Zebrafish pitx3 is necessary for normal lens and retinal development

Zebrafish pitx3 is necessary for normal lens and retinal development

Xiaohai Shia,1, D.V. Bosenkob,1, N.S. Zinkevichb, S. Foleya, D.R. Hydea,E.V. Seminab,*, Thomas S. Vihtelica,*

aDepartment of Biological Sciences, Center for Zebrafish Research, Galvin Life Sciences Center, University of Notre Dame, Notre Dame, IN 46556, USAbDepartment of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA

Received 2 June 2004; received in revised form 11 November 2004; accepted 19 November 2004

Available online 10 December 2004

Abstract

The human PITX3 gene encodes a bicoid-like homeodomain transcription factor associated with a variety of congenital ocular conditions,

including anterior segment dysgenesis, Peter’s anomaly, and cataracts. We identified a zebrafish pitx3 gene encoding a protein (Pitx3) that

possesses 63% amino acid identity with human PITX3. The zebrafish pitx3 gene encompasses approximately 16.5 kb on chromosome 13 and

consists of four exons, which is similar to the genomic organization of other pitx genes. Expression of the zebrafish pitx3 gene was studied by

in situ mRNA hybridization and RT-PCR. The pitx3 transcripts were detected throughout development with the greatest level of expression

occurring in the developing lens and brain at 24 hpf. In adults, the highest expression was detected in the eye. Morpholinos were used to

knockdown expression of the Pitx3 protein and a control morpholino that contains five mismatched bases was used to confirm the specificity

of the phenotypes. The morphants had small eyes, misshapen heads and reduced jaws and fins relative to controls. The morphants exhibited

abnormalities in lens development and their retinas contained pyknotic nuclei accompanied by a reduction in the number of cells in different

neuronal classes. This suggests the lens is required for retinal development or Pitx3 has an unexpected role in retinal cell differentiation or

survival. These results demonstrate zebrafish pitx3 represents a true ortholog of the human PITX3 gene and the general function of the Pitx3

protein in lens development is conserved between mammals and the teleost fish.

q 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Pitx3; Lens; Retina; Zebrafish; Morpholino

1. Introduction

The generation of a functional vertebrate eye requires

coordinating the movement and differentiation of different

cell and tissue types throughout development (Chow and

Lang, 2001). The tissue interactions are also necessary for

maintaining some of the differentiated cell types in the eye

(Yamamoto et al., 2003; Yamamoto and Jeffery, 2000).

Historically, the ocular lens has served as a model to

identify factors required for embryological tissue induction

(Grainger, 1992; Henry and Grainger, 1990; Hirsch and

Grainger, 2000; Zygar et al., 1998). More recently, genetic

0925-4773/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.mod.2004.11.012

* Corresponding authors. Tel.: C1 574 631 2895; fax: C1 574 631 7413

(TSV) or Tel.: C1 414 456 4996; fax: C1 414 456 6516 (EVS).

E-mail addresses: [email protected] (E.V. Semina), [email protected]

(T.S. Vihtelic).1 These authors made equal contributions to this paper.

screens using zebrafish (Danio rerio) identified mutants

with disrupted retina and lens development (Brockerhoff

et al., 1998; Fadool et al., 1997; Heisenberg et al., 1996;

Malicki et al., 1996; Vihtelic and Hyde, 2002; Vihtelic et al.,

2001).

Zebrafish lens development proceeds rapidly, with the

morphologically identifiable stages being identical to the

stages observed in other vertebrates (Easter and Malicki,

2002). The lens placode is recognizable at approximately

16 h post-fertilization (hpf) and the lens vesicle detaches

from the surface ectoderm by 24 hpf (Malicki, 2000;

Schmitt and Dowling, 1999). By 36 hpf, the morphologi-

cally distinct epithelial and fiber cells are visible and a fully

functional lens is present by 72 hpf (Easter and Nicola,

1996; Schmitt and Dowling, 1994). Fiber cells are

continually added to the growing lens throughout the

life of mammals and fish, which requires the tight regulation

of cell proliferation, migration and differentiation

Mechanisms of Development 122 (2005) 513–527

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X. Shi et al. / Mechanisms of Development 122 (2005) 513–527514

(McAvoy et al., 1999). The lens epithelial cells transition

from a central quiescent zone into the germinative zone as

they enter mitosis. They migrate into the lens equatorial

zone or bow region, where the cells elongate and lose their

nuclei and other membrane-bound organelles as they

differentiate into fiber cells (Bassnett and Beebe, 1992;

Wride, 2000).

Pitx3 is one of three homeobox gene family members

(designated Pitx1, Pitx2 and Pitx3) found in a variety of

animal species (Gage et al., 1999a). The Pitx protein family

plays important roles during the development of a wide

variety of tissues (Gage et al., 1999a). Pitx1 knockout mice

exhibit hind limb abnormalities, anterior pituitary defects

and cleft palate, while mice homozygous for the Pitx2 gene

deletion die before birth with anomalies in the heart, gut,

lung, pituitary and eye (Kitamura et al., 1999; Lanctot et al.,

1999; Lin et al., 1999; Lu et al., 1999; Szeto et al., 1999). In

contrast, Pitx3 developmental expression in mammals is

largely restricted to the eye and midbrain (Semina et al.,

1997, 1998; Smidt et al., 1997), with mutations in the human

PITX3 gene being responsible for congenital ocular

phenotypes such as Anterior Segment Ocular Dysgenesis

(ASOD), Peter’s anomaly and autosomal dominant con-

genital cataract (Semina et al., 1998). In addition, mutations

in the mouse Pitx3 gene are responsible for the aphakia (ak)

phenotype, which is characterized by small eyes that lack

lenses (Rieger et al., 2001; Semina et al., 2000; Varnum and

Stevens, 1968). These phenotypes are consistent with the

Pitx3 expression patterns in these species. During the initial

stages of eye development, Pitx3 is expressed throughout

the presumptive lens ectoderm and becomes further

restricted to the epithelial cells at the lens equator as lens

fiber differentiation begins (Semina et al., 1997). The Pitx3

expression pattern suggests it is important for both lens

development and continuous growth. While lens induction

and the accompanying morphological changes during

differentiation have been correlated with a hierarchy of

transcription factors (Ashery-Padan et al., 2000; Grindley

et al., 1995; Henry et al., 2002; Kamachi et al., 1998, 2001;

Ogino and Yasuda, 2000; Oliver et al., 1996; Wigle et al.,

1999), the role of Pitx3 in these processes has not been

precisely defined.

