© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1417 doi:10.1242/dev.108969 CORRECTION Abnormal embryonic lymphatic vessel development in Tie1 hypomorphic mice Xianghu Qu, Kevin Tompkins, Lorene E. Batts, Mira Puri and H. Scott Baldwin There was an error published in Development 137, 1285-1295. Author name H. Scott Baldwin was incomplete. The correct author list appears above. The authors apologise to readers for this mistake. 1417 RESEARCH ARTICLE 1285 Development 137, 1285-1295 (2010) doi:10.1242/dev.043380 © 2010. Published by The Company of Biologists Ltd Abnormal embryonic lymphatic vessel development in Tie1 hypomorphic mice Xianghu Qu1, Kevin Tompkins1, Lorene E. Batts1, Mira Puri2 and Scott Baldwin1,3,* SUMMARY Tie1 is an endothelial receptor tyrosine kinase that is essential for development and maintenance of the vascular system; however, the role of Tie1 in development of the lymphatic vasculature is unknown. To address this question, we first documented that Tie1 is expressed at the earliest stages of lymphangiogenesis in Prox1-positive venous lymphatic endothelial cell (LEC) progenitors. LEC Tie1 expression is maintained throughout embryonic development and persists in postnatal mice. We then generated two lines of Tie1 mutant mice: a hypomorphic allele, which has reduced expression of Tie1, and a conditional allele. Reduction of Tie1 levels resulted in abnormal lymphatic patterning and in dilated and disorganized lymphatic vessels in all tissues examined and in impaired lymphatic drainage in embryonic skin. Homozygous hypomorphic mice also exhibited abnormally dilated jugular lymphatic vessels due to increased production of Prox1-positive LECs during initial lymphangiogenesis, indicating that Tie1 is required for the early stages of normal lymphangiogenesis. During later stages of lymphatic development, we observed an increase in LEC apoptosis in the hypomorphic embryos after mid-gestation that was associated with abnormal regression of the lymphatic vasculature. Therefore, Tie1 is required for early LEC proliferation and subsequent survival of developing LECs. The severity of the phenotypes observed correlated with the expression levels of Tie1, confirming a dosage dependence for Tie1 in LEC integrity and survival. No defects were observed in the arterial or venous vasculature. These results suggest that the developing lymphatic vasculature is particularly sensitive to alterations in Tie1 expression. KEY WORDS: Tie1, Lymphatic development, Hypomorphic, Proliferation, Apoptosis, Mouse INTRODUCTION vein. These differentiating lymphatic endothelial cells (LECs) The lymphatic vascular system is a blind-ended network of sprout, migrate and proliferate to form primary lymph sacs in the endothelial cell-lined vessels essential for the maintenance of tissue jugular region (Oliver, 2004). Subsequently, several lymph sacs are fluid balance, immune surveillance and absorption of fatty acids in formed close to major veins in different regions of the embryo. The the gut. The lymphatic vessels are also involved in the pathogenesis primary lymphatic vascular plexus undergoes remodeling and of diseases such as tumor metastasis, lymphedema and various maturation to create a mature lymphatic network composed of large inflammatory conditions. Despite central roles in both normal and lymph vessels as well as an extensive lymphatic capillary network. disease physiology, our understanding of the development and Numerous signaling proteins have been identified as important in molecular regulation of the lymphatic vasculature lags far behind these later stages of the lymphatic development including ephrin B2 that of the parallel blood vascular system (Oliver, 2004; Oliver and (Makinen et al., 2005), neuropilin 2 (Yuan et al., 2002), angiopoietin Alitalo, 2005). 2 (Gale et al., 2002; Dellinger et al., 2008), podoplanin (Schacht et Many details about the development of the lymphatic vasculature al., 2003), integrin alpha 9 (Huang et al., 2000), and the transcription have been described only within the past decade, largely as a result factors Foxc2 (Petrova et al., 2004), Net (Ayadi et al., 2001), Vezf1 of studies of gene-targeted mice (Oliver and Srinivasan, 2008). For (Kuhnert et al., 2005), adrenomedullin (Fritz-Six et al., 2008) and mammals, the development of the lymphatic vessels in embryos is Aspp1 (Hirashima et al., 2008). initiated when a subset of endothelial cells in the cardinal vein sprout The orphan receptor tyrosine kinase (RTK) Tie1 shares a high to form the primary lymph sacs (Oliver and Detmar, 2002), degree of homology and is able to form heterodimers with Tie2, the confirming speculation of a venous origin made over a century ago receptor for the angiopoietins (Yancopoulos et al., 2000; Peters et (Sabin, 1909). In mice, the initiation of lymphatic differentiation al., 2004), and is known to play a major role in vascular is first discernible at embryonic day 10.5 (E10.5), when a development. Genetic studies in mice have demonstrated that Tie1 subpopulation of endothelial cells (ECs) expressing Lyve1, Prox1 is required for development and maintenance of the vascular system (Wigle and Oliver, 1999), Sox 18 (Francois et al., 2008; Hosking et as mice lacking Tie1 die in mid-gestation of hemorrhage and al., 2009) and Vegfr3 are detected on one side of the anterior cardinal defective microvessel integrity (Puri et al., 1995; Sato et al., 1995). Expression of Tie1 is restricted to ECs and to some hematopoietic cell lineages (Partanen et al., 1992; Korhonen et al., 1994; Dumont 1Division of Pediatric Cardiology, Vanderbilt University Medical Center, Nashville, et al., 1995; Hashiyama et al., 1996; Taichman et al., 2003; Yano et 2 TN 37232, USA. Sunnybrook and Women’s College Health Sciences Center, al., 1997). Interestingly, there is evidence that Tie1 is also expressed University of Toronto, Toronto, Ontario M4N 3M5, Canada. 3Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, by the initial and collecting lymphatic vessels in adult mice (Iljin et USA. al., 2002) and the first visible defect in the Tie1 mutants is edema (Sato et al., 1995). These observations led us to hypothesize that *Author for correspondence ([email protected]) Tie1 might serve a unique function in the development or Accepted 5 February 2010 maintenance of the lymphatic vasculature. DEVELOPMENT 1286 RESEARCH ARTICLE Development 137 (8) In this study, we found that Tie1 was expressed in the Prox1- mouse Vegfr3 antibody or rabbit anti-Prox1 and Alexa Fluor 594-conjugated positive venous LEC progenitors and lymphatic vessels donkey anti-goat IgG (Molecular Probes, A-11058) or Alexa Fluor 555 goat throughout embryonic and postnatal life. To circumvent early anti-rabbit IgG (Molecular Probes, A-21428). Lymphatic endothelial cell embryonic lethality observed in homozygous mutant animals, we (EC) proliferation was also determined by labeling with Prox1 and Ki67 generated mice with a conditional Tie1 allele that fortuitously (BD Biosciences, 550609). Other primary antibodies used in tissue section resulted in hypomorphic expression of Tie1. We were able to take immunohistochemistry were rat anti-mouse CD34 (eBioscience, 14-0341), advantage of this Tie1 hypomorphic allele to demonstrate a unique rat anti-mouse Icam1 (eBioscience, 14-0542), rat anti-mouse endoglin role for Tie1 in lymphatic development that was not observed in (eBioscience, 14-1051) and rat anti-mouse VE-cadherin (BD Biosciences, 555289). The percentage of proliferative lymphatic ECs in the cardinal vein the arterial or venous vasculature. Furthermore, the severity of the and jugular lymph sac areas of embryos at E11.5 to E13.5 was defined as the phenotypes observed correlated with the expression level of Tie1. number of BrdU+/Prox1+ cells divided by the total number of Prox1+ cells Our studies show that Tie1 is required for the early stages of in each field at similar level. normal lymphangiogenesis and is also involved in the later Apoptosis in lymphatic ECs was assed by TUNEL assay using ApopTag remodeling and stabilization of lymphatic vessels in a dosage- Plus Fluorescein in situ Cell Death Detection Kit (CHEMICON dependent manner. International, S7111). In addition, cleaved caspase 3 (Cell Signaling Technology, 9661) and Vegfr3 double staining was used to detect apoptotic MATERIALS AND METHODS LECs on frozen sections. Images were acquired on an Olympus fluorescent Generation of Tie1 mutant alleles microscope and processed in Adobe Photoshop. Percent apoptotic cells was loxP/loxP To generate Tie1 mice, a Tie1 floxed targeting vector was constructed determined by blinded quantification of TUNEL-positive cells in the based on the 129-Sv mouse genomic fragment used by Puri et al. (Puri et al., lymphatic or vascular endothelium divided by total Prox1 or DAPI-stained 1995). A 940 bp HpaI-SacI fragment containing a Tie1 minimal promoter nuclei in each comparable field. (Korhonen et al., 1995; Iljin et al., 2002) and exon 1 (containing the initial ATG codon) was inserted into the floxed KpnI-ClaI sites of the pDELBOY In situ hybridization plasmid (pDELBOY-3X), which contains an Frt-site-flanked neomycin In situ hybridization was performed on paraffin sections of 6 mm with gene (see Fig. S1 in the supplementary material). The targeting vector was radiolabeled single-stranded RNA probes. Probes were 0.42 kb mouse Tie1 electroporated into 129 R1 embryonic stem (ES) cells (Nagy et al., 1993) fragments corresponding