Kenneth Campbell

Regional incorporation and site-specific differentiation of striatal precursors transplanted to the embryonic forebrain ventricle

The developmental potential of neural progenitors derived from the E13.5-E14 lateral or medial ganglionic eminences (LGE and MGE, respectively) or the E12 ventral mesencephalon (VM) was examined in cross-species transplantation model. After injection into the E15 rat forebrain ventricle, mouse LGE progenitors (unlike those of the MGE or VM) were consistently integrated into the host striatum, expressing neurochemical phenotypes and axonal projections characteristic of striatal projection neurons. Additionally, both LGE and MGE precursors displayed widespread incorporation into distinct forebrain and midbrain structures, whereas the more caudally derived VM cells were largely confined to midbrain structures. These results suggest that many LGE precursors are positionally specified for striatal incorporation, while a portion also possess greater potential reflected in more widespread integration following intraventricular injection.

Early specification of striatal projection neurons and interneuronal subtypes in the lateral and medial ganglionic eminence

The striatum is thought to be generated from two transient swellings in the ventral telencephalon, the lateral and medial ganglionic eminences, present at mid-stages of embryonic rat development. We have studied the relative contribution of these structures to the specific generation of striatal neuronal subtypes such as projection neurons and cholinergic and somatostatin-containing interneurons at an early stage and a mid stage in striatal neurogenesis. Dissociated progenitors isolated from the embryonic day 12.5 and embryonic day 15.5 rat lateral ganglionic eminence grafted into the previously ibotenic acid lesioned adult striatum, produce grafts containing extensive numbers of neurons expressing messenger RNA for the striatal projection neuron marker, DARPP-32, whereas grafts of the embryonic day 12.5 and embryonic day 15.5 medial ganglionic eminences do not. While preprosomatostatin messenger RNA-expressing neurons were observed in grafts from each of the lateral ganglionic eminence and medial ganglionic eminence at both embryonic day 12.5 and embryonic day 15.5, choline acetyltransferase messenger RNA-expressing cholinergic neurons were largely found in grafts derived from the embryonic day 12.5 medial ganglionic eminence. These results suggest that the neuronal diversity of the adult striatum may derive both from the lateral ganglionic eminence, providing DARPP-32-expressing projection neurons as well as somatostatin-containing interneurons, and the early stage medial ganglionic eminence specifically contributing the cholinergic interneurons.

Projection neurons in fetal striatal transplants are predominantly derived from the lateral ganglionic eminence

In the present study, we have characterized aspects of integration, growth and phenotypic differentiation of embryonic grafts derived from the selective dissection of either the lateral or medial portion of the ganglionic eminences of the rodent forebrain. Donor tissues were derived from embryonic day 15 rat, or embryonic day 14 mouse embryos, and injected, as single cell suspensions into the striatum or substantia nigra of adult rats previously subjected to an intrastriatal ibotenic acid lesion. Two to six weeks following grafting, immunocytochemical detection of DARPP-32, the 32,000 mol. wt dopamine- and cyclic AMP-regulated phosphoprotein, was used to identify areas with a striatum-like phenotype within both the intrastriatal and the intranigral grafts. It was thus revealed that all the lateral ganglionic eminence grafts, irrespective of their placement, were dominated by striatum-like tissue (up to 90% of the total graft volume), while the medial ganglionic eminence transplants were only sparsely positive (< 10% of the total graft volume). These striatum-like regions of the grafts were selectively innervated by tyrosine hydroxylase immunopositive fibres from the host substantia nigra. Furthermore, axons derived from the lateral ganglionic eminence mouse grafts placed in the striatum, as detected by the mouse-specific neuronal marker M6, showed a more extensive and directed outgrowth towards the globus pallidus when compared to fibres emanating from the medial ganglionic eminence grafts. Mouse lateral and medial ganglionic eminence grafts placed into the substantia nigra exhibited similar fibre outgrowth patterns; both types of grafts thus innervated the substantia nigra-pars reticulata and extended axons into the cerebral peduncle. These results show that DARPP-32-positive striatal projection neurons are derived, for the most part, from the lateral ganglionic eminence and that the restricted lateral ganglionic eminence dissection provides a more optimal source of striatal tissue for grafting in the rat Huntington model.

Sonic Hedgehog Signaling Confers Ventral Telencephalic Progenitors with Distinct Cortical Interneuron Fates

Interneurons in the cerebral cortex regulate cortical functions through the actions of distinct subgroups that express parvalbumin, somatostatin, or calretinin. The genesis of the first two subgroups requires the expression of NKX2.1, which is maintained by SHH signaling during neurogenesis. In this paper, we report that mosaic elimination in the medial ganglionic eminence (MGE) of Smo, a key effector of SHH signaling, reveals that MGE progenitors retain a remarkable degree of plasticity during the neurogenic period. SHH signaling prevents the upregulation of GSX2 and conversion of some MGE progenitors to a caudal ganglionic eminence-like, bipolar calretinin-expressing cell fate that is promoted by GSX2. In addition, a higher level of SHH signaling promotes the generation of the somatostatin-expressing interneuron at the expense of parvalbumin-expressing subgroup. These results indicate that cortical interneuron diversity, a major determinant of cortical function, is critically influenced by differential levels of SHH signaling within the ventral telencephalon.

