Electrophysiology showed that sensitivity of GGA knockdowns was only
slightly diminished relative to appropriate controls out to 22 days post-eclosio
light-reared. Presence of ERG on-and off-transients showed that R1-6 synaptic
connections to the first order visual neuropil (the lamina ganglionaris)
were functional. In transmission electron microscopy, structure of the GGAkd
retina and first optic neuropil were strikingly normal with virtually no
signs of recrptor degeneration at 8 days post-eclosion light-reared. The
only obvious abnormality is that some cells had split rhabdomeres. Following
out to 37 days-post-eclosion in the light, receptors were still healthy
though there was an accumulation of membrane circles and intraretinular
pigment granules. Considering the overall normality in electrophysiology
and electron microscopy, the deep pseudopupil was non-existant from newly-emerged
dark-reared out to >40 days in the light. Using the optical neutralization
technique, oddly, though in disarray, rhabdomere tips were present and similar
in appearance from newly-emerged dark-reared out to >40 days in the light.
Based on histological observations, Eissenberg
et al. claimed that GGAkd flies had age-dependent retinal degeneration;
my electrophysiology, ultrastructure and optics contradict this conclusion.
ERGs of GGA knockdowns and controls
Animals. GGA knockdown - Female GGAE3 X Male eyGal4+GMRGal4.
The appropriate control was driver only. The control for the control was
Electrophysiology. Electrographic (ERG) methodology analysis
has been standard in this laboratory for decades (Stark, 1973) (McKay et
al, 1995). For this work, the stimulus was from a 150 W Xenon Arc in an
Opti-Quip housing and 1600 power supply and the readout was via a PowerLab
410 feeding into a Macintosh computer running operating system 9.2. To photograph
the deep pseudopupil (DPP) before running the ERG, the cover slip the flies
were fixed to was optically fused to a microscope slide using a drop of
water, and the DPP was viewed with a 10x air objective. For the ERG, a long
wavelength, 585 nm, was chosen for two reasons: (1) potential differences
between the effects of eye color pigments of experimental and control flies
would be minimized (Stark, 1973); and (2) visualization of on- and off transients
would be optimized (Stark & Wasserman, 1972). Data acquisition was expedited
by quickly obtaining responses to a defined sequence of seven stimuli of
1.0 s 0.3 to 0.6 log units apart (an intensity response sequence). After
obtaining ERGs, the cover slip was again placed on a slide, and rhabdomeres
were photographed using oil immersion and a 40x objective.
ERGs of GGA knock-downs were surprisingly consistent with the expectation
for "normal" flies. Near threshold, i.e. when the ERG component
attributed to receptor depolarization was 0-3 mV, there were on- and off-transients
that were fairly large. For higher intensities, the receptor wave was larger,
and the transients were diminished. At the highest intensities, the receptor
component sometimes settled back rather than just swing up to a steady state.
Newly emerged GGA knockdown flies were run as well as flies of various ages
out to 22 days, and they all had similar ERG responsivities and sensitivities.
ERG on- and off-transients not only imply a high level of R1-6 function
but also that the synaptic connections of R1-6 in the first order optic
neuropile, the lamina ganglionaris, are functional. GGA knock downs, (top)
and driver-only controls (second row) are shown. Newly emerged (left), 6-8
days (second column), 15 days (3rd column) and 22 days (right). For comparison,
wild-type newly emerged is at the bottom left. The vertical scales have
been adjusted to be equivalent
Newly eclosed, knockdown and driver are about the same sensitivity and about
0.6 log units than wild type. With age, the knockdown stays about the same
while the driver only stock increases to wild type, consistent with the
increase in rhabdomere size in only the driver-only stock. Assuming the
lower sensitivity of the GGA knockdown were based on strictly on a lower
amount of rhodopsin, then the inference is that the GGA knockdown has about
0.25 x the normal rhodopsin amount since sensitivity and visual pigment
content were shown to be linearly related in the invertebrate (Hamdorf &
Schwemer, 1975). Contrast the approximate 0.6 log unit diminution of the
GGA knockdown's sensitivity with the over 2 log unit decrease of sensitivity
of vitamin A deprived flies (Stark & Zitzmann, 1976).