We identified and began characterizing a zebrafish pitx3

gene to complement our genetic analyses of vertebrate lens

and anterior segment development. The zebrafish pitx3

ortholog displays striking conservation with the Pitx3 genes

in human, mouse and frog at several different levels

(Pommereit et al., 2001; Semina et al., 1997, 1998; Smidt

et al., 1997). First, the zebrafish Pitx3 protein possesses over

62% amino acid identity with other vertebrate Pitx3

proteins. Second, the zebrafish pitx3 gene is localized to a

region of chromosome 13 that is syntenic to the human

chromosome 10 region that contains PITX3. Third, the

zebrafish pitx3 gene is initially expressed in the developing

lens, diencephalon and the pituitary region and later its

expression in the eye is restricted to the lens equator. Fourth,

morpholino-mediated protein reduction demonstrates the

zebrafish Pitx3 protein plays a critical role during lens

development. Surprisingly, the morphants also exhibit the

loss of differentiated neurons in the retina, which is different

from the mouse ak mutant eye phenotype. While our results

indicate that Pitx3 functions in lens development were

conserved during evolution, the Pitx3 requirement in retinal

development or maintenance may reveal either a previously

unknown function of the Pitx3 orthologs or a novel function

specific to zebrafish Pitx3. Thus, the zebrafish pitx3 gene

provides avenues to further elucidate the molecular

mechanisms of ocular development.

2. Results

2.1. Cloning and characterizing the zebrafish pitx3 gene

The zebrafish pitx3 cDNA sequences were amplified by

PCR from adult zebrafish eye cDNA using primers

corresponding to the Xenopus pitx3 sequence. The amino

acid sequence deduced from the cDNA (Fig. 1A) showed

high amino acid identity with the Pitx family of proteins

from different species, but the greatest identity was to Pitx3.

The zebrafish sequence shared 63% (Pitx1), 63% (Pitx2)

and 90% (Pitx3) amino acid identity with the Xenopus Pitx

proteins. The zebrafish sequence also showed 62 and 63%

amino acid identity with the mouse and human Pitx3

proteins, respectively (Fig. 1A). The previously identified

conserved functional domains in this protein family, the

homeobox and OAR domains, shared nearly 100% amino

acid identity between the zebrafish and all other Pitx3

proteins (Amendt et al., 1999; Brouwer et al., 2003;

Furukawa et al., 1997; Semina et al., 1996). The only

other known zebrafish Pitx protein, Pitx2, showed 100%

identity in the homeobox domain, 75% identity in the

C-terminal region and 17% identity in the N-terminal region

with this new zebrafish Pitx sequence. Based on these

homologies, and in consultation with the Zebrafish Nomen-

clature Committee, this zebrafish gene was named pitx3

(Fig. 1A). Although the zebrafish genomic sequence is not

complete, searches of the nucleotide and protein databases

failed to identify a duplicated pitx3 gene, which suggests

that zebrafish possess only one pitx3 orthologue.

The pitx3 cDNA sequence was compared with the

genomic sequences in the zebrafish database (BLAST;

http://www.sanger.ac.uk/Projects/D_rerio/). Genomic con-

tig ctg11787 contained all of the pitx3 gene sequence except

for the first exon and part of the second intron (Fig. 1B). We

PCR amplified these two regions (p3x_1 and p3x_2;

Fig. 1B) from genomic DNA and determined that the

pitx3 gene encompasses approximately 16.5 kb, with four

exons (240, 129, 208 and 704 bp) separated by introns of

5.6, 2.6 and 7.4 kb (Fig. 1B). This genomic structure is

conserved with the Pitx3 genes from other species (Semina

et al., 1997, 1998).

http://www.sanger.ac.uk/Projects/D_rerio/

Fig. 1. Zebrafish Pitx3 protein sequence comparison and genomic structure. (A) The zebrafish Pitx3 amino acid sequence is aligned with the mouse, human and

Xenopus Pitx3 sequences. The homeobox and OAR domains are shown in bold and italics, respectively. The amino acid identity between the zebrafish and

mammals are highlighted in green, while the identity between zebrafish and Xenopus is shown in yellow. The peptide sequence used to generate the Pitx3

polyclonal antiserum (amino acids 221–238) is underlined. (B) The zebrafish pitx3 gene is composed of four exons and three introns. The exon sizes (in bp) are

shown in the four boxes, while intron sizes are indicated below in kilobases (kb). The locations of the translation start and stop codons (marked by arrows) are

within the second and fourth exons, respectively. The relative locations of the homeobox and OAR domains are shown in rust and blue, respectively. The

ctg11787 genomic contig and the two PCR products (p3x_1 and p3x_2) that we generated to assemble the pitx3 gene are also shown.

X. Shi et al. / Mechanisms of Development 122 (2005) 513–527 515

The zebrafish pitx3 gene was mapped to chromosome 13

using the mouse-zebrafish LN54 radiation hybrid panel

(Hukriede et al., 1999, 2001). The pitx3 gene mapped

closest to SSLP marker Z13611 (LOD 12.5), with the

second best linked marker possessing a lower LOD score of

4.2. This region of zebrafish chromosome 13 is syntenic to

human chromosome 10, which also contains PITX3

(Barbazuk et al., 2000; Semina et al., 1998).

2.2. Gene expression during development and adulthood

Zebrafish pitx3 gene expression was examined by RT-

PCR using RNA extracted from embryos and adult tissues

(Fig. 2A). While low levels of pitx3 mRNA were detected as

early as 3–8 hpf, high levels of pitx3 expression were first

identified at 24 hpf and remained high through 120 hpf. In

the adult, high levels of pitx3 expression were detected in

the eye and much lower levels in the internal organs, which

included a mixture of liver, gastrointestinal system, heart,

genitourinary system and pancreas (Fig. 2A).

To examine the spatial expression of pitx3 during

development, we performed in situ mRNA hybridization

on whole-mount embryos using a full-length pitx3 ribop-

robe. At 24 hpf, pitx3 expression was largely restricted to

the forebrain and lens (Fig. 2B (a, e)). High levels of

expression were also detected in the pectoral fin buds at

24 hpf (not shown). At 48 hpf, the strongest pitx3 expression

was detected in the diencephalon and pituitary, while the

lens expression became restricted to a subpopulation of cells

at the lens equator (Fig. 2B (b, f)). At 72 hpf, expression

became evident in cartilage surrounding the mouth (Fig. 2B

(c, g)). At 96 hpf, pitx3 expression was detected in the iris of

the eye, nests of cells within Meckel’s cartilage of the

developing lower jaw and the branchial arches (Fig. 2B

(h–j)). In addition, expression persisted in the pectoral fins

and was identified within the developing musculature along

the trunk at 96 hpf (Fig. 2B (k, i)).

