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DEVELOPMENTAL DYNAMICS 228:414 – 423, 2003
ARTICLE
Histology-Based Screen for Zebrafish Mutants with
Abnormal Cell Differentiation
Manzoor-Ali P.K. Mohideen,1 Lee G. Beckwith,1 Gladys S. Tsao-Wu,2 Jessica L. Moore,1 Andrew C.C. Wong,3
Mala R. Chinoy,2 and Keith C. Cheng1,4*
The power of histology to define states of cell differentiation was used as the basis of a mutagenesis screen in
zebrafish. In this screen, 7-day-old parthenogenetic half-tetrad larvae from potential carrier females were screened
for mutations affecting cell differentiation in hematoxylin and eosin-stained tissue sections. Seven,
noncomplementing, recessive mutations were found. Two mutations affect only the retina: segmented
photoreceptors (spr) show a discontinuous photoreceptor cell layer; vestigial outer segments (vos) has fewer
photoreceptor cells and degenerated outer segments within this cell layer. Three mutants have gut-specific defects:
the epithelial cells of kirby (kby) are replaced by ballooned cells; the intestines of stuffy (sfy) and stuffed (sfd) contain
increased luminal mucus. Two mutations affect multiple organs: disordered neural retina (dnr) has disrupted retinal
layering and mild nuclear abnormalities in the gut and liver; and in huli hutu (hht), the retinal cell layers are
disorganized and multiple organs have mild to severe nuclear abnormalities that are reminiscent of the atypia of
human neoplasia. Each mutation appears to be homozygous lethal. This screen is proof of principle for the feasibility
of histologic screens to yield novel mutations, including potential models of human disease. The throughput for this
type of screen may be enhanced by automation. Developmental Dynamics 228:414 – 423, 2003.
© 2003 Wiley-Liss, Inc.
Key words: zebrafish mutant; larval arrays; histology; cell differentiation; retina; cancer
Received 14 May 2003; Accepted 4 August 2003
INTRODUCTION
Infinite combinations of architectural and cytologic features of tissue
cells are detectable by the light microscopic study of stained tissue
sections. Histologic studies distinguish a multitude of cell types, cell
processes, and abnormalities. This
power is routinely exploited in the
field of pathology, where histologic
features are used to assess biologi-
cal behavior, identify abnormalities
in cell differentiation, and evaluate
cell injury in human diseases, including cancer (Demay, 1996; Cotran et
al., 1999). We reasoned that the
small size of zebrafish larvae containing primordia of most adult tissues,
combined with the high resolution
and sensitivity associated with light
microscopy of tissue sections, could
be harnessed to detect subtle
changes in cellular structure. Cloning of such mutations would allow
the dissection of their genetic basis.
The subtlety of many lesions routinely
assessed in pathology indicate that
many abnormalities in organogenesis and/or cytology would be difficult
or impossible to detect by microscopy of live larvae, even when
transparent. Pigmentation of the larvae further impairs detection of cel-
1
The Jake Gittlen Cancer Research Institute, Department of Pathology, Pennsylvania State University College of Medicine, Hershey,
Pennsylvania
2
The Jake Gittlen Cancer Research Institute, Department of Surgery, Pennsylvania State University College of Medicine, Hershey, Pennsylvania
3
Department of Human Genetics, University of Chicago, Chicago, Illinois
4
The Jake Gittlen Cancer Research Institute, Department of Biochemistry and Molecular Biology, Pennsylvania State University College of
Medicine, Hershey, Pennsylvania
Grant sponsor: National Institutes of Health; Grant numbers: RO1-CA73935; RO1-HD040179; NRSA IF32GM19794; Grant sponsor: NSF; Grant
number: MCB-93198174.
*Correspondence to: Keith C. Cheng, Jake Gittlen Cancer Research Institute H059, Pennsylvania State University College of Medicine,
500 University Drive, Hershey, PA 17033. E-mail: [email protected]
DOI 10.1002/dvdy.10407
© 2003 Wiley-Liss, Inc.
ZEBRAFISH HISTOLOGIC MUTANT SCREEN 415
lular changes. We expected a histology-based screen to find previously
undiscovered mutants for two reasons. First, stained tissue sections provide a higher resolution than unsectioned, unstained, whole embryos,
as used in two large published
screens (Haffter et al., 1996; Driever
et al., 1996). Second, organogenesis
is more advanced in 7-day larvae
than in embryos or younger larvae,
which permits identification of mutations affecting later stages of cell differentiation. The ability to embed zebrafish larval arrays in agarose
consisting of 64 larvae per block, developed in our laboratory (Tsao-Wu
et al., 1998) made the screen possible. The first histology-based genetic
screen, performed in Drosphila, was
reported by Heisenberg and Bohl
(1978). Here, we report the first histology-based genetic screen in a vertebrate.