Hamdorf K, Schwemer J (1975) Photoregeneration and the adaptation process
in insect photoreceptors of invertebrates. In Photoreceptor optics,
Snyder AW, Menzel R (eds), pp 263-289. Berlin: Springer
McKay RR, Chen DM, Miller K, Kim S, Stark WS, Shortridge RD (1995) Phospholipase
C rescues visual defect in norpA mutant of Drosophila melanogaster.
J Biol Chem 270: 13271-13276
Stark WS (1973) The effect of eye colour pigments on the action spectrum
of Drosophila. J Insect Physiol 19: 999-1006
Stark WS, Wasserman GS (1972) Transient and receptor potentials in the electroretinogram
of Drosophila. Vision Res 12: 1771-1775
Stark WS, Zitzmann WG (1976) Isolation of adaptation mechanisms and photopigment
spectra by vitamin A deprivation in Drosophila. J Comp Physiol
work completed Summer 2009
Transmission Electron Microscopy
microscopy (TEM). Control (left) and GGA knockdown (right). Flies were
aged eight days in normal room lighting after emergence as adults from the
pupa case. Roughly equivalent fields of distal retina (top) and receptor
axons projecting to the first optic neuropil ("brain") (bottom)
were selected for comparison. There is only one striking abnormality, and
that concerns the rhabdomeres, the rhodopsin-containing photoreceptive organelles
(dark circles in the retina at the top): The number, shape, size and organization
for the GGA knockdown (right) does not match the tidy organization of 7
rhabdomeres per ommatidium (facet in the compound eye) for the control (left).
Considering how bad the retina looks optically, in the deep pseudopupil
and with optical neutralization of the cornea (see this
text), it is astounding how many untrastructural features of the visual
system in eight day post-eclosion adults are close to normal. Retinula cells
appear healthy and have normal nuclei and mitochondria (1)(3).
They are connected to their neighboring retinula cells with the appropriate
belt desmosomes (2)(3).
Rhabdomeres have the customary submicrovillar cisternae (3).
Within microvilli, there is the usual electron dense rod (3).
Also retinula cells have normal intraretinular pigment granules (5)
and rhabdomere caps (6).
The intraretinular pigment granules are known to migrate toward the rhabdomere
in the light, and their position is indicative that the cell was responding
to light at the time of fixation (5).
Axons from the retina proceed through the basement membrane to the first
optic neuropil, the lamina ganglionaris, where the R1-6 terminals form into
the appropriate synaptic glomeruli, optic cartridges (7,
control left, GGA kd right, bottom of figure). The reason this was considered
to be important was that, in rdgB (which has light-induced retinal degeneration),
R1-6 terminals in the optic cartridges showed gross degeneration under the
minimal dim red light conditions sufficient for dissection for fixation
(Stark & Sapp, 1989), well before the retinula cells in the retina fill
with a dense reticulum and lipid droplets (Stark & Carlson, 1982). Close
examination reveals the membrane specializations that characterize functional
synapses, the T-bars (8).
left, control right) It is tempting to speculate that the rich investment
of membrane circles in the retinula cell cytoplasm near the rhabdomere is
a characteristic feature of the GGA kd; however, the control also shows
these structures, and it is not realistic to compare them quantitatively.
Such circles were posited as the vehicles to carry rhodopsin and/or membrane
to rhabdomeres during vitamin A replacement (Stark et al, 1988); also they
were plentiful in ora (outer rhabdomeres absent) where there were no rhabdomeres
to receive membrane intended for rhabdomeres (Stark & Sapp, 1987) and
after retinoic acid feeding (Lee et al, 1996). Importantly, they fill the
retinula cell cytoplasm in Rab11 mutant cells that lack rhodopsin transport
to the rhabdomeres (Satoh et al, 2005). Such vesicles also fill the cytoplasm
in Drosophila Rip11 (Rab11 interacting protein) mutants (Li et al, 2007).
