Early eye morphogenesis in the zebrafish is similar to that of other vertebrates, though some differences exist that we will try to highlight.

 Specification of the eye field

The first step in eye formation is the specification of a single field of coherent cells within the anterior neural plate. This domain, the eye field, is the precursor of the neural retina, retinal pigmented epithelium and optic nerve of both eyes.

The eye field is defined by the overlapping expression of a number of transcription factors, collectively known as eye field transcription factors(EFTFs). These genes are highly conserved across the animal kingdom, and form a cross-regulatory network essential for eye development.  In zebrafish, the earliest EFTF to be expressed is rx3, which can be detected in the future eye field from mid-gastrulation (or 8 hpf, Fig1). It is unclear how the expression of these genes is first induced, but their expression is at least modulated by the same signals that are involved in the induction of other anterior neural plate territories. Thus, eye field specification is coordinated with the formation of the telencephalon, dorsal thalamus and hypothalamus.


Splitting of the eye field and evagination of the optic vesicles 

The eye field is specified as a primordium straddling the midline, and subsequently it undergoes extensive reorganisation that leads to the evagination of two optic vesicles from the lateral walls of the forming neural tube. This process has been poorly analysed and the mechanisms underlying optic vesicle evagination are largely unknown, but it is thought that a morphogenetic program instructed by the EFTFs, in combination with signals derived from the ventral midline tissues result in eye field cells moving laterally and evaginating to give rise to the left and right optic vesicles.

Contrary to what happens in other vertebrates, where the optic vesicles evaginate as hollow structures, in the zebrafish the optic primordia evaginate as solid masses of cells with the optic stalk at an anterior position. Subsequently, rotation of the eye primordium about the optic stalk brings the latter into a ventral position.


Patterning of the optic vesicles

Concomitant with their evagination, the optic vesicles are patterned in the proximo-distal axis. The proximal part of the optic vesicle gives rise to the optic stalk while distal regions give rise to the optic cup. The complementary expression of pax2 (proximal) and pax6 (distal) is the earliest readout of this regionalisation, and mutual repression between these two transcription factors stabilises the boundary between proximal and distal domains (fig3?). The Shh signalling molecule, released by the ventral midline tissues is essential to induce pax2 and vax1 in the prospective proximal part of the optic vesicle and triggers the establishment of pax2/pax6 complementary domains.

The function pax2 and vax1 is essential for the specification of the proximal part of the evaginating optic vesicles that narrows and elongates forming the optic stalk. The optic stalk is a transient tubular structure precursor of the optic nerve.

At their distal-most side, the optic vesicles make close contact with the overlying surface ectoderm. This contact is essential for the subdivision of the distal part of the optic vesicle into RPE and neural retina. The surface ectoderm releases signals, among them Fgfs, which are essential for induction and maintenance of the neural retina. The region of the distal optic vesicle that is not in close apposition with the surface ectoderm is specified as RPE. The RPE is in direct contact with the periocular mesenchyme (POM) and its specification is greatly influenced by signals derived from this tissue.

Formation of the optic cup

The close apposition of optic vesicle and surface ectoderm also triggers the transformation of the optic vesicle into an optic cup. This transformation is tightly coordinated with lens induction and invagination (see below), and extends during a period of several hours, from 16 to 24hpf.

The distal vesicle invaginates to give rise to a double-layered optic cup, and provides the first indication of the final shape of the eye. The inner layer of the cup, closer to the lens, gives rise to the neural retina, and the outer layer becomes the RPE.

The invagination of the optic vesicle around the lens begins in its ventral part, and, at the end of the invagination process, a narrow opening, known as the optic fissure, remains in the ventral most part of the cup. The invagination process and formation of the optic fissure (or choroid fissure) occur not only in the optic vesicle, but also in the optic stalk. The optic fissure provides a channel for the entry of blood vessels and the exit of projecting axons.


NT and DV patterning of the neural retina

By optic cup stages, a number of markers (transcriptor factors, see below) are expressed in restricted patterns in the neural retina reflecting its subdividsion in nasal-temporal (NT) and dorsal-ventral (DV) territories. This patterning process resembles the patterning of the neural tube, and requires a balance of different extracellular signaling molecules. Among those, Fgf signaling is required to establish the N/T axis; Fgf promotes nasal and represses temporal retinal identity (Picker et al. 2005). Bmp, Shh, Fgf and RA signaling are required for the specification of the D/V axis, with higher levels of Bmp in the dorsal retina and higher levels of RA in the vental retina. Shh signaling plays a key role in P/D patterning with high levels promoting proximal identity.