Heterozygous mutations in carrier
females were rendered homozygous in their progeny by gynogenesis (Streisinger et al., 1981; Cheng
and Moore, 1997). Seven recessive
mutations with novel phenotypes
were obtained from this initial screen,
demonstrating that histology-based
screens can detect interesting mutants that do not otherwise exhibit
any easily identifiable nonhistologic
phenotypes. The mutants reported
here include two with defective retinal photoreceptor differentiation,
three with abnormal differentiation
of the intestinal epithelium, and two
with multiorgan defects. The latter
two show disruption of retinal layers
in addition to cytologic abnormalities in multiple organs. The mutants
found include models of retinal dysplasia and of cytologic dysplasia
seen in human cancer. The mutations identified in this screen are different from those reported in earlier
zebrafish screens, further validating
the utility of histology-based screens.
RESULTS AND DISCUSSION
Half-tetrad progeny were produced
from F1 females carrying N-ethyl-Nnitrosourea (ethylnitrosourea, ENU)
-induced mutations and were
screened for histologic defects at 7
days of development (Fig. 1). The
small size of zebrafish larvae (⬃5mm-long at 7 days postfertilization)
made it possible to fit a section from
an array of 64 larvae on one microscope slide (Tsao-Wu et al., 1998).
The screen was performed at 7 days,
because most organs have distinct
cell patterns and appearances by
that age (Kimmel et al., 1995). Larvae with gross abnormalities noted
under a dissection microscope were
discarded, based on the asumption
that mutations causing such defects
would have been found in past genetic screens. We chose not to use
older larvae because gut mutations
affecting absorption would be expected to be lost beginning on day
8, when yolk is normally depleted. A
total of 3,281 larvae from 72 histologic arrays of up to 7-day-old parthenogenetic half-tetrad larvae per
F1 female were initially screened at
⫻25–⫻400 magnification, and as
necessary, examined at ⫻1,000
magnification for additional cytologic detail. Among the embedded
larvae, 2,038 (62%) larvae were
screened for eye morphology and
1,670 (51%) were screened for intestinal morphology in at least one of
three stained sections. Seven putative mutants were obtained, all of
which showed transmission to at
least one succeeding generation
and exhibited Mendelian inheritance. Five organ-specific and two
pleiotropic mutations were found
(Table 1). Of these, vos and hht are
described in greater detail.
Eye-Specific Mutants
The two eye mutants vestigial outer
segments (vos) and segmented
photoreceptors (spr) appear normal
under the dissecting microscope.
Histologic examination of these two
mutants revealed defects specific to
the photoreceptor cell layer (PCL) of
the retina. The photoreceptor cells
of the wild-type 7-day zebrafish eye
are elongated and are oriented perpendicular to the outer circumference of the eye (Fig. 2A,B). The PCL
consists of the outer nuclear layer,
and inner and outer segments. The
outer nuclear layer stains blue with
Fig. 1. Genetic screen for histologic mutants in zebrafish. N-Ethyl-N-nitrosourea (ethylnitrosourea, ENU) was used to mutagenize adult
males, which were outcrossed with homozygous goldenb1 (gol) females to generate F1 progeny for screening. Early pressure parthenogenesis (EP) was used to generate half-tetrads from F1 females. At 7 days of age, half-tetrad larvae were fixed in 10% neutral buffered
formalin, and up to 64 sibs from individual EP families were arrayed in individual agarose blocks. After embedding in paraffin, 6-␮m-thick
sections were cut and stained with hematoxylin and eosin for histologic screening. F1 carriers that generated half-tetrad progeny with
interesting phenotypes were then outcrossed for further study, including confirmation of Mendelian inheritance. m, mutant.
Fig. 2. vos specifically affects the photoreceptor cell layer of the developing zebrafish retina. Figure depicts coronal sections through the
eye. A–D: Wild-type retina. E–H: vos mutant retina. A,B,E,F: hematoxylin and eosin–stained sections; C,D,G,H are scanning electron
photomicrographs. B,F: Enlarged views of the photoreceptor cell layer (indicated between arrowheads in A and E, respectively). C,D,G,H:
Outer segments of the photoreceptor cells; D and H are the magnified images of the boxed area in C and G, respectively. rpe/c, retinal
pigment epithelium/cells; is/os, inner segment/outer segment of the photoreceptor cells; onl, outer nuclear layer; inl, inner nuclear layer;
ipl, inner plexiform layer; gcl, ganglion cell layer; opl, outer plexiform layer; the space between the white arrowheads indicates the
thickness of the photoreceptor cell layer; asterisks, photoreceptor outer segment; red arrows, vacuolar spaces; black arrowheads,
mitochondria. Scale bars ⫽ 10 ␮m in A,B,E,F, 1 ␮m in C,D,G,H.
