Paula Peltopuro

Fibroblast growth factor receptors cooperate to regulate neural progenitor properties in the developing midbrain and hindbrain.

Fibroblast growth factors (FGFs) secreted from the midbrain–rhombomere 1 (r1) boundary instruct cell behavior in the surrounding neuroectoderm. For example, a combination of FGF and sonic hedgehog (SHH) can induce the development of the midbrain dopaminergic neurons, but the mechanisms behind the action and integration of these signals are unclear. We studied how FGF receptors (FGFRs) regulate cellular responses by analyzing midbrain–r1 development in mouse embryos, which carry different combinations of mutant Fgfr1, Fgfr2, and Fgfr3 alleles. Our results show that the FGFRs act redundantly to support cell survival in the dorsal neuroectoderm, promote r1 tissue identity, and regulate the production of ventral neuronal populations, including midbrain dopaminergic neurons. The compound Fgfr mutants have apparently normal WNT/SHH signaling and neurogenic gene expression in the ventral midbrain, but the number of proliferative neural progenitors is reduced as a result of precocious neuronal differentiation. Our results suggest a SoxB1 family member, Sox3, as a potential FGF-induced transcription factor promoting progenitor renewal. We propose a model for regulation of progenitor cell self-renewal and neuronal differentiation by combinatorial intercellular signals in the ventral midbrain.

Cell-autonomous FGF signaling regulates anteroposterior patterning and neuronal differentiation in the mesodiencephalic dopaminergic progenitor domain

The structure and projection patterns of adult mesodiencephalic dopaminergic (DA) neurons are one of the best characterized systems in the vertebrate brain. However, the early organization and development of these nuclei remain poorly understood. The induction of midbrain DA neurons requires sonic hedgehog (Shh) from the floor plate and fibroblast growth factor 8 (FGF8) from the isthmic organizer, but the way in which FGF8 regulates DA neuron development is unclear. We show that, during early embryogenesis, mesodiencephalic neurons consist of two distinct populations: a diencephalic domain, which is probably independent of isthmic FGFs; and a midbrain domain, which is dependent on FGFs. Within these domains, DA progenitors and precursors use partly different genetic programs. Furthermore, the diencephalic DA domain forms a distinct cell population, which also contains non-DA Pou4f1+ cells. FGF signaling operates in proliferative midbrain DA progenitors, but is absent in postmitotic DA precursors. The loss of FGFR1/2-mediated signaling results in a maturation failure of the midbrain DA neurons and altered patterning of the midbrain floor. In FGFR mutants, the DA domain adopts characteristics that are typical for embryonic diencephalon, including the presence of Pou4f1+ cells among TH+ cells, and downregulation of genes typical of midbrain DA precursors. Finally, analyses of chimeric embryos indicate that FGF signaling regulates the development of the ventral midbrain cell autonomously.

Distinct requirements for Ascl1 in subpopulations of midbrain GABAergic neurons.

Midbrain GABAergic neurons regulate multiple aspects of behavior and play important roles in psychiatric and neurological disease. These neurons constitute several anatomical and functional subpopulations, but their molecular heterogeneity and developmental regulatory mechanisms are poorly understood. Here we have studied the involvement of the proneural gene Ascl1 in the development of the midbrain GABAergic neurons. Analysis of Ascl1 mutant mice demonstrated highly region-specific requirements for Ascl1 for development of different GABAergic neuron subpopulations. Ascl1 is dispensable for the development of the ventral-most midbrain GABAergic neurons associated with dopaminergic nuclei substantia nigra pars reticulata (SNpr) and ventral tegmental area (VTA) GABAergic neurons. In the ventrolateral midbrain, loss of Ascl1 results in markedly delayed neurogenesis in the midbrain domains m3-m5. Within this region, Ascl1 has a unique role in m4, where it also regulates glutamatergic neurogenesis. Our results suggest that the m3-m5 midbrain neuroepithelium gives rise to the GABAergic neuron groups located in the midbrain reticular formation and ventrolateral periaqueductal gray. In contrast to m3-m5, Ascl1 is absolutely required in the dorsal midbrain domains m1-m2, for generation of the GABAergic neurons populating the superior and inferior colliculi as well as dorsal periaqueductal gray. These studies demonstrate different molecular regulatory mechanisms for the distinct midbrain GABAergic neuron subpopulations. Also, our results have implications on understanding the origins of the various midbrain GABAergic neuron groups in the embryonic neuroepithelium.

