ZFIN Figure: De Angelis et al., 2017, Fig. 2

Fig. 2

Tmem2 Expression Is Enriched in the Vasculature, the C Terminus Is Extracellular, and a Secreted C-Terminal Fragment Can Rescue the Angiogenesis Defect

(A) ISH of the zebrafish trunk showing tmem2 expression is ubiquitous at 24 hpf and becomes enriched in venous endothelium at 32 hpf. (A′) tmem2 expression is also observed in angiogenic sprouts (black arrows).

(B) Lateral trunk views of Tg(tmem2^BAC:tmem2-mCherry) embryos showing comparable expression with tmem2 RNA. Right-side panels showing overlay with Tg(fli1a:EGFP). Tmem2-mCherry protein was observed in angiogenic sprouts (white asterisks; white arrowheads depict iridophore autofluorescence).

(C) Single z scans of two venous sprouts showing Tmem2-mCherry enrichment. Closer analysis (right panels) indicate mCherry tag is extracellular (white arrowheads). Scale bar, 10 μm and 5 μm for zoom.

(D) Western blot analysis for mCherry showing bands consistent with full-length Tmem2-mCherry protein (red asterisk) as well as variable-sized fragments that form over developmental time. Staged Tg(kdrl:mCherry) lysates are controls.

(E) Column scatter graph representing the number of ISVs at 48 hpf per embryo in uninjected siblings (n = 13) and tmem2^hu5935 mutants (n = 89) as well as tmem2^hu5935 mutants injected with full-length tmem2 mRNA (yellow squares; n = 49), c-tmem2 mRNA (gray triangles; n = 51), and sc-tmem2 mRNA (red diamonds; n = 66).

(F) Schematic of Tmem2 protein and blow-up of the region where three newly identified tmem2 mutant alleles derived from forward genetic screens are located (tmem2^hu4800, tmem2^uq1ks, and tmem2^uq2ks) as well as mutations generated for analysis in (G).

(G) Scatterplots representing the number of ISVs at 48 hpf per embryo in uninjected siblings (n = 25), tmem2^hu5935 mutants (n = 68), and tmem2^hu5935 mutants injected with full-length tmem2 mRNA (yellow squares; n = 38) or mRNA of the PbH1 domain mutants tmem2^W429A (gray triangles; n = 43), tmem2^R439A (red triangles; n = 17), and tmem2^F608A (blue diamond; n = 14), where none of the PbH1 mutants showed capability to rescue. ^∗∗∗∗p < 0.0001; ns, not significant. Error bars represent ±SD.

Expression Data

Genes:	cemip2 EGFP mCherry
Fish:	WT uq3ksTg; y1Tg
Anatomical Terms:	angiogenic sprout caudal vein plexus endothelial cell plasma membrane intersegmental vein posterior cardinal vein posterior cardinal vein endothelial cell trunk vasculature whole organism
Stage Range:	Prim-5 to Long-pec

Expression Detail

Antibody Labeling

Phenotype Data

Fish:	cemip2^{hu5935/hu5935}
Observed In:	intersegmental vessel intersegmental vessel sprouting angiogenesis
Stage:	Long-pec

Phenotype Detail

Acknowledgments

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Reprinted from Developmental Cell, 40, De Angelis, J.E., Lagendijk, A.K., Chen, H., Tromp, A., Bower, N.I., Tunny, K.A., Brooks, A.J., Bakkers, J., Francois, M., Yap, A.S., Simons, C., Wicking, C., Hogan, B.M., Smith, K.A., Tmem2 Regulates Embryonic Vegf Signaling by Controlling Hyaluronic Acid Turnover, 123-136, Copyright (2017) with permission from Elsevier. Full text @ Dev. Cell