Dorsal-ventral patterning in the mammalian telencephalon

The telencephalon is the most diverse region of the brain with respect to both morphology and neuronal subtypes. This fact makes the task of unraveling the mechanisms underlying the development of this brain region rather daunting. Recent attempts to subdivide the embryonic telencephalon into distinct progenitor domains along the dorsal-ventral axis have provided an important framework on which to begin this process. These progenitor domains are defined by the restricted expression of transcriptional regulators and are proposed to give rise to specific subtypes of neurons. Work over recent years has provided important insights into the establishment and maintenance of these progenitor domains in the developing telencephalon.

Early forebrain wiring: genetic dissection using conditional Celsr3 mutant mice.

Development of axonal tracts requires interactions between growth cones and the environment. Tracts such as the anterior commissure and internal capsule are defective in mice with null mutation of Celsr3. We generated a conditional Celsr3 allele, allowing regional inactivation. Inactivation in telencephalon, ventral forebrain, or cortex demonstrated essential roles for Celsr3 in neurons that project axons to the anterior commissure and subcerebral targets, as well as in cells that guide axons through the internal capsule. When Celsr3 was inactivated in cortex, subcerebral projections failed to grow, yet corticothalamic axons developed normally, indicating that besides guidepost cells, additional Celsr3-independent cues can assist their progression. These observations provide in vivo evidence that Celsr3-mediated interactions between axons and guidepost cells govern axonal tract formation in mammals.

Striatal c-fos Induction by Cocaine or Apomorphine Occurs Preferentially in Output Neurons Projecting to the Substantia Nigra in the Rat

Fluorogold or rhodamine‐labelled latex beads were injected in the substantia nigra (SN) or the globus pallidus (GP) in order retrogradely to label striatal output neurons that project to the two target structures. Ten days later, striatal c‐fos was induced by systemic administration of cocaine (five normal rats; 25 mg/kg cocaine i.p. 2 h before killing) or apomorphine (five unilaterally dopamine‐denervated rats; 0.25 mg/kg apomorphine s. c. 2 h before killing), and detection of the Fos protein in the striatum was achieved by immunofluorescence. Sections through the caudate‐putamen that displayed good labelling from both SN and GP were selected for a quantitative analysis: the number of retrogradely labelled cells that exhibited Fos immunoreactivity, as well as the total number of retrogradely labelled cells located within a grid (0.16 mm2 in size) were counted manually at 25 x magnification. Cocaine induced a proportionally higher c‐fos expression in striate‐nigral compared to striate‐pallidal neurons, whereas apomorphine activated Fos almost exclusively in striate‐nigral neurons. The present findings are consistent with the idea that striatal c‐fos induction by dopaminergic agents is primarily mediated by an interaction with D1 ‐receptors, which are thought to be selectively localized on neurons projecting to SN.

Regional specification of neurosphere cultures derived from subregions of the embryonic telencephalon.

We have studied the molecular specification of precursor cells in expanded neurosphere cultures derived from distinct subregions of the embryonic mouse telencephalon. These regionally derived cultures exhibited differential responses to the mitogens EGF and bFGF, suggesting that the precursors in these cultures were differentially specified as is the case in situ. To examine this further, cultures from each of the telencephalic subregions were expanded in both EGF and bFGF before differentiation. The neurons produced displayed molecular phenotypes similar to those normally derived from each of these regions in vivo. Moreover, analysis of gene expression in the undifferentiated cultures showed that the regionally derived neurospheres express many of the same developmental control genes as their in vivo counterparts. Taken together, the present findings suggest that precursor cells in neurosphere cultures, derived from distinct subregions of the embryonic telencephalon, maintain at least certain aspects of their molecular specification, even after significant expansion in vitro.

Expression of Meis and Pbx genes and their protein products in the developing telencephalon: implications for regional differentiation

The Meis and Pbx genes encode for homeodomain proteins of the TALE class and have been shown to act as co-factors for other homeodomain transcription factors (Mann and Affolter, 1998. Curr. Opin. Genet. Dev. 8, 423-429). We have studied the expression of these genes in the mouse telencephalon and found that Meis1 and Meis2 display region-specific patterns of expression from embryonic day (E)10.5 until birth, defining distinct subterritories in the developing telencephalon. The expression of the Meis genes and their proteins is highest in the subventricular zone (SVZ) and mantle regions of the ventral telencephalon. Compared to the Meis genes, Pbx genes show a broader expression within the telencephalon. However, as is the case in Drosophila (Rieckhof et al., 1997. Cell 91, 171-183; Kurrant et al., 1998. Development 125, 1037-1048; Pai et al., 1998. Genes Dev. 12, 435-446), nuclear localized PBX proteins were found to correlate highly with Meis expression. In addition, DLX proteins co-localize with nuclear PBX in distinct regions of the ventral telencephalon.

Generation of mice with a conditional null allele for Wntless.

The Wnt‐signaling pathway is necessary in a variety of developmental processes and has been implicated in numerous pathologies. Wntless (Wls) binds to Wnt proteins and facilitates Wnt sorting and secretion. Conventional deletion of Wls results in early fetal lethality due to defects in body axis establishment. To gain insight into the function of Wls in later stages of development, we have generated a conditional null allele. Homozygous germline deletion of Wls confirmed prenatal lethality and failure of embryonic axis formation. Deletion of Wls using Wnt1‐cre phenocopied Wnt1 null abnormalities in the midbrain and hindbrain. In addition, conditional deletion of Wls in pancreatic precursor cells resulted in pancreatic hypoplasia similar to that previously observed after conditional β‐catenin deletion. This Wls conditional null allele will be valuable in detecting novel Wnt functions in development and disease. genesis 48:554–558, 2010.