Transcriptional regulatory mechanisms underlying the GABAergic neuron fate in different diencephalic prosomeres

Diverse mechanisms regulate development of GABAergic neurons in different regions of the central nervous system. We have addressed the roles of a proneural gene, Ascl1, and a postmitotic selector gene, Gata2, in the differentiation of GABAergic neuron subpopulations in three diencephalic prosomeres: prethalamus (P3), thalamus (P2) and pretectum (P1). Although the different proliferative progenitor populations of GABAergic neurons commonly express Ascl1, they have distinct requirements for it in promotion of cell-cycle exit and GABAergic neuron identity. Subsequently, Gata2 is activated as postmitotic GABAergic precursors are born. In P1, Gata2 regulates the neurotransmitter identity by promoting GABAergic and inhibiting glutamatergic neuron differentiation. Interestingly, Gata2 defines instead the subtype of GABAergic neurons in the rostral thalamus (pTh-R), which is a subpopulation of P2. Without Gata2, the GABAergic precursors born in the pTh-R fail to activate subtype-specific markers, but start to express genes typical of GABAergic precursors in the neighbouring P3 domain. Thus, our results demonstrate diverse mechanisms regulating differentiation of GABAergic neuron subpopulations and suggest a role for Gata2 as a selector gene of both GABAergic neuron neurotransmitter and prosomere subtype identities in the developing diencephalon. Our results demonstrate for the first time that neuronal identities between distinct prosomeres can still be transformed in postmitotic neuronal precursors.

Distinct developmental origins and regulatory mechanisms for GABAergic neurons associated with dopaminergic nuclei in the ventral mesodiencephalic region

GABAergic neurons in the ventral mesodiencephalic region are highly important for the function of dopaminergic pathways that regulate multiple aspects of behavior. However, development of these neurons is poorly understood. We recently showed that molecular regulation of differentiation of the GABAergic neurons associated with the dopaminergic nuclei in the ventral midbrain (VTA and SNpr) is distinct from the rest of midbrain, but the reason for this difference remained elusive. Here, we have analyzed the developmental origin of the VTA and SNpr GABAergic neurons by genetic fate mapping. We demonstrate that the majority of these GABAergic neurons originate outside the midbrain, from rhombomere 1, and move into the ventral midbrain only as postmitotic neuronal precursors. We further show that Gata2, Gata3 and Tal1 define a subpopulation of GABAergic precursors in ventral rhombomere 1. A failure in GABAergic neuron differentiation in this region correlates with loss of VTA and SNpr GABAergic neurons in Tal1 mutant mice. In contrast to midbrain, GABAergic neurons of the anterior SNpr in the diencephalon are not derived from the rhombomere 1. These results suggest unique migratory pathways for the precursors of important GABAergic neuron subpopulations, and provide the basis for understanding diversity within midbrain GABAergic neurons.

The role of Tal2 and Tal1 in the differentiation of midbrain GABAergic neuron precursors

Summary Midbrain- and hindbrain-derived GABAergic interneurons are critical for regulation of sleep, respiratory, sensory-motor and motivational processes, and they are implicated in human neurological disorders. However, the precise mechanisms that underlie generation of GABAergic neuron diversity in the midbrain–hindbrain region are poorly understood. Here, we show unique and overlapping requirements for the related bHLH proteins Tal1 and Tal2 in GABAergic neurogenesis in the midbrain. We show that Tal2 and Tal1 are specifically and sequentially activated during midbrain GABAergic neurogenesis. Similar to Gata2, a post-mitotic selector of the midbrain GABAergic neuron identity, Tal2 expression is activated very early during GABAergic neuron differentiation. Although the expression of Tal2 and Gata2 genes are independent of each other, Tal2 is important for normal midbrain GABAergic neurogenesis, possibly as a partner of Gata2. In the absence of Tal2, the majority of midbrain GABAergic neurons switch to a glutamatergic-like phenotype. In contrast, Tal1 expression is activated in a Gata2 and Tal2 dependent fashion in the more mature midbrain GABAergic neuron precursors, but Tal1 alone is not required for GABAergic neuron differentiation from the midbrain neuroepithelium. However, inactivation of both Tal2 and Tal1 in the developing midbrain suggests that the two factors co-operate to guide GABAergic neuron differentiation in a specific ventro-lateral midbrain domain. The observed similarities and differences between Tal1/Tal2 and Gata2 mutants suggest both co-operative and unique roles for these factors in determination of midbrain GABAergic neuron identities.

