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Sonic hedgehog (Shh) signaling regulates multiple morphogenetic processes during embryonic neurogenesis and craniofacial skeletal development. Gpr161 is a known negative regulator of Shh signaling. Nullizygous Gpr161 mice are embryonic lethal, presenting with structural defects involving the neural tube and the craniofacies. However, the lineage specific role of Gpr161 in later embryonic development has not been thoroughly investigated. We studied the Wnt1-Cre lineage specific role of Gpr161 during mouse embryonic development. We observed three major gross morphological phenotypes in Gpr161 cKO (Gpr161 f/f; Wnt1-Cre) fetuses; protrusive tectum defect, encephalocele, and craniofacial skeletal defect. The overall midbrain tissues were expanded and cell proliferation in ventricular zones of midbrain was increased in Gpr161 cKO fetuses, suggesting that protrusive tectal defects in Gpr161 cKO are secondary to the increased proliferation of midbrain neural progenitor cells. Shh signaling activity as well as upstream Wnt signaling activity were increased in midbrain tissues of Gpr161 cKO fetuses. RNA sequencing further suggested that genes in the Shh, Wnt, Fgf and Notch signaling pathways were differentially regulated in the midbrain of Gpr161 cKO fetuses. Finally, we determined that cranial neural crest derived craniofacial bone formation was significantly inhibited in Gpr161 cKO fetuses, which partly explains the development of encephalocele. Our results suggest that Gpr161 plays a distinct role in midbrain development and in the formation of the craniofacial skeleton during mouse embryogenesis.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) causes complete inhibition of rat embryonic midbrain (CNS) cell differentiation in the micromass in vitro test when applied at a concentration of 5 μg/ml under conditions where MX is rapidly degraded in culture medium with a half-life of 56 min. This study investigated whether or not degradation products of MX have inhibitory effects on CNS cell differentiation following pre-incubation of MX in culture medium for 0.5, 1 or 2 hr. When MX was pre-incubated for 0.5 hr, the mean number of differentiated foci was 0.2 against 62.5 for the control. However, the number increased to 44.7 when pre-incubation time was extended to 2 hr. These results suggest that MX, but not its degradation products, is a teratogen in vitro. MX manifested almost complete inhibitory effects on CNS cell differentiation by 0.5 hr of exposure.

In a previous study of the embryonic midbrain radial glia in a lizard, we observed that these cells define boundaries and regional subdivisions in a pattern that largely supports the adult model [C. Diaz, C. Yanes, C.M feminine. Trujillo, L. Puelles, Cytoarchitectonic subdivisions in the subtectal midbrain of the lizard Gallotia galloti, J. Neurocytol. 29 (2000) 569-593]. With the goal to check whether the midbrain of chick embryos has a similar pattern, we examined the radial glia distribution in this model using a lipophilic dye (DiI) injected intraventriculary. As in the lizard, chick radial glia distribution and fasciculation defines at least six regional subdivisions in the midbrain, five of which are alar and one basal. Each territory corresponds to a particular cytoarchitectonic area, recognized previously. The rostral mesencephalic limit (m/d) is underlined in the alar zone by a thick band of fasciculated radial glia intercalated between the griseum tectale and the posterior commissure. A fasciculated radial glia band also defines the caudal mesencephalic limit (m/r) across the alar and basal zones. These glial specializations, not described previously in the chick, give physical entity to the limits deduced from gene expression studies and suggested by descriptive cytoarchitectonic analysis. We conclude that the midbrain of sauropsides shares a common subdivision pattern co-defined by radial glia arrangements.

Segmentation is a mechanism that controls spatial organization along the anteroposterior axis of the neural tube and is particularly well characterized for the hindbrain region [1]. The generation of distinct and regionally specific structures from each rhombomere is achieved with the almost complete absence of cell mixing between neighboring rhombomeres [2, 3]. Here, we have examined cell mingling at the isthmus, where Otx2-expressing midbrain cells abut Gbx2-expressing hindbrain cells [4]. The sharp line of demarcation between the two expression domains suggests that this interface would be a compartment boundary, with no intermixing of cells, but this has not been directly tested. We have used short-term reaggregation assays to compare the adhesive properties of cells derived from midbrain and anterior hindbrain and cell labeling in vivo directly to monitor cell behavior at the midbrain/hindbrain boundary. Interestingly, our data demonstrate that, in contrast to the rhombomeres, differential adhesion does not seem to operate between the midbrain and anterior hindbrain and that cells move between the two territories. We conclude that these two subdivisions are not maintained by cell lineage restriction but by cells maintaining labile fates.

Neuronal migration, a key event during brain development, remains largely unexplored in the mesencephalon, where dopaminergic (DA) and GABA neurons constitute two major neuronal populations. Here we study the migrational trajectories of DA and GABA neurons and show that they occupy ventral mesencephalic territory in a temporally and spatially specific manner. Our results from the Pitx3-deficient aphakia mouse suggest that pre-existing DA neurons modulate GABA neuronal migration to their final destination, providing novel insights and fresh perspectives concerning neuronal migration and connectivity in the mesencephalon in normal as well as diseased brains.

We have studied the organization of the midbrain radial glia in embryos of Gallotia galloti using the fluorescent lipophilic dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) and the antibodies H5 and RC2. Our goal was to verify if the radial glia takes part in the midbrain boundaries formation and if it defines different zones. Our exam reveals two clear limits, anterior or mesencephalic–diencephalic (m/d) and posterior or mesencephalic–rhombencephalic (m/r), that can be defined as the borders where the midbrain radial glia processes end. Moreover, fasciculate radial glia processes characterize these limits totally or partially. They coincide with gene expression limits and with cytoarchitectonic limits defined by other criteria. Six different subdivisions, five alar and one basal, can be defined according to radial glia distribution, fasciculation, and immunohistochemical features. The ventral part of the alar region is defined by an RC2-positive bundle of radial glial cells. This bundle supposes a trustworthy landmark to point out the tectal/tegmental boundary. We hypothesize that this pattern of midbrain radial glia represents a basic model in amniota. J. Comp. Neurol. 473:162–176, 2004. © 2004 Wiley-Liss, Inc.

Rohrbacher, Jutta, Katja Sauer, Andrea Lewen, and Ulrich Misgeld. Enhancement of synaptic excitation by GABAA receptor antagonists in rat embryonic midbrain culture. J. Neurophysiol. 79: 1113–1116, 1998. Alterations of synaptic excitation induced by exposure to γ-aminobutyric acid-A (GABAA) receptor antagonists were investigated employing tight-seal whole cell recording from single neurons or pairs of neurons in rat embryonic midbrain culture. Application of GABAA receptor antagonists led to sustained depolarizations followed by synchronous paroxysmal depolarization shifts (PDSs). PDSs induced a transient increase in miniature excitatory postsynaptic currents in the presence as well as in the absence of a N-methyl-d-aspartate receptor antagonist. The increase in glutamate release supports the excitatory drive required to reinitiate PDSs from the quiescent interburst intervals. After washout of GABAA receptor antagonists, synaptic activity remained grouped, regardless of the presence or absence of PDS blockade by tetrodotoxin (TTX). Impediment of action potential-triggered transmitter release by Cd2+ or TTX also induced grouped activity. We conclude that changes in synaptic excitation are produced by the impaired GABAA inhibition per se and by the initiation of PDSs.