2.3. Morpholino-mediated knockdown of zebrafish

Pitx3 expression

To examine the functions of Pitx3 during eye develop-

ment, we injected three different morpholino anti-sense

oligos (Morph1, 2 and 3) into 1–4 cell stage embryos. The

morpholinos were designed to hybridize either the 5 0

untranslated sequences of the pitx3 mRNA near the

inititation codon (Morph1 and 2) or the splice donor site

at the 5 0 end of intron 3 (Nasevicius and Ekker, 2000). A

control morpholino containing five mismatched bases

relative to Morph1 was also tested. To verify

Fig. 2. Temporal and spatial expression patterns of the pitx3 gene. (A) RT-PCR results from cDNA isolated from various staged embryos and larvae.

Additionally, RT-PCR products resulting from different adult tissues are shown. Molecular weight markers and a control PCR reaction lacking DNA template

are shown in the first and last lanes, respectively. The lower panel shows amplification of b-actin as a control. (B) The in situ hybridization of embryos and

larvae using the full-length pitx3 riboprobe. Whole-mount embryos at 24, 48, 72 and 96 hpf (a–d). Frontal sections of 24, 48 and 72 hpf embryos at the level of

the eye (e–g). The arrowheads in (e) and (f) point to lens, while expression surrounding the mouth is shown in (g). At 96 hpf, expression is shown within the iris

(h), Meckel’s cartilage (i), branchial arch (j), pectoral fin (k) and lateral trunk (l). Abbreviations: de, diencephalon; le, lens; pi, pituitary.


the morpholino reduced Pitx3 protein levels, we developed

a polyclonal antiserum that recognizes the zebrafish 32 kDa

Pitx3 protein (Fig. 3A, lane 1). All the anti-pitx3

morpholinos produced the same morphological phenotypes

including small eyes, malformed pectoral fins and mild to

moderate body axis deviation. In addition, histological

analysis demonstrated nearly identical lens and retinal

phenotypes in the 5 0 UTR morpholino-injected fish

compared to the morphants generated by splice donor

interference (not shown). We will document the anti-pitx3

Morph1 hereafter.

The relative levels of the Pitx3 protein in the morphants

were compared to uninjected and five base mismatched

morpholino-injected control larvae (Fig. 3A). The Pitx3

protein was nearly undetectable in the morphant larvae at 3,

4, 5 and 7 dpf (lanes 2, 5, 8 and 11) compared to the five

Fig. 3. Morpholino knockdown of Pitx3 protein and the morphant phenotypes. (A) Immunoblot analysis of protein extracts from Morph1-injected embryos

(M), a five base mismatch control (mmC) and an uninjected control (uiC) at 3 dpf (lanes 2–4), 4 dpf (lanes 5–7), 5 dpf (lanes 8–10) and 7 dpf (lanes 11–13).

Lane 1 reveals the 32 kDa Pitx3 protein in 24 hpf whole embryo extract using the anti-Pitx3 polyclonal serum. Pitx3 protein expression was reduced in the

morphant extracts compared to the mismatch and uninjected controls. Immunodetection of actin revealed that equivalent amounts of protein were loaded for all

three samples from each time point (not shown). (B–Q) The pitx3 morphant phenotypes. Lateral views of wild type, morphants and mismatch control fish at

3 dpf (B–E). The small morphant eyes are shown in lateral views relative to the wild type and mismatch controls at 3 and 5 dpf (F–H and I–K, respectively). At

5 dpf, the morphants lack normal lower jaws (J; arrow) and pectoral fins (M; arrow). At 7 dpf, the morphant eyes are further reduced in size compared to the

wild type and mismatch control (O–Q).


base mismatched morpholino control larvae (lanes 3, 6, 9

and 12). The Pitx3 expression in the uninjected control

larvae (lanes 4, 7, 10 and 13) was indistinguishable from

the five base mismatched morpholino control larvae. Thus,

the reduction in Pitx3 protein expression is specific to the

injection of the pitx3 morpholinos.

The first detectable morphological phenotype observed

after injecting the anti-pitx3 morpholinos was a small eye,

which was evident at 3 dpf (Fig. 3B–H). Some of the

morphants exhibited an abnormal body axis (Fig. 3D) and/or

lens opacity (Fig. 3G). The percentage of morphant larvae

exhibiting the small eye phenotype was related to the

concentration of the injected morpholino and ranged from

46 to 92% (0.5–2.0 ng/nl). The injection of the five base

mismatched control morpholino produced larvae that were

indistinguishable from wild type (compare Fig. 3E, H to B,

F, respectively). At 5 dpf, the visible area of the morphant

eyes was 10,830.4G2604.3 mm2 (nZ83 eyes), compared to

18,286.4G1714.0 (nZ31) and 18,355.3G1512.8 mm2 (nZ50), for the uninjected and five base mismatched morpholino

control eyes, respectively. In addition, the morphant jaws

appeared abnormal relative to the control larvae (Fig. 3I–K)

and development of the pectoral fins was disrupted

(Fig. 3L–M). By 7 dpf, the reduced eye size in the morphants

relative to the controls was severe (Fig. 3O–Q). Therefore,

the reduction in Pitx3 protein expression resulted in a

progressive reduction in eye size, which was accompanied

by abnormalities in jaw and pectoral fin development.

2.4. Histological analysis of the anti-pitx3 morphant

eye phenotypes

To examine the eye defects, we compared the histology

of the anti-pitx3 morphant eyes to wild type (Fig. 4A–I). To

confirm that the morphant eye phenotypes were specifically

due to inhibition of pitx3 translation, we also examined

Fig. 4. Histological analysis of the morphant lens and retina phenotypes. Stained eye sections of wild type (left column) and anti-pitx3 morphants at low and

high magnification (center and right columns, respectively) at 3, 5 and 7 dpf. Sections of control eyes (wild type and mismatch control) are shown in the bottom

row of panels (J–L). Retinal cell pyknosis (long arrows in B, E, F, H and I) and retention of the lens fiber cell nuclei (short arrows in B and C) are visible in the

morphants, but absent in the controls. At 5 dpf, the epithelial cells in the morphant lens are irregularly shaped and overlie one another in the lens cortex (E, F).

