orthodenticle

EVOLUTIONARY HOMOLOGS

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Orthodenticle homologs in birds

The avian equivalent of Spemann's organizer, Hensen's node, begins to lose its ability to induce a nervous system from area opaca epiblast cells at stage 4+, immediately after the full primitive streak stage. From this stage, the node is no longer able to induce regions of the nervous system anterior to the hindbrain. Stage 4+ is marked by the emergence from the node of a group of cells, the prechordal mesendoderm. Does the prechordal region possesses the lost functions of the organizer? The prechordal region does not have neural inducing ability, as it is unable to divert extraembryonic epiblast cells to a neural fate. However, it can confer more anterior character to prospective hindbrain cells of the host, making them acquire expression of the forebrain markers tailless and Otx-2. It can also rescue the expression of Krox-20 and Otx-2 from nervous system induced by an older (stage 5) node in extraembryonic epiblast. These properties reflect a true change of fate of cells rather than recruitment from other regions. The competence of neuroectoderm to respond to anteriorizing signals declines by stages 7-9, but both posteriorizing signals and the ability of neuroectoderm to respond to them persist after this stage (Foley, 1997).

Beads containing recombinant FGF8 (FGF8-beads) were implanted in the prospective caudal diencephalon or midbrain of chick embryos at stages 9-12. This induces the neuroepithelium rostral and caudal to the FGF8-bead to form two ectopic, mirror-image midbrains. Furthermore, cells in direct contact with the bead form an outgrowth that protruded laterally from the neural tube. Tissue within such lateral outgrowths developed proximally into isthmic nuclei and distally into a cerebellum-like structure. These morphogenetic effects are apparently due to FGF8-mediated changes in gene expression in the vicinity of the bead, including a repressive effect on Otx2 and an inductive effect on En1, Fgf8 and Wnt1 expression. The ectopic Fgf8 and Wnt1 expression domains form nearly complete concentric rings around the FGF8-bead, with the Wnt1 ring outermost. These observations suggest that FGF8 induces the formation of a ring-like ectopic signaling center (organizer) in the lateral wall of the brain, similar to the one that normally encircles the neural tube at the isthmic constriction, which is located at the boundary between the prospective midbrain and hindbrain. This ectopic isthmic organizer apparently sends long-range patterning signals both rostrally and caudally, resulting in the development of the two ectopic midbrains. Interestingly, the data suggest that these inductive signals spread readily in a caudal direction, but are inhibited from spreading rostrally across diencephalic neuromere boundaries. These results provide insights into the mechanism by which FGF8 induces an ectopic organizer and suggest that a negative feedback loop between Fgf8 and Otx2 plays a key role in patterning the midbrain and anterior hindbrain (Martinez, 1999).

The patterns of the Gbx2, Pax2, Wnt1, and Fgf8 gene expression were analyzed in the chick with respect to the caudal limit of the Otx2 anterior domain, taken as a landmark of the midbrain/hindbrain (MH) boundary. The Gbx2 anterior boundary is always concomitant with the Otx2 posterior boundary. The ring of Wnt1 expression is included within the Otx2 domain and Fgf8 transcripts included within the Gbx2 neuroepithelium. Pax2 expression is centered on the MH boundary with a double decreasing gradient. A new nomenclature is proposed to differentiate the vesicles and constrictions observed in the avian MH domain at stage HH10 and HH20, based on the localization of the Gbx2/Otx2 common boundary (Hidalgo-Snachez, 1999).

The expression pattern of Otx2, a homeobox-containing gene, was analyzed from the beginning of eye morphogenesis until neural retina differentiation in chick embryos. Early on, Otx2 expression is diffuse throughout the optic vesicles but becomes restricted to their dorsal part when the vesicles contact the surface ectoderm. As the optic cup forms, Otx2 is expressed only in the outer layer, which gives rise to the pigment epithelium. This early Otx2 expression pattern is complementary to that of PAX2, which localizes to the ventral half of the developing eye and optic stalk. Otx2 expression was always observed in the pigment epithelium at all stages analyzed but was extended to scattered cells located in the central portion of the neural retina around stage 22. The number of cells expressing Otx2 transcripts increases with time, following a central to peripheral gradient. Bromodeoxyuridine labeling in combination with immunohistochemistry with anti-OTX2 antiserum and different cell-specific markers were used to determine that OTX2-positive cells are postmitotic neuroblasts undergoing differentiation into several, if not all, of the distinct cell types present in the chick retina. These data indicate that Otx2 might have a double role in eye development. First, it might be necessary for the early specification and subsequent functioning of the pigment epithelium. Later, OTX2 expression might be involved in retina neurogenesis, defining a differentiation feature common to the distinct retinal cell classes (Bovolenta, 1997).

A mature inner ear is a complex labyrinth containing multiple sensory organs and nonsensory structures in a fixed configuration. Any perturbation in the structure of the labyrinth will undoubtedly lead to functional deficits. Therefore, it is important to understand molecularly how and when the position of each inner ear component is determined during development. To address this issue, each axis of the chick otocyst at embryonic day 2.5 (E2.5), stage 16-17, was changed systematically at an age when axial information of the inner ear is predicted to be fixed based on gene expression patterns. Transplanted inner ears were analyzed at E4.5 for gene expression of BMP4 (bone morphogenetic protein), SOHo-1 (sensory organ homeobox-1), Otx1 (cognate of Drosophila orthodenticle gene), p75NGFR (nerve growth factor receptor) and Msx1 (muscle segment homeobox), or at E9 for their gross anatomy and sensory organ formation. The results show that axial specification in the chick inner ear occurs later than expected and patterning of sensory organs in the inner ear is first specified along the anterior/posterior (A/P) axis, followed by the dorsal/ventral (D/V) axis. Whereas the A/P axis of the sensory organs is fixed at the time of transplantation, the A/P axis for most non-sensory structures is not specified and is still able to be re-specified according to the new axial information from the host. The D/V axis for the inner ear was not fixed at the time of transplantation. The asynchronous specification of the A/P and D/V axes of the chick inner ear suggests that sensory organ formation is a multi-step phenomenon, rather than a single inductive event. The expression patterns of BMP4 in rotated ears is consistent with a role for BMP4 in the specification of sensory organs. Similarly, Otx1 and SOHo-1 gene expressions are always found associated with the formation of the cochlear and semicircular canals, respectively, suggesting that their gene products may play a role in the specification of these inner ear structures (Wu, 1998).

The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. These data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates (Matsumoto, 2004).

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