Andreas van Impel1, Zhonghua Zhao2,*, Dorien M. A. Hermkens1,3,*, M. Guy Roukens1,*, Johanna C. FischerM4, Josi Peterson-Maduro1, Henricus Duckers3, Elke A. Ober4,6, Philip W. Ingham2,7 and Stefan Schulte-Merker1,5,‡.
1 Hubrecht Institute – KNAW & UMC Utrecht, 3584 CT Utrecht, The Netherlands.
2 A-STAR Institute of Molecular and Cell Biology, 61 Biopolis Drive, 138673, Singapore.
3 Erasmus MC, 3015 CE Rotterdam, The Netherlands.
4 Developmental Biology, MRC National Institute for Medical Research, London, NW7 1AA, UK.
5 Experimental Zoology Group, Wageningen University, 6708 PB Wageningen, The Netherlands.
6 The Danish Stem Cell Centre, University of Copenhagen, Blegdamsvej 3B, DK-2200, Copenhagen N, Denmark.
7 Lee Kong Chian School of Medicine, Imperial College London/Nanyang Technological University, 138673 Singapore.
*These authors contributed equally to this work.
‡Author for correspondence
Published online in Development ePress on 12 February 2014.
In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis. Murine Prox1-null embryos lack lymphatic structures, and sustained expression of Prox1 is indispensable for the maintenance of lymphatic cell fate even at adult stages, highlighting the unique importance of this gene for the lymphatic lineage. Whether this pre-eminent role of Prox1 within the lymphatic vasculature is conserved in other vertebrate classes has remained unresolved, mainly owing to the lack of availability of loss-of-function mutants. Here, we re-examine the role of Prox1a in zebrafish lymphangiogenesis. First, using a transgenic reporter line, we show that prox1a is initially expressed in different endothelial compartments, becoming restricted to lymphatic endothelial cells only at later stages. Second, using targeted mutagenesis, we show that Prox1a is dispensable for lymphatic specification and subsequent lymphangiogenesis in zebrafish. In line with this result, we found that the functionally related transcription factors Coup-TFII and Sox18 are also dispensable for lymphangiogenesis. Together, these findings suggest that lymphatic commitment in zebrafish and mice is controlled in fundamentally different ways.
Figure Legend: Lymphangiogenesis in prox1a mutant embryos. (A) Schematic of the homeodomain (HD) containing Prox1a protein, indicating the predicted effect of the 10 bp deletion in the prox1ai278 allele, leading to a frame-shift (red amino acids) and a truncated protein after 153 amino acids. (B,C) Prox1a immunostaining of slow muscle fibers in sibling (B) and homozygous mutant prox1ai278 (C) embryos demonstrates a complete loss of wild-type Prox1a protein (green) at 30 hpf (slow myosin heavy chain-1 is shown in red). (D,E) Brightfield pictures of 5 dpf sibling (D) and homozygous prox1ai278 mutant (E) embryos. Note the strong edema formation around the eye and gut area (arrowheads), which can be even more pronounced in other prox1a mutants at this stage. (F,G) In both heterozygous siblings (F) and homozygous prox1ai278 mutants (G), PLs appear at the level of the horizontal myoseptum at 2 dpf (arrows). (H) Average PL numbers per embryo are mildly reduced in prox1a mutants at 2 dpf (Student's t-test, *P=0.025). Error bars indicate s.d. of wild-type (green), heterozygous (orange) and mutant (red) groups in embryos from a prox1a+/- incross. (I-K) flt4:mCit; flt1enh:tdTom double transgenic embryos highlighting arterial ISVs in red and venous and lymphatic structures in green. Compared with heterozygous siblings (I), most homozygous prox1ai278 mutants do not display TD defects at 5 dpf (J), whereas others display a mild reduction (K) in some areas of the trunk (arrows point at TD; asterisks mark the lack of TD). Note the overall unaffected ratio of venous and arterial ISVs in mutants (J,K). (L) Average number of segments positive for TD cells, scored in the first ten segments above the yolk extension at 5 dpf. Error bars indicate the s.d. for the respective genotypic class from a prox1a+/- incross. ***P=2.3E–08 (Student’s t-test, comparison of wild-type and mutant population). (M,N) The average percentage of intersegmental veins (M) and arteries (N) does not differ between genotypic classes in an incross of prox1ai278 carriers. (O) In prox1ai278 mutants, the average number of ISVs is not altered. Error bars represent s.d. n.s., not statistically significant.
For more information on Philip INGHAM’s laboratory, please click here.