[The knockdown figure (left) shows a split R7 rhabdomere and several gross
abnormalities at the 11 and 2 o'clock positions, presumably in secondary
This list of healthy features focuses our attention on the most striking
abnormalities: the size, orientation and number of rhabdomeres in each ommatidium
are irregular (2);
also there are gaps between ommatidia (9);
some gaps may represent fused or fragmented ommatidia, others may have resulted
from damage during tissue preparation for fixation. Upon examination of
ommatidia with too many rhabdomeres, the retinula cell count is usually
correct; thus retinula cells often have too many rhabdomeres (2)
(4). While autophagic
bodies, large endosomes, abnormal lysosome-related bodies, or disrupted
biosynthetic machinery (Golgi apparatus or rough endoplasmic reticulum)
might have been expected, no striking alterations from control were present.
TEM of 37 day ey GMR GGA knockdown
Many of the descriptions for 8
day apply here
like MVBs but with double membrane bounded vesicles and pits
Here is shown
a tremendous quantity of membrane circles and intraretinular pigment granules
in a plane near the basement membrane; see discussion of circles here.
Here we saw one
(and only one) retinula cell that appeared to be dead or dying.
Here is shown
a peculiar basement membrane, very swolen
Here is a distal section,
oblique (nearly longitudinal) showing the rhabdomere split and looking like
beads on a string
The optic cartridge is shown here
with fairly normal structure and conspicuous T-synapses
TEM of 24 day ey GMR GGA knockdown
is the abnormal accumulation of circles and pigment granules
like MVBs but with double membrane bounded vesicles and pits (as in 37 day)
is an indication of normal synaptic structure
Lee RD, Thomas CF, Marietta RG, Stark WS (1996) Vitamin A, visual pigments
and visual receptors in Drosophila. Micros Res Tech 35:
Li BX, Satoh AK, Ready DF (2007) Myosin V, Rab11, and dRip11 direct apical
secretion and cellular morphogenesis in developing Drosophila photoreceptors.
J Cell Biol 177: 659-669
Satoh AK, O'Tousa JE, Ozaki K, Ready DF (2005) Rab11 mediates post-Golgi
trafficking of rhodopsin to the photosensitive apical membrane of Drosophila
photoreceptors. Development 132(7): 1487-1497
Stark WS, Carlson SD (1982) Ultrastructural pathology of the compound eye
and optic neuropiles of the retinal degeneration mutant (w rdgBKS222) Drosophila
melanogaster. Cell Tissue Res 225: 11-22
Stark WS, Sapp RJ (1987) Ultrastructure of the retina of Drosophila melanogaster:
The mutant ora (outer rhabdomeres absent) and its inhibition of degeneration
in rdgB (retinal degeneration-B). J Neurogenet 4: 227-240
Stark WS, Sapp RJ (1989) Retinal degeneration and photoreceptor maintenance
in Drosophila: rdgB and its interaction with other mutants. In Inherited
and Environmentally Induced Retinal Degenerations, LaVail MM, Anderson
RE, Hollyfield JG (eds), pp 467-489. New York: Liss
Stark WS, Sapp RJ, Schilly D (1988) Rhabdomere turnover and rhodopsin cycle:
maintenance of retinula cells in Drosophila melanogaster. J Neurocytol
In landmark papers, Franceschini and Kirschfeld (Franceschini & Kirschfeld,
1971a) (Franceschini & Kirschfeld, 1971b) revolutionized the study of
fly photoreceptor optics. Because the index of refraction of water or immersion
oil approximate that of the cornea, the focusing function of these facet
lenslets is eliminated when the eye is viewed with an immersion objective.
