Clinical embryology and development of the eye

Differentiation

Differentiation is the process whereby collections of cells develop into a multicellular organism with tissues of particular functions. Multicellular organisms are made of 3D spatiotemporal arrays of different cell types that develop over time. The fate map defines the individual intrinsic (gene) steps to achieve this. Increasingly recognized are the environmental factors (pH, metabolites, morphogens).

Cell migration

Collective cell migration plays essential roles in embryogenesis to facilitate the dramatic shape changes that occur over short periods of time. Differential rates of cell division within and between tissues contribute to these shape changes. Regulatory processes include self-generating gradients, internal chemotaxis or mechanotaxis and contact-dependent polarization within migrating cell groups. Neural crest cells (NCC) are one of the most striking examples of cell migration moving from their birthplace to a distant region including the developing eye.

Programmed cell death

Development includes processes of construction as well as remodeling (destruction). Apoptosis is critical for tissue remodeling enabling normal development.

Signaling

Embryonic development depends on accurate, timely, and specific communication between cells. Cell communication involves several components, including ligands which are molecules that bind another specific target molecule, termed a receptor. Receptors can be on the cell surface or intracellular. The ligand–receptor interaction will often lead to a signal transduction cascade. Some ligand–receptor interactions can activate several different signal transduction pathways. By convention, the most commonly used pathway is known as canonical (e.g. β-catenin activation in Wnt signaling) and the alternative pathway is known as the non-canonical pathway. Highly conserved core pathways, including the Notch, Transforming Growth Factor beta (TGF-β), Wnt, Hedgehog (HH), and Receptor Tyrosine Kinase (RTK) signaling systems, play pivotal roles across a broad range of developmental processes.

Transcription factor codes

Transcription factors (TFs) are proteins that bind DNA by recognizing specific sequence motifs located at regulatory elements, such as promoters and enhancers. In turn, this TF binding controls downstream processes such as recruitment of RNA polymerases, DNA methylation, and nucleosome chemical modifications and displacement. The result is the activation or repression of gene expression. The expression of combinations of TFs can be used to mark a region of undifferentiated tissue that will become a specific structure. RAX is an example that defines the eye field.

Eye embryogenesis

Early vertebrate eye formation follows a conserved sequence of events. Following gastrulation, the eye field is specified in the anterior neural plate extending across the midline. Studies in vertebrate model systems indicate that eye specification is defined by discrete and overlapping expression domains of different transcription factors (TFs) (see Table 2.1 ). The eye field transcription factors (EFTF) include: Otx2 , Tbx3 , Rax , Pax6 , Six3 , Six6 , Lhx2 , and Nr2e1 . Rax is the EFTF most used as a marker of the eye field ( Fig. 2.1 ). Mutations in the human Rax gene are a rare cause of human microphthalmia. Otx2 is the earliest molecular delineator of the eye field and mutations in Otx2 cause severe ocular malformations in humans. The eye field must split in the midline to form two separate eyes. Both Shh and Six3 are critical regulators of eye field bifurcation. Following splitting of the eye field, the first morphological landmarks are bilateral indentations (optic sulci or pits) in the cranial end of embryonic neural folds at approximately 22 days.

Following the establishment of the two eye primordia, the optic vesicle (OV) precursor cells continue migrating, initiating vesicle evagination. A subset of eye field transcription factors, including Rax and Pax6 , control OV evagination and formation (see Fig. 2.1 ). The OVs are connected to the forebrain by the optic stalk (a short tube that eventually forms the optic nerve) ( Fig. 2.2A–B ).

Eye organogenesis (4th to 8th week gestation, human)

Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development.

Fifth week

Further invagination of the OV to form the OC predominates in the 5th week. Invagination involves the retinal disc, lens plate, and the ventrocaudal wall of the optic vesicle ( Fig. 2.2C–D ). A critical developmental step is the patterning of the dividing OV progenitor cells to establish dorsal–ventral, nasal–temporal, and distal–proximal identity. The establishment of these molecular boundaries directs the OC to regionalize along several axes: dorsal–ventral, anterior (cornea)–posterior (retinal pigment epithelium, RPE), OS (ventral) nasal–temporal, and central (central neural retina)–peripheral (ciliary epithelium). Shh, retinoic acid (RA), and bone morphogenetic protein 4 (BMP4) are the key factors in patterning the developing OV. Bmp4 is expressed in the distal OV and subsequently within dorsal regions of the OC. The midline gradient of Shh signaling differentiates the OS from the OC through induction of the OS markers, Pax2, Vax1, and Vax2 ( Fig. 2.2D ).

Networks of transcription factors act by induction and repression to form the boundaries between the OS, RPE, and the presumptive neural retina (PNR). Key transcription factors include Pax2 , Pax6 , Vax1 , Vax2 , Tbx2 , Tbx3 , Tbx5 , Vsx2 , and Mitf . The establishment of these molecular boundaries is vital for subsequent steps in eye formation, as different regions of the OV give rise to the RPE (dorsal), OS (ventral), and PNR ( Fig. 2.2D ).

Invagination of the retinal disc of the OV leads to formation of the inner layer of the OC which becomes the PNR, while the external layer of the OC is the forerunner of the RPE ( Fig. 2.2D ). The OC is not continuous and forms a fold inferiorly and ventrally that is continuous with the OS. This fold is called the embryonic (optic) fissure.

Lens development from the lens placode is controlled by signals emanating from both the OV (positive) and periocular mesenchyme (POM) (negative). This occurs along the head surface ectoderm through Pax6 inhibition by TGFβ/Smad3 and Wnt/beta-catenin signaling from the POM to the ectoderm.

The lens pit deepens to become the lens vesicle. This occurs synchronously with optic cup invagination ( Fig. 2.2C–D ). The lens placode cells constrict apically leading to lens vesicle separation from the surface ectoderm which will become the corneal epithelium. The resulting lens vesicle is relatively large and fills the OC ( Fig. 2.3 ). Two key regulators of lens development are Pitx3 and Foxe3 . Pitx3 expression in the lens placode is more refined than Foxe3 but becomes expressed throughout the lens vesicle and is another key regulator of lens development.