Fig. 3. The loss of outer segments of the photoreceptor cells in vos mutants occurs at 5 days postfertilization (dpf). Embryos (from 3 to 6
dpf) from pair-wise crosses of previously identified vos heterozygotes were fixed, embedded, sectioned, stained with hematoxylin and
eosin, and were examined under the light microscope for any abnormalities in the developing retina. At 3 and 4 dpf, the developing
retina looked normal in all embryos (data not shown). At 5 dpf, in approximately 25% of the embryos (vos), the outer segments are not
apparent (A,B, wild-type; C,D, vos). In addition, the retinal pigment epithelium layer in vos appears to be thicker than the wild-type. At 6
dpf, approximately 25% of the embryos (vos) continue to exhibit the loss of outer segments (data not shown). A,C are low-power views
showing all the cell layers of the developing retina; B,D, are enlarged views of the photoreceptor cell layer (between red arrowheads),
Scale bars ⫽ 10 ␮m.
Fig. 1.
Fig. 2.
Fig. 3.
ZEBRAFISH HISTOLOGIC MUTANT SCREEN 417
TABLE 1. Summary of Mutants Found in Zebrafish Histologic Screena
Mutant (abbreviation)
segmented
photoreceptors (spr)
vestigial outer
segments (vos)
Linkage group
*
LG23**
(Z23370;0.6 cM)
kirby (kby)
LG18**
(Z13692;2.3 cM)
stuffy (sfy)
LG23**
(Z20039;1.1 cM)
LG6**
(Z9738;4.6 cM)
Not determined
stuffed (sfd)
disordered neural
retina (dnr)
huli hutu (hht)
LG12**
(Z22103;2.3 cM)
Eye defects
Gut and other defects
Photoreceptor layer discontinuous,
normal to reduced thickness
Reduced number of
photoreceptor cells;
photoreceptor outer segments
fragmented and parallel to
circumference
None
None
None
None
Small eyes; poorly formed lens;
disorganized retina layers
Severe retinal disorganization,
decreased cell number, nuclear
fragments
None
Ballooned cells, mild nuclear
pleomorphism, nuclear
fragments
Increased luminal mucus;
clusters of mucous cells
Increased luminal mucus
Mild nuclear pleomorphism,
hyperchromasia, enlargement,
and irregularity in intestine and
liver
Severe atypia of esophagus,
intestine, liver, exocrine
pancreas, pancreatic duct,
and swim bladder; enlarged,
dark, irregular nuclei, nuclear
pleomorphism, multinucleation;
rare tripolar mitosis (seen in
brain and pancreas)
a
Linkage group data presented as nearest tested linked marker and distance from its centromere.
*, spr is not linked to LG05, 06, 12, 18, and 23; **, vos mapped to the south arm, while kby, sfy, sfd, and hht mapped to the
north arm of their respective linkage groups.
hematoxylin and corresponds ultrastructurally to the nucleus. The inner
and outer segments stain pink with
eosin (Fig. 2A,B). Ultrastructurally, the
inner segment includes mitochondria and Golgi apparatus, whereas
the outer segment contains long
stacks of membranes (Fig. 2C,D)
containing the light-sensitive opsins
responsible for light detection (Adler
and Hewitt, 1994; Schmitt and Dowling, 1999). The PCL in wild-type is approximately 20 ␮m thick, including
nuclei and outer segments (Fig.
2A,B). In contrast, the PCL in vos is
only 7–10 ␮m thick, consisting primarily of a nuclear layer (Fig. 2E,F).
Furthermore, the number of photoreceptor cells in vos is decreased by
approximately 50% (Fig. 2E), and the
outer segments of the remaining
photoreceptor are essentially undetectable by light microscopy (Fig.
2F). Vacuolar spaces suggestive of
cellular degeneration are also scattered near the nuclei of the PCL in
vos (Fig. 2E,F). The photoreceptor
cells that remain have outer segments that are misoriented and diminished in size (Fig. 2G,H). Low-
power electron photomicrographs
of vos retina were used to determine
that the volume of outer segments
Fig. 4. The photoreceptor cell layer in spr mutants is formed in patches. At 7 days postfertilization, spr mutants do not have a continuous photoreceptor cell layer (PCL). The PCL is
present as patches (indicated by white arrowheads). There is also no distinct outer plexiform layer in spr mutants (compare with wild-type in Fig. 2A). Scale bar ⫽ 10 ␮m.