Also, rhabdomeres carry light like a fiber optic (light pipe). Hence, this
"optical neutralization of the cornea" technique allows viewing
of rhabdomere tips when light is transmitted up (antidromically) through
the eye. Because of the regularity of rhabdomeres in each ommatidium and
the regularity of the arrangement of ommatidia, a low power air objective
can be used to image the "deep pseudopupil," a virtual image of
the rhabdomere tips, magnified deep (about 150 microns beneath the cornea)
and superimposed from about 25 ommatidia (that number depending on the numerical
aperture of the microscope objective). Stark & Thomas (2004) is a good
(Here is a plate
made from GGA kd 43 days (left) [F1 from male eyGMRdriver x female GGA.E3],
control eyGMR 42 days (middle) [eyGMRdriver;boss/+], GGA control 40 days
Because the deep pseudopupil and the optical neutralization of the cornea
techniques are relatively straightforward, they have long served as a convenient
entry point to diagnose the structural integrity of the retina. The deep
pseudopupil of GGA knockdowns (driven by ey GMR) was in such disarray that
there was no focal plane that showed any rhabdomere clearly. This was invariably
the case at every time point assayed ranging from zero days (in the dark
or in the light) out to 4 weeks. Focusing up and down, the eye color pigment
seemed mottled and uneven.
a selected depth series to demonstrate these points from GGA kd 43 days
post-eclosion where 1 = distal, 6 = deeper than the deep pseudopupil
In optical neutralization, rhabdomeres, though abnormal, were present (Figure)
and were the same on day zero dark reared and 29 days light reared.
Under optical neutralization of the cornea, rhabdomeres were present at
all time points. However, rarely was a single rhabdomere imaged clearly.
The number, size, and orientation of rhabdomeres that could be imaged per
ommatidium was always variable. In contrast, rdgB (retinal degeneration
B, even amid a wasteland of dead R1-6 cells and debris) and ora (outer rhabdomeres
absent, lacking R1-6 rhabdomeres) gave clear images of R7 (and clearly lacking
R1-6) (Harris et al, 1976).
Why were the GGA knockdown's pseudopupils so disrupted? The diagnoses were
straightforward when Harris et al (1976) utilized the pseudopupil techniques
developed by (Franceschini & Kirschfeld (1971a; b): R1-6 was
clear but R 7 was missing in sevenlses (sev); R7 was clear but R1-6 was
missing in outer rhabdomeres absent (ora in what Washburn & O'Tousa,
(1989) later showed to be a rhodopsin mutation) and in retinal degeneration
With hindsight, it is obvious that the optical neutralization of the cornea
presaged the subsequent histological and ultrastructural findings of variable
numbers, sizes, counts and configurations of rhabdomeres in the ommatidia.
Since the deep pseudopupil is formed by the magnified, superimposed images
of the rhabdomere tips collected from many ommatidia, it is reasonable to
conclude that all the factors of rhabdomere disarray preclude a good deep
Franceschini N, Kirschfeld K (1971a) Etude optique in vivo des elements
photorecepteurs dans l'oeil compose de Drosophila. Kybernetik
Franceschini N, Kirschfeld K (1971b) Les phenomenes de pseudopupille dans
l'oeil compose de Drosophila. Kybernetik 9: 159-182
Harris WA, Stark WS, Walker JA (1976) Genetic dissection of the photoreceptor
system in the compound eye of Drosophila melanogaster. J Physiol
(Lond) 256: 415-439
Stark WS, Thomas CF (2004) Microscopy of multiple visual receptor types
in Drosophila. Mol Vis 10: 943-955
Washburn T, O'Tousa JE (1989) Molecular defects in Drosophila rhodopsin
mutants. J Biol Chem(264): 15464-15466
Acknowledgements. The work described above was done Summer 2009-Spring
2010 as part of a collaboration with Joel Eissenberg, Department of Biochemistry
and Molecular Biology, Saint Louis University School of Medicine. Dr Eissenberg
sponsored my SLU-funded sabbatical Spring 2010. The SLU Presidents Research
Fund and Beaumont grant funded some of these studies.The hairpin construct
allowing RNAi for GGA was provided to Prof. Eissenberg by Andre C. Dennes,
DanielaWaschkau and Regina Pohlmann at the UKM Munster. Electron microscopy
was done in collaboration with Dr. Jan Ryerse and Ms Barbara Nagel of SLU's
Imaging Core; the grids for the 8 day TEM analysis were from Prof. Eissenberg.
Many of the GGAkd flies were isolated and delivered to me by Anne Ilvarsonn.