј end of a newly synthesized transcript, confers stability and is required for nuclear export (Huang and Carmichael 1996;Jacobson and Peltz 1996). This occurs following cleavage of the newly synthesized pre-mRNA, typically 15-30 nucleotides downstream from a conserved hexamer motif (usually AAUAAA or AUUAAA) (Proudfoot and Brownlee 1976;Zhao et al. 1999). A large proportion of human genes utilize more than one polyadenylation (polyA) site (Tian et al. 2005), making alternative polyadenylation a major source of transcriptional diversity. The mechanisms governing alternative polyA site selection in mammalian cells have been studied in detail at only a small number of loci; notably the immunoglobulin heavy chain (Takagaki and Manley 1998) and calcitonin/CGRP genes (Lou et al. 1998). In both cases, polyA site selection is cell typespecific and is regulated by changes in the concentration of diffusible RNA processing factors (Lou et al. 1998;Takagaki and Manley 1998).Imprinted genes are differentially expressed on maternally and paternally derived alleles and are estimated to com...

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc-Vegfd-Ccbe1-Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.