Fig. 5. Histologic mutations affecting the intestine. Low-power (upper row) and high-power
(lower row) photographs of wild-type (A,B), kby (C,D), and sfy (E,F). Intestinal epithelial
cells are ballooned in kby (C) and lack brush border (D); the lumen is indicated by
asterisks. Mucus accumulates in sfy (E); abnormal clumps of mucous cells are occasionally found in sfy mutants (E, F, black arrowheads). wt, wild-type. Scale bars ⫽ 10 ␮m in A–F.
Fig. 6. Mutants with multiorgan defects. The mutations dnr (A,B,D) and hht (E–H) show
architectural and cytologic effects in multiple organs. At low power, disruption of the
neural retina is evident in both dnr (A) and hht (E). The dnr mutation shows cellular atypia
in the intestine (white arrowheads in B; compare nuclear size with normal intestine in Fig.
3A) and liver (white arrowhead in D; compare with normal liver shown in C). Atypical cells
of hht (F,G) include giant cells in the intestine (white arrowheads in F; a single giant cell is
shown in G), pancreas (F), central nervous system, and other endodermally derived
epithelia. An atypical mitosis in hht pancreas is shown in F (downward arrow, lower left)
and H (arrowhead). Cells with nuclear atypia were present in proximal intestine and
pancreas (F, black arrows). Scale bars ⫽ 10 ␮m in A–H.
Fig. 7. The distributions of nuclear areas in wild-type vs. hht intestine. The area of 100 hht
and 100 wild-type intestinal nuclei were obtained as described in Experimental Procedures
section and plotted by size, wild-type in black and hht in red.
Fig. 4.
Fig. 7.
Fig. 5.
Fig. 6.
ZEBRAFISH HISTOLOGIC MUTANT SCREEN 419
was less than 10% of that seen in
wild-type retina (not shown).
However, the electron photomicrographs show that vestigial outer
segments are present in vos (Fig.
2G,H); in the outer segments in vos
are oriented parallel, rather than
perpendicular, to the circumference of the eye (Fig. 2H). To determine the time of onset of the vos
phenotype, we examined larvae
obtained from a pair of vos heterozygous parents from 2 to 7 days
postfertilization (dpf). Histologically,
photoreceptors can be distinguished at 2 dpf with the outer segments beginning to appear by 2.5
dpf (Branchek and Bremiller, 1984).
The first defects were detected in
the photoreceptors of vos larvae at
day 5, when 25% of the larvae from
crosses between heterozygotes
lacked definitive outer segments
(Fig. 3D). The retinal pigmented epithelium (RPE) appears to be thicker
in vos (Figs. 2E–G, 3D) than wild-type
(Figs. 2A–C, 3C). We do not know at
this time whether the changes in the
RPE have any bearing on the PCL
defects of vos. In Xenopus laevis, retinal pigmented epithelium has been
reported to participate in the organization of photoreceptor outer segments (Stiemke et al., 1994), and in
mouse, it is required for the development and maintenance of the neural retina (Raymond and Jackson,
1995). In zebrafish, analyses of the
mosaic eyes mutant has provided
the first genetic evidence that signalling from the RPE to the retina is
required for the induction of proper
retinal organization (Jensen et al.,
2001). Furthermore, the gantenbein
zebrafish mutant shows both cone
dystrophy and RPE degeneration
(Biehlmaier et al., 2003). Adults from
crosses between vos heterozygotes
were genotyped for SSR markers
tightly linked to vos; no homozygotes
for the linked allele were identified,
indicating that the vos mutation is
lethal (data not shown).
In wild-type larvae, photoreceptors form a continuous, distinct layer
just inside the retinal pigmented epithelium (Fig. 2A,B). In contrast, spr
has a discontinuous photoreceptor
layer, with patches of variable size
and thickness (Fig. 4). The spr phenotypes varied in the size and number
of patches, and the thickness of the
discontinuous PCL. It also lacks a
clear outer plexiform layer. At 7 dpf,
the RPE also appears to be thicker
than those of wild-types. We do not
know whether spr mutants first develop an intact PCL that is then lost
in patches or whether the PCL forms
only in patches to begin with. This
mutant did not exhibit any other defects identifiable under the dissection microscope. This defect was
fully penetrant and followed Mendelian inheritance. Unfortunately,
the spr mutation was lost before we
could perform further characterization.
Gut-Specific Mutants
Three mutations, kirby (kby), stuffy
(sfy), and stuffed (sfd) affect only the
intestine. kby is characterized by ballooned gut epithelial cells, which also
display perinuclear clearing (Fig.
