ProxTom Lymphatic Vessel Reporter Mice Reveal Prox1 Expression in the Adrenal Medulla, Megakaryocytes, and Platelets

Abstract

Lymphatic vessels (LVs) are important structures for antigen presentation, for lipid metabolism, and as conduits for tumor metastases, but they have been difficult to visualize in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in ontogeny and the maintenance of LVs. To visualize LVs in the lymph node of a living mouse in real time, we made the ProxTom transgenic mouse in a C57BL/6 background using red fluorescent LVs that are suitable for in vivo imaging. The ProxTom transgene contained all Prox1 regulatory sequences and was faithfully expressed in LVs coincident with endogenous Prox1 expression. The progenies of a ProxTom × Hec6stGFP cross were imaged using two-photon laser scanning microscopy, allowing the simultaneous visualization of LVs and high endothelial venules in a lymph node of a living mouse for the first time. We confirmed the expression of Prox1 in the adult liver, lens, and dentate gyrus. These intensely fluorescent mice revealed the expression of Prox1 in three novel sites: the neuroendocrine cells of the adrenal medulla, megakaryocytes, and platelets. The novel sites identified herein suggest previously unknown roles for Prox1. The faithful expression of the fluorescent reporter in ProxTom LVs indicates that these mice have potential utility in the study of diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

“…Gene-targeted mice expressing a Cre-recombinase or a fluorophore under an LEC-specific promoter have recently been generated, [32][33][34][35][36] but only 2 IVM studies performed in such mice have been reported so far. 35,36 Therefore, to date, LVs have mainly been visualized in IVM by in vivo labeling with intracutaneously injected fluorescent antibodies.…”

“…35,36 Therefore, to date, LVs have mainly been visualized in IVM by in vivo labeling with intracutaneously injected fluorescent antibodies. 4,37,38 However, antibody labeling is considered less optimal for IVM than endogenous expression of fluorophores; it is typically restricted to the injection site and downstream regions and could interfere with leukocyte-endothelium interactions or lead to unwanted immune activation.…”

Angiopoietin-1 is required for Schlemm’s canal development in mice and humans

“…After staining, eyes were fixed overnight in 10% formalin before paraffin embedding and sectioning using standard methods. Prox1-tdTomato [Tg(Prox1-tdTomato)] mice were a gift from Guillermo Oliver (Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA) and were genotyped as previously described (40). Adult eyes were used for whole-mount immunostaining as described above.…”

Lymphatic vessels and tertiary lymphoid organs

“…Analysis of lymphocyte, DC, and antigen-trafficking patterns in TLOs in real time in vivo is now possible with the use of mice that express fluorescent markers for HEVs (80) and LVs (79,81). Although the technique of in vivo imaging is well established for analysis of trafficking in LNs (82), addressing this issue in TLOs is a greater challenge.…”