Is Senescence-Associated β-Galactosidase a Reliable in vivo Marker of Cellular Senescence During Embryonic Development?

Abstract

During vertebrate embryonic development, cellular senescence occurs at multiple locations. To date, it has been accepted that when there has been induction of senescence in an embryonic tissue, β-galactosidase activity is detectable at a pH as high as 6.0, and this has been extensively used as a marker of cellular senescence in vivo in both whole-mount and cryosections. Such senescence-associated β-galactosidase (SA-β-GAL) labeling appears enhanced in degenerating regions of the vertebrate embryo that are also affected by programmed cell death. In this sense, there is a strong SA-β-GAL signal which overlaps with the pattern of cell death in the interdigital tissue of the developing limbs, and indeed, many of the labeled cells detected go on to subsequently undergo apoptosis. However, it has been reported that β-GAL activity at pH 6.0 is also enhanced in healthy neurons, and some retinal neurons are strongly labeled with this histochemical technique when they begin to differentiate during early embryonic development. These labeled early post-mitotic neurons also express other senescence markers such as p21. Therefore, the reliability of this histochemical technique in studying senescence in cells such as neurons that undergo prolonged and irreversible cell-cycle arrest is questionable because it is also expressed in healthy post-mitotic cells. The identification of new biomarkers of cellular senescence would, in combination with established markers, increase the specificity and efficiency of detecting cellular senescence in embryonic and healthy mature tissues.

Keywords: cell death; cell senescence; development; histochemistry; limb; retina.

FIGURE 1

Areas segmenting SA-β-GAL activity and apoptosis during avian embryonic development. Detection of SA-β-GAL activity in embryonic day 3.5 (A,B) and E4 (C) chicken embryos, and E3.5 (D), E6 (E), E7 (F), and E8 (G) hindlimbs. Neutral red staining for cell death detection in E3.5 (H), E6 (I), E7 (J), and E8 (K) hindlimbs. TUNEL assay for apoptosis detection in E4 (L), E6 (M), E7 (N), and E8 (O) hindlimbs. Labeling of AER can be noted in (D,H, and L). Arrowheads in (E–G), (I–K), and (M–O) point to the third interdigital space during the establishment of cell senescence and the progression of interdigital programmed cell death. SA-β-GAL histochemistry (P) and QH1 immunostaining (R) label macrophages in the interdigital mesenchyme of quail at stage 36. DAPI staining (Q) shows the structure of the interdigital space. E, eye; Fl, forelimb; H, heart, HL, hindlimb, Le, lens; NT, neural tube, O, otic vesicle; PA, pharyngeal arches; T, tail bud.

FIGURE 2

The presence of SA-β-GAL activity in the postnatal day P3 mouse head tissue. Horizontal (A,B) and sagittal (C–E) cryosections were treated with SA-β-GAL histochemistry. (A,B) Intense SA-β-GAL signal is found in the intermediate layer of the olfactory epithelium (arrowheads). (C) Strong SA-β-GAL staining is detected in sensory neurons in the trigeminal ganglion (asterisks). (D,E) SA-β-GAL activity is detected in the cerebellum, mainly in the Purkinje cell layer (arrows). Scale bars: 200 μm (A,D), 50 μm (B,E), and 20 μm (C).

FIGURE 3

The presence of SA-β-GAL activity in the embryonic day E15 chicken retina. Cryosections of retinas were treated with SA-β-GAL histochemistry (A,B) and antibodies against p21 (B–D). DAPI staining shows the laminated structure of the retina (C). SA-β-GAL staining is found in the photoreceptor outer segments and in subpopulations of amacrine and ganglion cells (A,B). The horizontal cell layer appears faintly labeled (A,B). p21 immunostaining strongly correlates with the SA-β-GAL labeling pattern. ac, amacrine cells; gc, ganglion cells; GCL, ganglion cell layer; hc, horizontal cells; INL, inner nuclear layer; ONL, outer nuclear layer; pos, photoreceptor outer segments. Scale bars: 50 μm (A and B–D).

FIGURE 4

The presence of SA-β-GAL activity in the embryonic day E4 chicken retina. Cryosections of retinas were treated with SA-β-GAL histochemistry and antibodies against p21 (A–E), CatD (F–H), and TUJ1 (I–K). DAPI staining shows that the neural retina consists of a NbL (C,F,I). SA-β-GAL staining is detected in the scleral (asterisks in A,D,G,J) and vitreal (arrowheads in A,D,G,J) regions of the retina. p21 immunostaining correlates with the SA-β-GAL staining pattern in the undifferentiated retina (arrowheads and asterisks in B,E) and lens (arrows in B). CatD immunoreactivity (arrowheads and asterisks in H) is strongly coincident with the SA-β-GAL histochemistry signal (arrowheads and asterisks in G). TUJ1 immunoreactivity is intense in the vitreal surface of the NbL (arrowheads in K), coinciding with the vitreal SA-β-GAL histochemistry signal detected in the same region (arrowheads in J). Le, lens; NbL, neuroblastic layer. Scale bars: 150 μm (A,B), 50 μm (C–K).

FIGURE 5

SA-β-GAL activity and cell death in the embryonic day E9 (A) and E10 (B) chicken retina. Cryosections were doubly stained with SA-β-GAL histochemistry and TUNEL technique. TUNEL-positive nuclei are mainly detected in the GCL (arrowheads) and in the middle region of the INL (arrows). SA-β-GAL activity is observed in the GCL, amacrine cell layer, and horizontal cell layer. ac, amacrine cells; gc, ganglion cells; GCL, ganglion cell layer; hc, horizontal cells; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; pos, photoreceptor outer segments. Scale bar: 50 μm.