5C,D; compare with the corresponding wild-type sections in 5A,B). In addition, the epithelial cells are deficient
in brush borders (Fig. 5D). The ballooned cells suggest degenerative
changes, while brush border deficiency suggests a defect in cell differentiation. The nuclei in kby intestinal
epithelium (Fig. 5D) are larger and
more variable in size and shape than
wild-type (Fig. 5B). Occasional nuclear fragments and pyknotic nuclei
are also found in kby (not shown). Further study of this mutant may provide
insights into mechanisms of cell differentiation, nuclear atypia, and cell degeneration in the intestine.
The proximal intestine of wild-type
7-day larvae contains little to no mucus (Fig. 5A,B). In the distal intestine,
small amounts of mucus are associated with scattered, solitary mucus
cells surrounded by absorptive epithelium. However, in the two intestinal mutations, sfy and sfd, there is
increased luminal mucus in the proximal intestine (Fig. 5E, only sfy is
shown). In addition, in sfy, clusters of
mucus cells appear in the proximal
intestinal epithelium (Fig. 5E,F, indicated by arrowheads). The mucus
present in these mutants stains with
the same weak intensity as in wildtype with both mucicarmine and
colloidal iron mucus stains (Luna,
1960; not shown). All three gut mu-
tants are fully penetrant and show
Mendelian inheritance in outcrosses.
None of these phenotypes were described among the previously described zebrafish intestinal mutants
(Pack et al., 1996; Chen et al., 1996).
Pleiotropic Mutations
Two mutations showed multiorgan
and cytologic defects (Fig. 6). The
first, disordered neural retina (dnr),
exhibits defects in the eye, intestine,
and the liver (Fig. 6). The eyes of dnr
mutants show severely disrupted architecture of the neural retina (Fig.
6A). Histologically, the lens and retinal layers of dnr are poorly formed.
The different layers of the retina are
not distinct and have a disordered
appearance. The dnr retina contains scattered nuclear fragments
and vacuolar spaces. This eye phenotype is detectable as early as day
3 (not shown). The dnr gut phenotype is characterized by a consistent
nuclear pleomorphism of the intestine
(Fig. 6B) and nuclear enlargement in
the liver (Fig. 6D; compare with nuclei
of normal liver in 6C). The nuclear
atypia in the intestine and liver of dnr
mutants cosegregated consistently
with the eye phenotype, suggesting
that a mutation in the dnr gene is responsible for these defects. The dnr
mutants could be identified by their
smaller eye phenotype with the help
of a dissection microscope. However,
their gut defects could not be detected without histologic examination. The mutant larvae do not survive
to adulthood. Unfortunately, this mutant has also been lost before further
characterization was possible.
The second pleiotropic mutation,
huli hutu (hht), causes striking architectural and cytologic changes in
several organs (Fig. 6E–H). The eyes
of a variable fraction of mutants are
reduced in size. The cell types of different retinal layers are mixed together to varying degrees. In the
most extreme cases, no discernible
retinal layering is evident, similar to
what is seen in dnr. The eye phenotype is detectable at 2 dpf (not
shown) and can be mild to severe at
7 dpf. The retinal disorganization is
also associated with scattered nuclear fragments suggestive of apoptosis (Fig. 6E). Accordingly, the
420 MOHIDEEN ET AL.
number of nuclei per cross-section
of the eye was 25% to 75% of normal.
The decrease in cell number and severity of retinal disorganization were
roughly proportional to the degree
of disruption of layers and to eye
size, which ranged from normal to
approximately 75% of normal. The
nuclear defect is not evident in muscle, blood, gills, or blood vessels.
The most interesting of the hht
phenotypes is severe cytologic
atypia in the intestine, pancreas,
liver, swim bladder, and pneumatic
duct. This atypia includes a great
variability in nuclear size and shape,
frequent multinucleation, and architectural disorganization (Fig. 6F,G).
There were occasional giant cells in
the intestine and pancreas (Fig.
6F,G). Despite architectural disorganization of the intestine of 7-day-old
hht larvae, formation of microvilli
was preserved (not shown). In 100
randomly selected cells in hht, 19
had multiple nuclei (up to 3 per cell).
An intriguing but rare finding was tripolar mitosis (Fig. 6H); the significance and potential relationship of
this phenomenon to the other phenotypes of hht remains to be determined.
To quantify the degree of nuclear
enlargement in hht intestine, crosssectional nuclear areas were measured digitally for the intestinal epithelial nuclei of 100 cells of hht
larvae and wild-type siblings. The
area of hht nuclei vary in size and
are significantly larger than wildtype (Fig. 7); mean nuclear areas
were 34.8 ␮m2 for hht and 12.1 ␮m2
for wild-type. Because the density
of stain is at least as dark as wildtype, we expect this difference to
reflect differences in DNA content.