The lens epithelial cell layer contains gaps that lack nuclei or exhibits overlapping cells (B and C, arrowheads). Gaps exist between the lens epithelial cells and

the underlying fiber cells in the distal lens (E and F, asterisks). Central lens fiber degeneration (double arrows) is observed in the morphant lens (E, F) at 5 dpf,

but not in the wild-type lens (D). At 7 dpf, the retinal regression and lens degeneration in the morphant (H, I) are severe relative to wild type (G). The lens and

retina of the mismatch control (K, L) appear identical to the wild type (J). Abbreviations: PL, photoreceptor layer; INL, inner nuclear layer; GCL, ganglion cell

layer; OpN, optic nerve; LEC, lens epithelial cells; LN, lens nucleus; LC, lens cortex; IPL, inner plexiform layer. The scale bars represent 50 mm.


embryos injected with the five base mismatched control

morpholino, which failed to produce any detectable

phenotypes (Fig. 4K, L). At 3 dpf, the laminated wild-type

zebrafish retina is composed of the photoreceptor, inner

nuclear and ganglion cell layers (Fig. 4A). The lens contains

a single layer of epithelial cells and the majority of the lens

fibers have differentiated and lost their nuclei (Fig. 4A).

The nuclei of the newly differentiating fiber cells, which are

located proximally in the lens (near the retinal ganglion cell

layer), can be identified by their elongated morphology. At

3 dpf, the morphant retina lacked the laminar organization

seen in wild type and contained pyknotic nuclei that were

not detected in the control retinas (Fig. 4B, long arrow). The

morphant lens at 3 dpf exhibited two abnormalities. First,

the lens epithelial cell layer contained gaps that lacked

nuclei (Fig. 4B, C; arrowheads). Second, many of the

morphant lens fiber cells, including those in the lens core,

retained their nuclei (Fig. 4B, C; short arrows).

Fig. 5. Morphology and spatial organization of the lens nuclei. Propidium

iodide-stained wild type (A, B), morphant (C, D) and mismatch control

(E, F) frozen eye sections. At 3 dpf, the normal retinal laminar arrangement

(A) and lens epithelial cell organization (B) is shown. The epithelial cells

covering the distal aspect of the lens (LEC) and the proximal elongating

fiber cells (eLFC) are indicated by arrows (A, B). The 3 dpf morphant lens

retains the central fiber cell nuclei (C) and the epithelial cell population

exhibits enlarged and irregularly shaped nuclei and multilayering at 3 and

5 dpf (C, D). The mismatch control retina and lens (E, F) are

indistinguishable from wild type (A, B). Abbreviations: PL, photoreceptor

layer; INL, inner nuclear layer; GCL, ganglion cell layer; LEC, lens

epithelial cells; eLFC, elongating lens fiber cells. Scale bars in A, B, E and

F represent 50 mm, while the scale bars in C and D are 20 mm.


Between 5 and 7 dpf, the morphant eyes exhibited a

reduction in size, increased retinal disorder and lens

degeneration. At 5 dpf, the wild-type photoreceptor outer

segments were observed in both the ventral and dorsal retina

(Fig. 4D, double arrowheads). In contrast, the 5 dpf

morphant photoreceptor cells largely lacked outer segment

structures (Fig. 4E, double arrowheads). In addition,

pyknotic nuclei were observed in the photoreceptor and

inner nuclear layers of the morphant retina and also in the

proximal lens cortical region (Fig. 4E, F; arrows). The 5 dpf

morphant lenses were much smaller than wild type and were

characterized by central fiber cell degeneration (Fig. 4E, F;

double arrows). The lenses also displayed gaps between the

fiber and epithelial cell layers (Fig. 4E, F; asterisk). At 7 dpf

the morphants largely lacked retinal lamination (Fig. 4H)

and continued to exhibit pyknotic nuclei within the lens and

retina (Fig. 4H, I; arrows). Furthermore, the morphant eyes

lacked anterior chambers and were characterized by severe

lens degeneration (Fig. 4H, I; double arrows). Thus, the

morphant eyes exhibited progressive degeneration of both

the lens and retina from 3 to 7 dpf.

2.5. Morphant lens and retinal phenotypes are further

defined by immunohistochemistry

We examined the cellular organization of the lens and

retina in control and morphant frozen eye sections by

labeling the nuclei with propidium iodide (Fig. 5). At 3 dpf,

the wild-type retina possessed the characteristic retinal

lamination pattern, with three nuclear layers separated by

plexiform layers lacking nuclei (Fig. 5A). The wild-type lens

epithelial cell nuclei were uniformly arranged in a single

layer that covered the distal and lateral lens surfaces (Fig. 5B;

LEC). In addition, the elongating lens fiber cells were

observed proximal to the lens equator (Fig. 5B; eLFC). As

expected, the terminally differentiated fiber cells in the center

of the wild-type lens lacked nuclear staining (Fig. 5B). At

3 dpf, the variable nuclear staining pattern of the morphant

lens was consistent with the histological analysis. The

morphant lenses displayed large irregularly shaped epithelial

cell nuclei that were layered upon one another (Fig. 5C).

Similarly, the 5 dpf morphant lenses exhibited epithelial

cells with a variety of nuclear shapes and sizes that were

several layers thick (Fig. 5D). The absence of propidium

iodide-stained nuclei within the lens nucleus was consistent

with the central lens fiber degeneration observed in histogical

sections at 5 dpf (compare to Fig. 4E, F). The retinal and lens

nuclear patterns in the mismatch control embryos appeared

identical to the wild-type patterns (Fig. 5E, F).

We also examined the pitx3 morphant lenses by

localizing actin and the lens-specific antigen zl-1 (Fig. 6).

Actin was localized in a concentric pattern within the central

lens and the cortex of the wild-type lens at 3 dpf

(Fig. 6A, B), which is consistent with fiber cell expression.

Elongating fiber cells within the proximal lens cortex were

also stained, which coincides with the region of fiber cell

differentiation. The elongating fiber cells appeared to extend

from the proximal lens surface and conferred an ellipsoid

shape to the growing wild-type lens (Fig. 6B; eLFC). Actin

also localized to the distal and lateral membranes of the lens

epithelial cells (Fig. 6A, B; arrowheads). While the

morphant lens exhibited robust actin staining in the central

and cortical regions (Fig. 6C; asterisks), the distal epithelial

cell membrane staining was diffuse and the lateral epithelial

cell staining was not detected (Fig. 6D, arrowheads). At

3 dpf, the actin-stained morphant elongating fiber cells

appeared shorter and more stout than wild type (Fig. 6D,

arrows), which produced a smaller and more spherical lens

rather than ellipsoid-shaped. The actin staining within the

wild-type retinal outer (OPL) and inner (IPL) plexiform

layers (Fig. 6A, B) was also absent in the 3 dpf morphant

Fig. 6. Actin fails to localize properly in the morphant lens epithelial cells.

Phalloidin-AlexaFluor 488 (A–D) was used to stain the filamentous actin in

lenses and retinas of wild type (A, B) and morphants (C, D) at 3 dpf. In the

control lens, actin is localized in a concentric pattern associated with the

fiber cell membranes in both the nuclear and cortical regions (A, B). Lens

epithelial cells (A, LEC) exhibit actin localization to both the distal (basal)

and lateral aspects of the individual epithelial cells (B, arrowheads). Actin

staining of the morphant lens reveals poorly defined elongating fiber cells

(D, arrows) and a diffuse actin network in the lens epithelial cells (D,

arrowheads). The sections also reveal intense actin staining within the

retinal inner (IPL) and outer (OPL) plexiform layers (A, B), which is absent

in the morphant (C). The lens-specific zl-1 monoclonal antibody labels the

cortical region of the wild-type lens (E, arrowheads) and is localized near

the apical–apical interface between the epithelial and fiber cells (E, arrows).

In the morphant (F), the zl-1 protein is scattered throughout the lens and

appears to be associated with the nucleated fiber cells (arrowheads).

Abbreviations: PL, photoreceptor layer; INL, inner nuclear layer; GCL,

ganglion cell layer; OPL, outer plexiform layer; IPL, inner plexiform layer;

LEC, lens epithelial cells; eLFC, elongating lens fiber cells; OpN, optic

nerve. Scale bars represent 50 mm.


retina (Fig. 6C). This is consistent with the absence of

lamination that was observed by histology in the 3 dpf

morphant (Fig. 4B).

The zl-1 monoclonal antibody detects an unknown lens-

specific antigen. During zebrafish eye development, the zl-1

antigen is first detected at 21–22 hpf and becomes localized

to the lens cortical region as fiber cell differentiation

proceeds (Vihtelic et al., 2001). The wild-type lens

exhibited zl-1 within the outer lens cells and near the apical

region of the epithelial cell layer at 3 dpf (Fig. 6E,

arrowheads and arrows, respectively). In contrast, the zl-1

expression in the morphant was dispersed in a punctate

pattern throughout the lens and appeared to be associated

with the nucleated fiber cells in the central region (Fig. 6F,

arrowheads). While fiber cell elongation and actin

expression in the morphant lens suggest that some elements

of fiber cell maturation are proceeding normally, the delay

in fiber cell nuclear regression and attenuated fiber cell

elongation reveals that other elements must be disrupted.

The actin-staining pattern also suggests that lens epithelial

cell differentiation is affected.

The presence of pyknotic nuclei in the morphant retinas

(Fig. 4) suggested that some retinal cells were dying. In

addition, the lack of outer segments suggested photo-

receptor maturation or survival was affected in the

morphants. To examine the presence and patterning of

the different neuronal types, we performed immunohisto-

chemical localization experiments on frozen morphant eye

sections at 5 dpf (Fig. 7). At 5 dpf, the double cone

photoreceptors are spread along the entire retinal length in

wild type and the mismatch control, but are dramatically

reduced in the morphant retinas (Fig. 7A–C). The short

single cones, identified by immunodetection of the UV

opsin, were also greatly reduced in the morphant retina

compared to the wild type and mismatch control retinas

(not shown). The rod photoreceptors were identified by

immunodetection of the rhodopsin protein within the cell

outer segments (Vihtelic et al., 1999). The morphant

retinas exhibited a very sparse rhodopsin-positive cell

population, with the photoreceptor layer displaying large

gaps in rhodopsin expression (Fig. 7D–F). The rod and

cone photoreceptors that are present in the morphant retina

are primarily localized to the central region, with the

margins largely lacking photoreceptors (Fig. 7B, E). The

retinal amacrine and ganglion cells were identified using a

monoclonal antibody to detect the HuC/D RNA binding

protein (Marusich et al., 1994). Wild-type HuC/D

expression is restricted to the distal inner nuclear (INL)

and the ganglion cell (GCL) layers and extends to the

retinal peripheral margin (Fig. 7G). While the morphant

retinas displayed HuC/D-positive cells, the signal was less

intense and largely confined to the more central cells of the

retina’s inner layers, with no detectable HuC/D-labeled

cells near the peripheral margins (Fig. 7H). Thus, the

morphant retina exhibits greatly reduced numbers of

neurons in each of the retinal layers. The patchiness of

immunolabeling and the pyknotic nuclei suggest that the

different neuronal classes in the retina require Pitx3

expression for survival, which is surprising because pitx3

expression was not detected in the retina or retinal

pigmented epithelium. Therefore, the retinal phenotype is

likely secondary to the lens defect.

Fig. 7. Reduction in retinal neurons revealed by immunohistochemistry at 5 dpf. Wild type (left column), morphant (center column) and mismatch control

(right column) retinal cell types were identified by immunolocalization of zpr-1 (double cone photoreceptor cells, A–C), rhodopsin (rod photoreceptor outer

segments, D–F), and HuC/D (amacrine and ganglion cells, G–I) in frozen eye sections. While some double cone cells are identified in the morphant (B,

arrowheads), large regions of the retina lack this cell type (B, arrows). Similarly, only small numbers of rhodopsin-positive photoreceptors were detected in the

morphants (E, arrowheads) compared to the wild type (D) and mismatch control retinas (F). The amacrine and ganglion cells (G–I) were also reduced in the

morphants and were characterized by an absence of immunolabeling towards the retinal margins (compare H to G and I). Abbreviations: PL, photoreceptor

layer; INL, inner nuclear layer; GCL, ganglion cell layer; OpN, optic nerve; L, lens. The scale bars represent 50 mm.


