Results and discussion
Based on the embryonic stages of axolotl outlined by
Armstrong and Malacinski [7], we histologically evaluated
lens development in embryos as early as stage 36. Our evaluation
showed that by stage 44, the lenses had developed
well with full differentiation of the globe having a clear
lens epithelium and lens fibers (Figure 1). Since this is the
stage at which hatching occurs (and no further staging is
available), we decided to remove the lens at different time
intervals after stage 44 starting with day 1 to day 27. Each
group was examined at different times after lentectomy to
evaluate if animals were able to regenerate their lenses.
We found that lens regeneration was possible within a
particular time window after lens removal in animals up
to 14 days after hatching (stage 4; Table 1, Figure 2). After
that window of time, axolotls were found incompetent of
regenerating their lenses. The regeneration process was
very fast. Within one to two days after lentectomy, a well
differentiated lens was present. The frequency of lens
regeneration was highest when lentectomy was performed
3 or 7 days after stage 44; there were 10/16 (62.5%) and
8/10 (80%) regenerated lenses, respectively. We believe
that the failed cases were most likely the result of trauma
to the eye due to lentectomy. The eye of the axolotl at
these stages is very small and no matter how carefully lentectomy
is performed some injury is unavoidable. When
we examined later stages, such as 27 days past stage 44,
axolotls were no longer able to regenerate the lens. In
Table 2 in parentheses we include staging beyond stage
44, according to limb development [8].
Histological sections at day 1 after lentectomy hinted
that the lens regenerates from the iris; however, it could
be formed from either the dorsal or the ventral iris.
Prompted by these findings we decided to analyze lens
regeneration in more detail. For this, we removed the lens
at stage 44 + 8 days and analyzed the process of regeneration
3, 6, 12, 24 and 48 hours post-lentectomy. Three
hours before collection, animals were injected with BrdU
to examine cell proliferation. The collected animals were
embedded, sectioned and incubated with BrdU antibody
as well as with g-crystallin antibody that marks lens fibers.
The histological series are presented in Figure 3. In the
top panel, sections at 0 hour, just after removing the lens,
are shown. BrdU staining was only observed at the ciliary
margin, where proliferating retinal stem/progenitor cells
contribute to the growing retina. As expected, g-crystallin
Results and discussion
Based on the embryonic stages of axolotl outlined by
Armstrong and Malacinski [7], we histologically evaluated
lens development in embryos as early as stage 36. Our evaluation
showed that by stage 44, the lenses had developed
well with full differentiation of the globe having a clear
lens epithelium and lens fibers (Figure 1). Since this is the
stage at which hatching occurs (and no further staging is
available), we decided to remove the lens at different time
intervals after stage 44 starting with day 1 to day 27. Each
group was examined at different times after lentectomy to
evaluate if animals were able to regenerate their lenses.
We found that lens regeneration was possible within a
particular time window after lens removal in animals up
to 14 days after hatching (stage 4; Table 1, Figure 2). After
that window of time, axolotls were found incompetent of
regenerating their lenses. The regeneration process was
very fast. Within one to two days after lentectomy, a well
differentiated lens was present. The frequency of lens
regeneration was highest when lentectomy was performed
3 or 7 days after stage 44; there were 10/16 (62.5%) and
8/10 (80%) regenerated lenses, respectively. We believe
that the failed cases were most likely the result of trauma
to the eye due to lentectomy. The eye of the axolotl at
these stages is very small and no matter how carefully lentectomy
is performed some injury is unavoidable. When
we examined later stages, such as 27 days past stage 44,
axolotls were no longer able to regenerate the lens. In
Table 2 in parentheses we include staging beyond stage
44, according to limb development [8].
Histological sections at day 1 after lentectomy hinted
that the lens regenerates from the iris; however, it could
be formed from either the dorsal or the ventral iris.
Prompted by these findings we decided to analyze lens
regeneration in more detail. For this, we removed the lens
at stage 44 + 8 days and analyzed the process of regeneration
3, 6, 12, 24 and 48 hours post-lentectomy. Three
hours before collection, animals were injected with BrdU
to examine cell proliferation. The collected animals were
embedded, sectioned and incubated with BrdU antibody
as well as with g-crystallin antibody that marks lens fibers.
The histological series are presented in Figure 3. In the
top panel, sections at 0 hour, just after removing the lens,
are shown. BrdU staining was only observed at the ciliary
margin, where proliferating retinal stem/progenitor cells
contribute to the growing retina. As expected, g-crystallin
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