Lens formation

Non-neural ectoderm surrounding the anterior neural plate becomes competent to develop as lens already during gastrulation. Planar signals derived from the anterior neural plate are essential to bias the head ectoderm to lens fate.

Upon neural retina specification, signals from the neural retina induce lens fate in the overlying surface ectoderm. The lens placode then invaginates to give rise to the lens vesicle, which buds out at around 24hpf. The lens vesicle keeps growing throughout adulthood and fibre cells differentiation is maintained from a pool of undifferentiated proliferating epithelial cells located at the most anterior region of the vesicle.


Mesenchyme and neural crest derivatives

The POM gives rise to various extraocular structures, including the corneal endothelium, keratocytes of the corneal stroma, ciliary muscle, iris stroma, the sclera, choroidal pericytes, the trabecular meshwork, orbital cartilages and bones, blood vessels, as well as connective tissue associated with the extraocular muscles.

In addition, the POM has important signalling roles during the specification of the RPE and the closure of the choroid fissure. During these processes, the POM is source of signals that induce the expression of downstream genes in the retinal tissues, and lead to the establishment of differentiation and morphogenetic programs essential for the formation of a functional eye.


 Closure of the choroid fissure and optic nerve formation

Closure of the optic fissure marks the end of the morphogenetic phase. Soon after the POM cells entrance into the developing eye chamber, the fissure begins to fuse from its central region and proceeding anteriorly and posteriorly. Later on, the tight interaction of the growing axons with the stalk cells leads to the differentiationof the optic stalk cells into astrocytes that will populate the body of the mature optic nerve. (Horsburgh and Sefton, 1986).

Optic nerve glial cells originate from the in situ proliferation and differentiation of optic stalk cells, except for the oligodendrocytes, which migrate into the optic nerve from the diencephalons.


Formation of a functional eye

The closure of the choroid fissure happens during an extended period of time, and overlaps with the differentiation of the central neural retina to give rise to the seven different types of neurons and the glial cells that compose the mature retina.

The final stages of RPE differentiation also happen simultaneously, and lead to the formation of a cuboidal epithelium of pigmented cells at the interface between the neural retina and the vascular choroid layer.

Key Publications

Gestri G, Bazin-Lopez N, Scholes C, Wilson SW.
Cell Behaviors during Closure of the Choroid Fissure in the Developing Eye.
Front Cell Neurosci. 2018 Feb 20;12:42. 

Valdivia LE, Lamb DB, Horner W, Wierzbicki C, Tafessu A, Williams AM, Gestri G, Krasnow AM, Vleeshouwer-Neumann TS, Givens M, Young RM, Lawrence LM, Stickney HL, Hawkins TA, Schwarz QP, Cavodeassi F, Wilson SW, Cerveny KL.
Antagonism between Gdf6a and retinoic acid pathways controls timing of retinal neurogenesis and growth of the eye in zebrafish.Development. 2016 Apr 1;143(7):1087-98.

Hernández-Bejarano M, Gestri G, Spawls L, Nieto-López F, Picker A, Tada M, Brand M, Bovolenta P, Wilson SW, Cavodeassi F.
Opposing Shh and Fgf signals initiate nasotemporal patterning of the zebrafish retina.
Development. 2015 Nov 15;142(22):3933-42.

Bazin-Lopez N, Valdivia LE, Wilson SW, Gestri G.
Watching eyes take shape.
Curr Opin Genet Dev. 2015 Jun;32:73-9.

Lupo G, Gestri G, O'Brien M, Denton RM, Chandraratna RA, Ley SV, Harris WA, Wilson SW.
Retinoic acid receptor signaling regulates choroid fissure closure through independent mechanisms in the ventral optic cup and periocular mesenchyme.
Proc Natl Acad Sci U S A. 2011 May 24;108(21):8698-703.

Ivanovitch K, Cavodeassi F, Wilson SW.
Precocious acquisition of neuroepithelial character in the eye field underlies the onset of eye morphogenesis.
Dev Cell. 2013 Nov 11;27(3):293-305.

Cavodeassi F, Ivanovitch K, Wilson SW.
Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis.Development. 2013 Oct;140(20):4193-202.

Cavodeassi F, Carreira-Barbosa F, Young RM, Concha ML, Allende ML, Houart C, Tada M, WilsonSW.
Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/beta-catenin pathway.
Neuron. 2005 Jul 7;47(1):43-56.