In Vitro Differentiation of Mouse Embryonic Stem Cells into Neurons of the Dorsal Forebrain

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10–12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex

Lhx2 Is Required for Patterning and Expansion of a Distinct Progenitor Cell Population Committed to Eye Development

Progenitor cells committed to eye development become specified in the prospective forebrain and develop subsequently into the optic vesicle and the optic cup. The optic vesicle induces formation of the lens placode in surface ectoderm from which the lens develops. Numerous transcription factors are involved in this process, including the eye-field transcription factors. However, many of these transcription factors also regulate the patterning of the anterior neural plate and their specific role in eye development is difficult to discern since eye-committed progenitor cells are poorly defined. By using a specific part of the Lhx2 promoter to regulate Cre recombinase expression in transgenic mice we have been able to define a distinct progenitor cell population in the forebrain solely committed to eye development. Conditional inactivation of Lhx2 in these progenitor cells causes an arrest in eye development at the stage when the optic vesicle induces lens placode formation in the surface ectoderm. The eye-committed progenitor cell population is present in the Lhx2−/− embryonic forebrain suggesting that commitment to eye development is Lhx2-independent. However, re-expression of Lhx2 in Lhx2−/− progenitor cells only promotes development of retinal pigment epithelium cells, indicating that Lhx2 promotes the acquisition of the oligopotent fate of these progenitor cells. This approach also allowed us to identify genes that distinguish Lhx2 function in eye development from that in the forebrain. Thus, we have defined a distinct progenitor cell population in the forebrain committed to eye development and identified genes linked to Lhx2's function in the expansion and patterning of these progenitor cells

Candidate Gene Screen in the Red Flour Beetle Tribolium Reveals Six3 as Ancient Regulator of Anterior Median Head and Central Complex Development

Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly