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, 99 (14), 9248-53

Erk5 Null Mice Display Multiple Extraembryonic Vascular and Embryonic Cardiovascular Defects

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Erk5 Null Mice Display Multiple Extraembryonic Vascular and Embryonic Cardiovascular Defects

Christopher P Regan et al. Proc Natl Acad Sci U S A.

Abstract

Erk5 is a mitogen-activated protein kinase, the biological role of which is largely undefined. Therefore, we deleted the erk5 gene in mice to assess its function in vivo. Inactivation of the erk5 gene resulted in defective blood-vessel and cardiac development leading to embryonic lethality around embryonic days 9.5-10.5. Cardiac development was retarded largely, and the heart failed to undergo normal looping. Endothelial cells that line the developing myocardium of erk5-/- embryos displayed a disorganized, rounded morphology. Vasculogenesis occurred, but extraembryonic and embryonic blood vessels were disorganized and failed to mature. Furthermore, the investment of embryonic blood vessels with smooth muscle cells was attenuated. Together, these data define an essential role for Erk5 in cardiovascular development. Moreover, the inability of Erk5-deficient mice to form a complex vasculature suggests that Erk5 may play an important role in controlling angiogenesis.

Figures

Figure 1
Figure 1
Expression of erk5 during development and in adult tissues. Multitissue blots from CLONTECH were hybridized with erk5 cDNA probes as described in Materials and Methods. (A) A high level of erk5 mRNA was present at various embryonic stages, with a significant increase between E7 and E11 of development. (B) Ubiquitous but low-level erk5 expression was observed in various adult tissues: Ht, heart; Br, brain; Sp, spleen; Lu, lung; Sm, skeletal muscle; Kd, kidney; Ts, testis. (C and D) In situ hybridization of E9.5 was performed to localize erk5 expression. High levels of erk5 expression were localized to the developing heart and vascular structures. (E and F) Serial sections also were hybridized with an SM22α probe to highlight the developing cardiovascular system. Of note, regions that showed an SM22α signal were similar to those that showed an erk5 signal. FB, forebrain; BA, branchial artery; DA, dorsal aorta; OT, outflow tract; AS, aortic sac; A, atrium, V, ventricle.
Figure 2
Figure 2
erk5−/− embryos displayed multiple defects in cardiac development. Lateral view of the wild-type (A) and mutant (B) E9.5 embryos demonstrate retarded cardiac development in erk5−/− embryos. Arrows point to the heart region. Histological examination of +/+ (C) and −/− (D) hearts revealed that erk5−/− hearts failed to undergo normal looping. The common atrial chamber of the mutant hearts also appeared enlarged, the ventricular trabeculations (arrowheads) were attenuated and disorganized, and there was notable pericardial edema (asterisk). A, atrium; V, ventricle. High-power magnification of the hearts of wild-type (E) and erk5−/− (F) embryos revealed that the endothelial cells (arrowheads) of mutant hearts were rounded as compared with the thin, elongated cells present in the wild type.
Figure 3
Figure 3
Analysis of gene expression in E9.5 erk5 embryos. (A) Western blot of erk5+/+ and erk5−/− MEFs shows absence of functional Erk5 in knockout mice. Lane 1, +/+, unstimulated; lane 2, +/+, 100 ng/ml EGF; lane 3, −/−, unstimulated; lane 4, −/−, 100 ng/ml EGF. (B) RT-PCR of RNA isolated from erk5+/+ and erk5−/− E9.5 hearts showed decreased expression of a potential Mef2C-dependent gene, cripto, in mutant embryos. L7 was used as an internal control. (C) RNA extracted from +/+, +/−, and −/− embryos was used to amplify a panel of genes known to play important roles for vascular development (flt-1 and flt-4, tie-1 and tie-2, agpt-1 and agpt-2, flk-1, and vegf) and hematopoiesis (IL-3R and βH-1). L7 was used as an internal control. No obvious expression differences among genotypes were noted.
Figure 4
Figure 4
erk5−/− embryos displayed defects in yolk-sac and placental angiogenesis. Whole-mount visualization of wild-type (A) and mutant (B) E9.5 yolk sacs demonstrated that the large collecting vessels present in wild-type yolk sacs (arrow) were nearly absent in erk5−/− mice. Further histological analysis revealed the well defined collecting vessels in wild-type yolk sacs (arrow in C) were collapsed and contained fewer blood cells in erk5−/− yolk sacs (arrow in D). Placental vasculature was analyzed in hematoxylin/eosin-stained sections of wild-type (E) and mutant (F) placentas. Embryonic vessels, as defined by the presence of nucleated red cells, are outlined in red and maternal vessels are outlined in green to better highlight differences. (F) erk5−/− placentas had fewer embryonic vessels, and the vessels failed to invade deep into the labyrinth layer as compared with wild type.
Figure 5
Figure 5
Embryonic vascular defects were present in erk5−/− embryos. Whole-mount PECAM staining of +/+ (A) and −/− (B) E9.5 embryos revealed defects in vascular maturation/angiogenesis in erk5−/− mice. Intersomitic vessels (wild-type, Lower Right Inset in A; mutant, Lower Right Inset in B) appeared normal in erk5−/− embryos. However, the angiogenic pruning and remodeling that was present in the head region of wild-type embryos (Upper Right Inset in A) was absent in erk5−/− embryos (Upper Right Inset in B). Note the disorganized vasculature and lack of complex branching in the erk5−/− embryos. Further analysis of the embryonic vasculature revealed that SmαA-positive cells did not invest the vessels of mutant embryos efficiently. (C) Dorsal aorta. (D) Branchial artery, wild type. (E) Dorsal aorta. (F) Branchial artery, erk5−/−.

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