3. Discussion

The development of the ocular lens is precisely

regulated and directly affects other eye tissues, including

anterior segment structures and the retina. Mutations in

the Pitx3 transcription factor result in anterior segment

mesenchymal dysgenesis and cataract in humans and

aphakia in mice (Rieger et al., 2001; Semina et al., 1998,


2000). To study the roles of Pitx3 in eye development, we

cloned a zebrafish pitx3 ortholog and characterized its

developmental expression pattern and functions in zebra-

fish embryos and larvae. Our analyses demonstrated the

zebrafish pitx3 gene is expressed in both ocular and

extraocular tissues, which partially overlaps with the

mammalian expression pattern. Morpholino-mediated

Pitx3 protein knockdown resulted in abnormal lens

development and suggests that Pitx3 is required for lens

differentiation and growth. The pitx3 morphants also

exhibited retinal defects that were characterized by cell

death and significant reductions in some retinal cell types.

While our findings support a conserved function for

zebrafish Pitx3 in lens development, the retinal defects

highlight an important difference with the mouse aphakia

(ak) mutant phenotype.

The formation of a functional lens occurs in two phases

and pitx3 activity appears to be critical during both.

Initially, the lens vesicle cells differentiate into anterior

epithelial cells and primary fiber cells, which will

compose the lens nucleus (McAvoy et al., 1999, 2000).

The pitx3 temporal and spatial expression patterns suggest

it may be involved in the early stages of lens

differentiation. Pitx3 transcripts are detected at the

beginning of the lens placode stage in mouse (Semina et

al., 2000, 1997), Xenopus (Pommereit et al., 2001) and in

zebrafish (Fig. 2). The zebrafish pitx3 expression levels

increased by 24 hpf, which corresponds to the initial

differentation of lens primary fibers and epithelial cells

(Easter and Malicki, 2002; McAvoy et al., 1999). The

pitx3 morphant phenotypes are consistent with disruptions

in the normal differentiation of the lens epithelial and

fiber cells and the proper formation and maintenance of

the lens nuclear fibers (Figs. 4–6).

The second phase of lens formation is characterized

by lens growth through the continual addition of

secondary fiber cells, which differentiate from the

epithelial cell population. A germinative ring of mitoti-

cally active epithelial cells is maintained around the lens

central region (Graw and Loster, 2003). The daughter

cells emerging from this germinative zone migrate or are

displaced into the equatorial or bow region of the lens

where they elongate and differentiate into the secondary

lens fibers (Graw and Loster, 2003; McAvoy et al.,

2000). Therefore, lens fiber differentiation is continually

initiated in the equatorial zone during lens growth. In

zebrafish, pitx3 expression is detected throughout the lens

at 24 hpf, but the expression becomes restricted to the

lens equatorial region by 48 hpf (Fig. 2B), which is

equivalent to the restriction of the mouse Pitx3

expression to the lens equator region (Semina et al.,

1997, 1998). Thus, pitx3 initially exhibits widespread

lens expression during the primary differentiation phase

of lens formation and is then restricted to the region of

secondary fiber cell differentiation during the phase of

lens growth.

We examined the function of zebrafish Pitx3 during

eye development by selective reduction in Pitx3 protein

levels using morpholino anti-sense oligonucleotides. We

used three different anti-pitx3 morpholinos that all

produced small eyes and very similar lens and retinal

histological phenotypes. Two of the morpholinos were

designed to prevent translation of the Pitx3 protein by

hybridizing in the 5 0 untranslated region of the transcript,

while the third morpholino prevented proper pitx3

splicing. We also used a control morpholino that differed

by five bases from the Morph1 sequence. This control

morpholino failed to cause any macroscopic or histologi-

cal abnormalities in the injected embryos. Finally, we

used a polyclonal antibody generated against a zebrafish

Pitx3 peptide sequence to confirm that the Pitx3 protein

was reduced in the morphants relative to the controls.

Thus, the morphant phenotypes are likely specific for the

reduced Pitx3 protein expression. Mammalian Pitx3

expression is largely restricted to the developing eye

and midbrain (Gage et al., 1999a; Nunes et al., 2003;

Semina et al., 1997; Smidt et al., 1997), although

transcripts were detected during development in other

tissues such as the mesenchyme around the hyoid bones

and femur (Semina et al., 1998). In addition to the eye

phenotype, we identified abnormalities in mandible and

pectoral fin formation (Fig. 3J and M). Although zebrafish

pitx3 expression was detected in the diencephalon,

branchial arches and trunk muscle (Fig. 2B), we have

concentrated this phenotypic analysis on the eye.

Examination of the morphant lens morphology

suggests that the reduced Pitx3 expression affects lens

growth, rather than lens induction or the initial differen-

tiation of epithelial and primary fiber cells. Although the

morphant lenses were severely degenerated at 7 dpf, both

lens epithelial and fiber cells were identified at 3 and

5 dpf based on histological and immunohistochemical

analyses. However, the morphant fiber cell morphology

suggested that terminal fiber cell differentiation was

incomplete. In addition, the morphant lens epithelial

cells were disordered and lacked the morphology and

actin localization pattern that characterized the wild-type

cells. Transgenic expression of dominant negative forms

of the mouse FGF receptor-1 driven by the aA crystallin

promoter resulted in similar lens phenotypes (Chow et al.,

1995; Robinson et al., 1995). The failure to complete

some aspects of cell differentiation in the morphant could

affect cell viability and lead to the degeneration of the

morphant lenses.

Pitx3 mutations cause the mouse aphakia (ak) mutant

phenotype, which consists of small eyes that lack lenses

(Rieger et al., 2001; Semina et al., 2000; Varnum and

Stevens, 1968). While the pitx3 morphant lens defects

resemble the ak phenotype, notable differences suggest

that different functional phases of lens development are

affected in each organism. The first abnormality in the

ak eyes occurs slightly before closure of the lens pit.


Cells that are released by the lens epithelium progress-

ively accumulate in the lens vesicle (Varnum and

Stevens, 1968; Zwaan, 1975). The lens vesicle also

fails to detach from the ectoderm and stays connected

through the lens stalk (Grimm et al., 1998; Semina et

al., 2000; Varnum and Stevens, 1968). In contrast, the

pitx3 morphant lens failed to exhibit either the

accumulating lens epithelial cells or the persistent lens

stalks. The ak lens vesicle cells also never elongate to

become lens fibers, which suggests that primary lens

differentiation fails. While the primary differentiation of

the pitx3 morphant lens occurred, the morphant fiber

cells failed to terminally differentiate based on their

retention of nuclei and stunted elongation. In addition,

the morphant lens epithelial cells exhibited aberrant

differentiation because they displayed disorganized actin

localization patterns. The morphant lenses also under-

went progressive degeneration, which suggests they

ultimately failed to maintain growth. While both the

ak and pitx3 morphant lenses exhibit defective lens

differentiation, the ak defect is manifested earlier in

development and results in the complete failure of lens

formation. These differences may reflect the relative

strength of the phenotypes (Gage et al., 1999b; Lehmann

et al., 2003; Ormestad et al., 2002), as the ak mutant

represents the null condition and the pitx3 morphant

may retain some functional Pitx3 protein. This residual

Pitx3 activity may be sufficient for the morphant lens to

develop further than the ak lens. While our failure to

identify a duplicated gene by in silico analysis does not

totally rule out the possibility of a pitx3 paralogue, the

Pitx3 protein may have an earlier role in mouse lens

development than in zebrafish. This does not preclude a

similar requirement for Pitx3 in terminal lens fiber and

epithelial cell differentiation in both organisms.