The degree of nuclear irregularity,
which correlates with malignant
potential in human cancer (Demay, 1996), was measured as the
ratio of circumference to nuclear
area (“circularity”); greater values
correlate with increased irregularity. The mean circularity was 16.9
for hht and 14.3 for wild-type. Thus,
the nuclei of intestinal epithelial
cells in hht are larger, more variable in size, and are irregular in outline compared with wild-type.
Complementation Analyses
To determine the number of genes
corresponding to the seven recessive mutations found, we performed
complementation crosses and halftetrad mapping. Histologic analysis
of reciprocal complementation
crosses between known carriers of
the four mutations with eye phenotypes (spr, vos, dnr, and hht) showed
only wild-type progeny, consistent
with these mutations occurring in
four different genes. Half-tetrad genetic mapping (Streisinger et al., 1986;
Mohideen et al., 2000) of the five mutants with intestinal phenotypes (kby,
sfy, sfd, dnr, and hht) showed that
they reside on different chromosomes. Each of the seven mutants
were obtained from a different ENUtreated G0 male. Taken together, the
data suggest that we have identified
seven different single locus mutations.
Genetic Map Positions
Five mutations (vos, kby, sfy, sfd, and
hht) were mapped to specific chromosome arms on the SSR genetic
map by centromere-linkage mapping (Table 1); fine mapping for vos
and hht are in progress. Mapping of
spr and dnr is incomplete due to loss
of these mutant lines.
Viability
The mutations identified in this
screen are lethal after 8 dpf. Up to
this time, the eye-specific mutants
spr and vos, as well as the gut mutants kby, sfy, and sfd and the multiorgan mutant dnr look normal, with
some hht mutants exhibiting small
eyes. However, they all die soon after 8 dpf. We speculate that the histologic defects in spr and vos might
render them blind, which inhibits
their feeding ability. After 8 dpf, the
yolk becomes depleted and the larvae depend on food from their environment for survival. We also believe that, in kby, sfy, and sfd, their
histologic gut defects may cause
them to die because of insufficient
food absorption. The combined defects in both the eye and gut in dnr
and hht confer both disadvantages.
Progeny from crosses between heterozygotes of these mutations have
been grown to adulthood. Homozy-
gotes have not been recovered. In
the case of vos and hht, we have
genotyped adults with tightly linked
SSR markers and have not detected
any homozygotes among progeny
derived from known heterozygotes.
Frequency of Mutants
In this screen, half-tetrad progeny
from heterozygous mothers were
screened for homozygous mutations
affecting their histologic appearance. Because centromeric mutations are more frequently homozygous than are telomeric mutations in
half-tetrads (Streisinger et al., 1986),
half-tetrad screens have a bias toward detecting mutations that tend
to be close to centromeres (Grunwald and Streisinger, 1992; Henion et
al., 1996). Thus, the method used in this
report will underestimate the total
number of mutations that were induced and that could be detected in
the histologic screen. Three levels of
histologic sections were examined
per larva in this screen. Examining additional levels per block, while increasing the amount of work per block,
might be expected to yield additional
mutants. Under our conditions, approximately 1 in 10 mutagenized F1
individuals had a histologically detectable mutation, whereas 1 in 1,000
F1s had a mutation at a single predefined locus, golden. We estimate
that approximately 100 genes exist
that, when mutated, will yield defects
not seen in the dissecting scope but
seen with hematoxylin and eosin.
Relevance to Human Disease
The mechanisms responsible for defective photoreceptor layers in spr
and vos may relate to one or more
human genetic retinal diseases (Hewitt and Adler, 1994). In particular, the
histologic phenotypes of photoreceptor cell layer and pigment cells of vos
are similar to human retinal degenerative diseases such as retinitis pigmentosa. For example, a similar deficiency
of outer segments is seen in mice with
mutations in the otx-related homeobox gene crx (Furukawa et al.,
1997, 1999). Mutant alleles of crx in
man are associated with retinal degeneration syndromes, including
cone–rod dystrophy, Leber congeni-
ZEBRAFISH HISTOLOGIC MUTANT SCREEN 421
tal amaurosis, and retinitis pigmentosa
(Furukawa et al., 1997, 1999; Freund et
al., 1997; Swain et al., 1997; Sohocki et
al., 1998; Swaroop et al., 1999). The zebrafish crx homolog has been cloned
and subsequently mapped to LG05 on
the zebrafish genetic linkage map (Liu
et al., 2001). However, we have confirmed that vos and spr are not alleles of crx, because the vos locus is
on LG23 and spr is not linked to
LG05.
