Expression patterns of astrocyte elevated gene-1 (AEG-1) during development of the mouse embryo

Abstract

Expression of astrocyte elevated gene-1 (AEG-1) is elevated in multiple human cancers including brain tumors, neuroblastomas, melanomas, breast cancers, non-small cell lung cancers, liver cancers, prostate cancers, and esophageal cancers. This gene plays crucial roles in tumor cell growth, invasion, angiogenesis and progression to metastasis. In addition, over-expression of AEG-1 protects primary and transformed cells from apoptosis-inducing signals by activating PI3K-Akt signaling pathways. These results suggest that AEG-1 is intimately involved in tumorigenesis and may serve as a potential therapeutic target for various human cancers. However, the normal physiological functions of AEG-1 require clarification. We presently analyzed the expression pattern of AEG-1 during mouse development. AEG-1 was expressed in mid-to-hindbrain, fronto-nasal processes, limbs, and pharyngeal arches in the early developmental period from E8.5 to E9.5. In addition, at stages of E12.5-E18.5 AEG-1 was localized in the brain, and olfactory and skeletal systems suggesting a role in neurogenesis, as well as in skin, including hair follicles, and in the liver, which are organ sites in which AEG-1 has been implicated in tumor development and progression. AEG-1 co-localized with Ki-67, indicating a role in cell proliferation, as previously revealed in tumorigenesis. Taken together, these results suggest that AEG-1 may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation, and that temporal regulation of AEG-1 expression may be required during specific stages and in specific tissues during development.

Figure 1. Expression of AEG-1 in the mouse embryo

(A) Real-time PCR at embryonic stages E8.5 to E10.5 and (B) whole mount immunostaining with anti-AEG-1 at E9.5 and E10.5 were performed. Real-time PCR was performed at least three times in duplicate or triplicate, and the minimal number of embryos (n) used at each stage was 9. The expression of AEG-1 was normalized by GAPDH. Error bars indicate SEM.

Figure 2. Expression of AEG-1 in the developing brain

(A) Brains were collected from E10.5 to E18.5, and total RNA was prepared. Real-time PCR was performed at least three times in duplicate or triplicate (n=3) and the expression of AEG-1 was normalized by GAPDH. Error bars indicate SEM. (B) The section of the embryonic brain at E10.5 was stained with AEG-1 (Red). The scale bar represents 0.2 mm. OV: optic vesicle, NE: neuroectoderm, and MC: mesenchyme. (C) Immunostaining of brain sections at E12.5 to E18.5 was performed using anti-AEG-1 antibody. The expression of AEG-1 in whole brain (a-d, 100X magnification), cortex (e-h), choroid plexus (i-l) and cerebral region (m-p) (e-p: 200X magnification) were shown. LV: lateral ventricle.

Figure 3. Expression of AEG-1 in the skeletal and olfactory systems

(A) The skeletal system and (B) olfactory system were stained with anti-AEG-1 in E12.5 to E18.5 embryos (left, 100X; middle, 200X; right, 400X). AF: annulus fibrosus, NP: nucleus pulposus, RG: root ganglia and SP: spinal column. White square: Y-shaped olfactory system at E12.5 and NC: nasal cavity.

Figure 4. Expression of AEG-1 in the skin and hair follicle

(A) The skin was thin layered at E12.5, and stratified dermis and epidermis were detected at E14.5 to E18.5. Distinct expression of AEG-1 was detected in the epidermis (left, 100X; middle, 200X; right, 400X). (B) AEG-1 was detected in most hair follicles at E14.5, with restricted expression to defined regions at E16.5, and barely detectable at E18.5 (left, 100X; middle, 200X; right, 400X).

Figure 5. Expression of AEG-1 in the developing liver

Immunohistochemistry was performed on sagittal sections (4 mm) at E12.5 to E18.5. The expression of AEG-1 in liver at each stage is shown (left, 100X; middle, 200X; right, 400X).

Figure 6. Expression of AEG-1 in the developing limb and its colocalization with Ki-67

(A) The expression of AEG-1 in the limb was examined at E13.5 and E15.5 and at post natal day 1 (D1). (B) The section of the embryonic skin at E14.5 was stained with DAPI (Blue), AEG-1 (Red), and Ki-67 (Green). The parts of dermis and epidermis are shown (upper panel, 250X magnification). The cells expressing AEG-1 and Ki-67 in the dermis are magnified (lower panel, 1000X).