While the pitx3 morphant lens phenotype is less

severe than the ak lens developmental arrest, the cell

death characterizing the morphant retinas is more severe

than the ak retinal folding that occurs (Graw and Loster,

2003; Grimm et al., 1998). The pitx3 morphant retinal

cell death was marked by pyknotic nuclei in both the

inner and outer nuclear layers of the retinas. In addition,

the morphant photoreceptors failed to produce outer

segments and mature. The morphants also exhibited a

reduction in number and continuity of rod photo-

receptors, double and single cone photoreceptors, ama-

crine cells and ganglion cells. It is interesting that the

retinal margins, which correspond to the youngest

neurons, are largely devoid of any neurons in the

morphant retina (Fig. 7B, E, H). This suggests that loss

of Pitx3 may either block the differentiation of neurons

from the marginal zone or may result in the preferential

death of younger neurons. This potential requirement for

Pitx3 in the proliferating retinal margin cells may be

analogous to the requirement for Pitx3 in the lens

equatorial zone. Pitx3 expression in the mammalian

retina was not reported previously and we did not detect

expression in the zebrafish retina or the retinal pigmented

epithelium. Thus, the morphant retinal phenotype is

likely a non-autonomous effect from the aberrant lens

development.

Secondary retinal effects due to lens abnormalities

were observed in zebrafish when diptheria toxin was

expressed in the developing lens under the control of

the aA-crystallin promoter (Kurita et al., 2003). In

addition, lens transplantations between surface-dwelling

and eyeless cavefish demonstrated a central role for the

lens in retinal development (Yamamoto and Jeffery,

2000) and toxin-mediated lens ablation or surgical

removal in mouse resulted in secondary abnormalities

in the surrounding eye tissues (Coulombre and Cou-

lombre, 1964; Kaur et al., 1989). Indeed, chick

embryonic lens epithelial cells are necessary for the

proper differentiation of neural crest mesenchyme cells

to form the corneal endothelium and the lens provides

signals for iris and ciliary body induction (Beebe and

Coats, 2000; Thut et al., 2001). Additional experiments

utilizing lens transplantations between morphant and

wild-type zebrafish embryos may further elucidate the

lens roles during eye development in fish. Also, we are

currently developing antibodies for immunolocalization

of Pitx3 in zebrafish tissue sections to further examine

for Pitx3 protein expression in the retina and more

precisely delineate the expression patterns in the other

eye tissues.

While the effects of Pitx3 mutations on lens and

anterior segment development have been characterized in

both humans and mice, the genetic pathway for Pitx3

function has not been determined. The zebrafish model

offers significant opportunities for both forward and

reverse genetic analysis of gene roles and the molecular

dissection of eye development pathways. Characterizing

zebrafish mutants exhibiting lens or other anterior segment

defects using molecular markers identified by cloning

orthologs of known mammalian eye development genes

will facilitate mutant gene identification and function

determination. Furthermore, high-throughput screening for

target-selected mutations may generate allelic series,

which allow the study of essential genes and are valuable

for protein domain analysis (Colbert et al., 2001;

Wienholds et al., 2002). We identified a zebrafish pitx3

ortholog and characterized its expression pattern. Based

on protein knockdown analysis using anti-sense morpho-

linos, we confirmed that Pitx3 functions in some aspects

of lens cell differentiation and may also be required for

neuronal differentiation and viability in the retina, which

is different from the retinal phenotype described in ak

mice. The further study of pitx3 in zebrafish will allow

more precise identification of Pitx3 functions and inter-

acting factors necessary for eye anterior segment

formation.


4. Experimental procedures

4.1. Animals

Zebrafish (Danio rerio) were raised and maintained on

a 14/10-h light/dark cycle. The embryos were obtained

by natural spawning, raised at 28.5 8C and the develop-

mental stage [indicated as either hours or days post-

fertilization (hpf or dpf)] determined by morphological

criteria (Kimmel et al., 1995). All experiments were

conducted in accordance with the guidelines set forth by

the animal care and use committees at the participating

institutions.

4.2. Gene cloning, genomic structure determination

and gene mapping

Partial zebrafish pitx3 cDNAs were obtained by PCR

amplification of zebrafish whole eye cDNA using primers

derived from the Xenopus pitx3 sequence. Two overlap-

ping amplification products were obtained using Taq DNA

polymerase and the primers: forward primer #1 5 0-

ATGGATTTCAATCTTCTGACAGA-3 0 and reverse pri-

mer #1 5 0-AAGCTGTTCTTGCAGAGCTC-3 0 for the 5 0

PCR product and forward primer #2 5 0-CATTTTAC-

CAGCCAGCAACT-3 0 and reverse primer #2 5 0-

GGATCGGCCAGTATGAAGGA-3 0 for the 3 0 PCR

product. The PCR parameters were denaturation at 94 8C

for 30 s, annealing at 48–55 8C for 45 s, and extension at

72 8C for 45 s. The PCR products were cloned into pCRII

Topo (Invitrogen, Carlsbad, CA) and sequenced. Two

overlapping sequences were assembled and used as a

BLAST query of the non-redundant database. This

nucleotide sequence is most homologous to the known

Pitx3 sequences in different species. A BLAST search of

the zebrafish genomic sequences (http://www.sanger.ac.

uk/Projects/D_rerio/) identified matching sequences in the

ctg11787 contig (Fig. 1B). Using this contig sequence, we

designed new primers that contained the predicted

translation initiation and stop codons (shown in bold) of

the zebrafish pitx3 gene (forward primer 5 0-CGGTA-

GAGGTGATGGATTTTA-3 0 and reverse primer 5 0-

GAAATCTAGACGCATCGCTTTCA-3 0) and amplifed

the entire zebrafish pitx3 open reading frame from

zebrafish adult eye mRNA using PfuUltra high-fidelity

DNA polymerase (Stratagene, La Jolla, CA). The genomic

contig ctg11787 contained most of the genomic pitx3

sequence except for two regions. We designed primers to

span these regions and used PCR to obtain the complete

pitx3 gene sequence. The zebrafish pitx3 sequences were

submitted to GenBank (BankIt Numbers 624871 and

624872 for the cDNA and genomic sequences,

respectively).