Histologic sections allow us to detect changes otherwise hidden beneath normal, opaque choroidal
pigments that appear after day 3 in
zebrafish. Both mutant phenotypes
are clearly different from those with
photoreceptor defects found in earlier screens (Malicki et al., 1996; Fadool et al., 1997; Neuhauss et al.,
1999). The photoreceptor mutants
identified in previous screens have
small eyes and are usually associated with additional phenotypes
such as poor touch response, brain
defects, and abnormal pigmentation. The vos and spr mutants have
normal-sized eyes and do not have
any other scoreable defects. Only
upon histologic examination are
their photoreceptor cell defects apparent. It is unclear whether the primary defect in either mutant lies in
the photoreceptor cells themselves
or surrounding cells, such as the pigmented epithelium, upon which differentiation or maintenance of the
photoreceptor outer segment depends (Hewitt and Adler, 1994).
The common histologic phenotypes of human cancer include disorganization of tissue architecture, invasion, and a variety of cytologic
features, including nuclear hyperchromasia, nuclear pleomorphism, irregular nuclear outlines and chromatin patterns, multinucleation, and
multipolar mitoses (Frost, 1986; Demay, 1996; Cotran et al., 1999). Despite the critical importance of these
histopathologic phenotypes in clinical
medicine, their genetic origins remains largely unknown. To our knowledge, the hht mutation is the first vertebrate mutant to be identified by its
atypical histologic phenotype. Because the hht phenotype includes
several of the cytologic features of
high grade dysplasia, which frequently progresses to cancer, further
studies may contribute to our understanding of cytologic cancer phenotypes. It will be interesting to determine whether hht heterozygotes are
susceptible to cancer.
Rudolf Virchow not only played a
key role in defining the cellular basis
of disease, largely as revealed by
histologic analysis, but also pointed
out the importance of genetics in
gaining understanding of normal
and abnormal cell physiology (Virchow, 1855). This screen reflects the
first application of a classic genetic
approach to these fundamental
ideas.
Potential Throughput of
Histologic Screens
A histology screen such as the one
reported here involves several steps,
some of which can be automated
to improve the throughput of the
screen. First, the embedding step involves careful positioning of individual larvae into each agarose well.
The maximum speed we have
achieved for embedding is approximately 10 min per block of 64 larvae, or approximately 10 sec per
larva. We imagine that a mold that
closely matches the shape of the
larva may allow more rapid positioning than presently possible. There is
potential for robotic positioning of
the embryos (Schneider, 2003) and
automated removal of excess water
from the well that otherwise causes
poor sections. The next step that
could be automated is that of scoring sectioned and stained embryos.
While screening of slides of larval arrays is most rapidly done by an experienced human operator, written
and image documentation of mutant phenotypes can be greatly facilitated by combining database
software with virtual slide scanning
software such as that offered by MicroBrightField (Williston, VT) or Aperio
Technologies (Vista, CA). The virtual
images may be digitized, individual
embryos recognized and labeled,
and linkage to appropriate database fields be automated. Our laboratory is currently developing such
automations to facilitate histologic
screening of zebrafish larvae. Increases in the throughput of histologic screens can be applied not
only for genetic screens but also for
toxicologic, forward genetic (Nasevicius and Ekker, 2000), and chemical screens (Peterson et al., 2000).
EXPERIMENTAL PROCEDURES
Zebrafish Strains and Fish Care
Wild-type male zebrafish used for
mutagenesis were obtained from
Ekkwill (Gibsonton, FL) and Liles
(Ruskin, FL); microsatellite alleles in
the two strains were similar at several
loci (unpublished data). Homozygous goldenb1 females used for
crosses with mutagenized males
were descendants of goldenb1 fish
obtained from the Zebrafish Resource Center, University of Oregon.
A strain obtained from India was initially used to introduce polymorphisms by crosses to identified female carriers; these were discarded
after poor breeding of the progeny
led to the loss of mutant lines. The fish
were raised in a recirculating system
as described (Beckwith et al., 2000).
Fish were maintained and bred for
generating larvae as previously described (Westerfield, 1995). Before
fixation for histology, larvae were
anesthetized using tricaine methane
sulphonate (Westerfield, 1995).
Mutagenesis
Males, 7- to 9-month-old, were used
for mutagenesis. The males were selected for high fertility by previous
pair-wise matings. Germline mutations
were induced in adult male zebrafish
with 2.5 mM ENU (Sigma), as described (Mullins et al., 1994; Beckwith
et al., 2000). Mutagenized males were
first crossed 1 week after mutagenesis
to enable expulsion of sperm with single-strand DNA damage. Beginning 2
weeks postmutagenesis, males were
outcrossed to homozygous goldenb1
females to determine germline mutation rates and to generate F1 for
screening.