Distinct requirements for Ascl1 in subpopulations of midbrain GABAergic neurons

PB-cadherin belongs to the classical cadherin family of homotypic cell adhesion molecules. PB-cadherin is expressed in forebrain, midbrain–hindbrain boundary (MHB) and limb buds (Kitijama et. al., 1999). Little is still known of its functions. Short type PB-cadherin promotes survival of gonocytes in neonatal rats (Wu et al., 2005). Therefore, Pb-cadherin might participate in maintaining cell survival and stem cell population in other tissues. In midbrain–hindbrain specific FGFR1 knock-out embryos the border between midbrain and hindbrain is disrupted. Signaling trough FGFR1 may thus regulate cell adhesion properties at the MHB (Trokovic et al., 2003). Furthermore, there is a specific FGFR1 -dependent slowly proliferating boundary cell population between midbrain and rhombomere 1. These boundary cells may be important for development of the isthmic construction and separation of the two brain regions (Trokovic 2005, Kala 2008). The expression of PB-cadherin started in MHB at E8.0 and PB-cadherin was expressed in the boundary cells together with cell cycle inhibitor p21. PB-cadherin, was downregulated in MHB in the FGFR1 mutants (Trokovic et al 2003). Later, PB-cadherin is expressed also in ventral midbrain; rostral population correlates with Pou4f1 expressing (glutamatergic) cells and posterior with Gad1 expressing (gabaergic) cells. At E18.5 PB-cadherin is expressed in specific regions in forebrain including indesium griseum, amygdala, hippocampus, midbrain and hindbrain. The function of PB-CAD in nervous system remains mainly unknown. To study this we generated a conditional mutant allele of PB-cad (Turakainen et al., 2009).The mice homozygous for a PB-cad null allele (PBCdel/del) are viable and fertile, although their number was less than expected at weaning. However, changes of compartmentalization in early embryos were lacking. Midbrainhindbrain neuronal populations were normal in E18 PBCdel mutants. As other type II cadherins such as OB-cadherin are co-expressed with PB-cad there might be functional redundancy among the type II cadherins.

Redundant functions of Fgfr1, Fgfr2 and Fgfr3 in the development of the mid- and hindbrain

Hesx1 that are involved in specifying the anterior neurectoderm are unknown. We have previously shown that mouse embryonic stem cells differentiated as aggregates in HepG2 cell conditioned medium, synchronously and homogeneously differentiate to a midbrainlike population of neurectoderm cells. Analysis of ES cellderived neurectoderm suggest that it has not been exposed to patterning signals, such as the ventralising signal Sonic Hedgehog, making it a powerful tool for identifying and characterising molecules involved in neural tube patterning. We have exploited these characteristics to understand the signalling molecules that emanate from the AVE and anteriorise the neurectoderm. Hesx1 was over-expressed in HepG2 cells (HepG2:Hesx1) and the conditioned medium tested for the ability to anteriorise ES cell-derived neurectoderm. After 2 days, in presence of HepG2:Hesx1 CM anterior markers were up-regulated in the neurectoderm, suggesting that the CM contained signals that anteriorise neurectoderm. Microarray analysis was performed on HepG2 and HepG2:Hesx1 cells to identify differentially expressed genes. Of the four secreted molecules identified one growth factor was chosen for further characterisation. Addition of recombinant growth factor to ES cell-derived neurectoderm resulted in up-regulation of anterior markders, indicating that this growth factor is involved in anteriorising neural progenitors. In addition, expression analysis showed this growth factor is present in the AVE of 6.5 and 7.5 dpc mouse embryos suggesting a mechanism by which Hesx1 expression in the AVE regulates the positional specification of the anterior neurectoderm and forebrain.