The pitx3 gene was mapped using the LN54 radiation

hybrid panel (Hukriede et al., 1999, 2001). Each DNA

clone in the panel was amplified in duplicate using

primers forward 5 0-TGAGCGTTTGTAGACGATGG-3 0

and reverse 5 0-ATGTCAGACTGCACTTCGC-3 0, which

are located in introns 1 and 2, respectively, and amplify a

293 bp fragment containing exon 2 of the pitx3 gene. The

amplification results were analyzed using RHVECTOR

(http://mgchd1.nichd.nih.gov:8000/zfrh/beta.cgi) with dis-

cordance of less than 5%.

4.3. RT-PCR and tissue in situ mRNA hybridization

Total RNA was extracted from different embryonic

stages and adult tissues (RNA Extraction Kit: Qiagen,

Valencia, CA). RT-PCR reactions of the full-length pitx3

cDNA open reading frame were amplified from 100 ng of

total RNA in each reaction (RT-PCR Kit; Stratagene,

LaJolla, CA) using the primers and conditions described

above. PCR amplification of b-actin was used in control

reactions. We performed tissue in situ hybridization as

previously described (Link et al., 2001) using a full-length

in vitro transcribed riboprobe.

4.4. Polyclonal antibody production

A zebrafish Pitx3 peptide (amino acids 221–238,

Fig. 1A) was conjugated to KLH and used to immunize

rabbits to generate a polyclonal antiserum (Proteintech

Group, Chicago, IL). The antiserum was purified by Protein

A column chromatography (Pierce, Rockford, IL).

For immunoblot detection of Pitx3 expression, zebra-

fish embryos (24 hpf) or larval heads (3, 4, 5 or 7 dpf)

were homogenized in 0.02 M NaPO4 (pH 7.4)/0.01 M

EDTA/0.5% Triton X-100. At least 20 embryos or larval

heads were homogenized for each time point that was

examined. The homogenates were microcentrifuged at

15,000!g for 3 min, the supernatants were mixed with

NuPAGE LDS Sample Buffer (Invitrogen) and heated at

70 8C for 10 min. The total protein equivalent of one

embryo or one larval head was loaded per lane,

electrophoresed through 4–12% Bis-Tris gels (NuPAGE

Novex; Invitrogen) and transferred to Hybond-P PVDF

membrane (Amersham, Piscataway, NJ). The primary

antibody was detected using the ECL system (Amersham)

as described (Vihtelic et al., 1999). The immunodetection

of actin in each sample was used as a control for protein

loading.

4.5. Morpholino oligomer injections

Morpholino oligomers were purchased from Gene Tools

(Corvallis, OR). Two different oligomers were designed to

hybridize to partially overlapping sequences in the 5 0

untranslated region of the zebrafish pitx3 transcript: 5 0-

CGGTCTAGTGGAGGTAATCCTCGAA-3 0 (Morph1) and

5 0-GGCTATGAATCCCGGTCTAGTGGAG-3 0 (Morph2).

In addition, an oligomer complementary to sequences

surrounding the intron three splice donor site was used:



http://mgchd1.nichd.nih.gov:8000/zfrh/beta.cgi


5 0-CTCACAGAAGCAAGAGTACGGGTAC-3 0 (Morph3).

A control morpholino, containing five mismatched nucleo-

tides relative to Morph1, 5 0-CGCTCTACTGGACG-

TAATGCTCCAA-3 0 (mismatched nucleotides in bold)

was also used. The morpholinos were resuspended in

water and injected into 1–4 cell-stage embryos as

described (Wei et al., 2004). The morpholino concentration

used to generate the morphants for immunoblot analysis

was 1 ng/nl.

4.6. Histology and immunohistochemistry

For histological analysis, morphant and control larvae

were fixed in 2% formaldehyde/2.5% glutaraldehyde/0.1 M

cacodylate (pH 7.4) and processed into Polybed 812 as

described (Vihtelic and Hyde, 2000; Vihtelic et al., 2001).

Immunohistochemistry was performed on frozen larval

sections. The morphants and controls were fixed, embedded

in agarose and frozen sections were cut as described

(Vihtelic et al., 2001). TBS (pH 7.4)/5% normal goat serum/

0.3% Triton X-100 was used for tissue blocking and

antibody dilutions.

Lenses were evaluated by immunodetection of the zl-1

antigen (1:500; University of Oregon Monoclonal Antibody

Facility) as described (Vihtelic et al., 2001). Filamentous

actin in the lens and retina was stained using phalloidin-

AlexaFluor 488 (1:50; Molecular Probes, Eugene, OR) in

TBS/5% normal goat serum/0.5% Triton X-100. The retinal

amacrine and ganglion cells were detected using a

monoclonal antibody to HuC/D (1:30; Molecular Probes),

while the rods and short single cone photoreceptors were

detected using affinity-purified rhodopsin (1:5000) and UV

opsin antisera (1:1000), respectively (Vihtelic et al., 1999).

The double cone photoreceptors were identified using the

monoclonal antibody zpr-1 (1:200; University of Oregon

Monoclonal Antibody Facility). All antibody incubations

were overnight at room temperature and coverslips were

mounted using Vectashield (Vector Laboratories, Burlin-

game, CA). In some cases, Vectashield containing propi-

dium iodide was used to stain the nuclei.

Acknowledgements

Dr Xiangyun Wei provided technical advice for the

morpholino knockdown experiments. Debbie Bang and the

Freimann Life Sciences Center were responsible for

zebrafish husbandry at the University of Notre Dame.

Dr Ryan Thummel offered manuscript criticisms. We also

thank Dr Brian Link for consultation, Becki Tyler for

excellent technical assistance and Dr Zoltan Varga and

Sunit Dutta for their collegiality and expert comments. This

work was supported by NIH grants R01 EY13606 to E.V.S.

and R01 EY014455 to T.S.V.

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Zebrafish pitx3 is necessary for normal lens and retinal development

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