Early Pressure Parthenogenesis
Mutations induced by ENU in G0
males and carried in ⬃6-month-old F1
females were made homozygous by
“early pressure” gynogenesis (Fig. 1;
Streisinger et al., 1981) using conditions described previously (Gestl et al.,
422 MOHIDEEN ET AL.
1997). The resulting half-tetrads were
examined for mutations in histologic
sections. The fraction of homozygous
half-tetrads from maternal heterozygous mutations ranges from 5% to
50%, depending upon distance from
the centromere (Streisinger et al.,
1986; Kauffman et al., 1995).
Histology
Seven-day-old larvae without any visible defects were fixed in 10% neutral
buffered formalin for 16 to 64 hr at
4°C. Arrays of 32 to 64 larvae per
clutch were embedded in individual
agarose blocks, processed into paraffin, and cut into 6-␮m-thick sections,
three sets at a time as described previously (Tsao-Wu et al., 1998). One
slide per set was stained with hematoxylin and eosin by using standard
protocols (Luna, 1960), and the remaining sections were left unstained
for further study or for extracting DNA
for genetic mapping. F1 females that
produced half-tetrad progeny with interesting histologic phenotypes were
outcrossed to the India or AB strains
for further study.
Genetic Mapping
Mutations were first mapped to a
chromosome arm by centromere
linkage analysis as used by Johnson
et al. (1995), using markers described previously (Mohideen et al.,
2000). For genetic mapping, DNA
from mutant half-tetrad larvae as
well as from wild-type sibs were extracted from corresponding unstained, deparaffinized sections as
described previously (Tsao-Wu et al.,
1998). Centromere-linkage analysis
was performed using SSR markers
that are linked to the centromeres of
the 25 linkage groups (Mohideen et
al., 2000). Upon establishing linkage
to a specific linkage group, markers
flanking the centromere were used
to determine the specific arm on
which the mutation is located.
Electron Microscopy
Larvae were fixed for electron microscopy in 2.5% glutaraldehyde
(Electron Microscopy Sciences, Fort
Washington, PA) in 0.1 M sodium cacodylate buffer (Sigma) at 4°C for 2
hr, or in 1% glutaraldehyde 4% Ultrapure formalin (EM Sciences) in 0.1 M
cacodylate buffer at 4°C for 2 hr.
Fixed larvae were washed in 0.1 M
cacodylate buffer on ice, post-fixed
with 2% osmium tetroxide in 0.1 M
cacodylate buffer for 1 hr, dehydrated in prechilled graded alcohol
solutions, and embedded in EMbed
812 (EM Sciences). Light microscopy
of methylene blue–stained 1-␮m
transverse sections were used to
screen sections for electron microscopy. Ultra-thin, silver– grey sections
(60 nm) were cut from preselected
blocks, stained with uranyl acetate
and lead citrate, and examined on
a Philips 400 transmission electron microscope.
Photography
Sections were photographed with
Kodak Ektachrome T160 slide film on a
Zeiss Axiophot with a Zeiss MC spot
camera, using a variety of objectives.
The 35-mm slides were scanned using
a Polaroid SprintScan 35 Plus scanner
(Cambridge, MA), and labeling and
adjustments were done with Adobe
Photoshop.
Morphometry
For cell counts in hht, nuclei were
counted manually on 8- ⫻ 10-inch
enlargements obtained by using a
color photocopier of full coronal
sections of normal and hht eyes. Nuclear size and irregularity were determined by digital photography of
images from 6-␮m thick, hematoxylin and eosin–stained sections of
7-day-old wild-type and hht siblings
derived from early pressure parthenogenesis or crosses. Digitized images were acquired from the slides
by using an Olympus BH-2 microscope with a Zeiss ⫻40 dry objective,
a Sony DKC-5000 digital camera,
and Adobe Photoshop. Outlines of
digital images of 100 nuclei, each
from several wild-type and mutant
larvae, were traced by using Optimas 4.2 software (Silver Spring, MD)
to calculate nuclear areas and circumference/area ratios.
ACKNOWLEDGMENTS
We thank Lynn Budgeon and XiaoHong Wang for histologic work, Dr.
Ian Zagon for help with determining
nuclear areas, Roland Meyers for
electron microscopy, Michele Aros
for technical assistance, and Peggy
Hubley for fish facility management.
This work was supported by the Jake
Gittlen Memorial Golf Tournament
and by grants from the NIH (to
K.C.C. and J.L.M.) and the NSF (to
K.C.C.).
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