Specification of neuronal identity
apl. Prof. Dr. Hermann Rohrer
Research goals

We are interested in the way in which different types of neurons are generated from pluripotent precursor cells in the embryo. Our studies aim to characterize the cell lineage from pluripotent neural stem cells to distinct neuronal phenotypes and to identify signalling molecules and transcriptional control mechanisms involved in neuron phenotype specification. These questions are analysed in different parts of the vertebrate nervous system, with special focus on the generation and specification of noradrenergic and cholinergic neurons in the autonomic nervous system.

Methods

To address the role of extrinsic signalling molecules and intracellular signal transduction cascades we are using a combination of molecular, cellular and embryological approaches. We use in vivo transgenic approaches in mouse and zebrafish, retroviral expression in chick embryos and in vitro cultures of avian neural crest and peripheral ganglia.

Recent results

In the peripheral nervous system, we have shown that signalling molecules of the family of bone morphogenetic proteins (BMPs) are essential for sympathetic neuron development. BMPs act directly on neuron precursor cells to control the expression of a group of transcription factors that in turn specify the identity of the differentiating neurons (Fig.1). These transcription factors consist of the bHLH transcription factors Mash1 and dHand, the paired homeodomain transcription factors Phox2a and Phox2b and the Zn-finger transcription factors Gata2/3. Mash1, Phox2a and Phox2b are sufficient to elicit the generation of sympathetic neurons from neural crest precursor cells. Our recent loss-of-function studies in mouse and chick embryos demonstrated an essential role of Gata2/3 for both noradrenergic differentiation and generic sympathetic neuron development.

At later stages of sympathetic neuron development, signals from the innervated target control the final differentiation, leading to functionally different types of sympathetic neurons, i.e. vasoconstrictor, pilomotor, secretomotor and sudomotor neurons (Fig. 1A). Sympathetic neurons that innervate sweat glands differentiate in response to factors released from this target to cholinergic sudomotor neurons. They start to express the cholinergic marker genes choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the neuropeptide vasoactive intestinal peptide (VIP) and elicit sweat secretion by release of acetlycholine. Using a conditional knockout of the cytokine receptor gp130 in sympathetic neurons we recently provided the first in vivo evidence for the essential role of gp130 cytokines in target-dependent cholinergic differentiation.

The development of cholinergic parasympathetic ciliary neurons is also initiated by BMPs (Fig. 2) and involves Mash1, Phox2a and Phox2b. Interestingly, these cells transiently express noradrenergic properties before aquiring the final cholinergic neurotransmitter phenotype. The inability to maintain noradrenergic properties correlates with the lack of Hand2 and Gata2/3 expression. As Hand2 overexpression maintains noradrenergic properties in parasympathetic ciliary neurons, a maintenance function of Hand2 is implied in developing and mature symapthetic neurons (Fig. 1A).

Fig. 1: This scheme illustrates the cell lineage and the signals involved in the generation of sympathetic (A) and parasympathetic (B) neurons from neural crest progenitor cells. (A) The development of noradrenergic sympathetic neurons is initiated by BMPs that in turn control the expression of the transcription factors Mash1, Phox2a/2b, Hand2 and Gata2/3. Together they form a network of transcription factors that control the expression of generic neuronal and noradrenergic (blue) marker genes. Hand2 and Gata2/3 are implicated in the selective activation of noradrenergic gene expression. After innervation of different peripheral targets, differetiation factors released from the target direct the final differentiation. Cytokines acting through gp130 receptors are essential for the cholinergic (red) differentiation of sweat gland innervating (sudomotor) neurons. (B) The development of parasympathetic ciliary neurons is initiated by BMPs. In contrast to sympathetic neurons, only Mash1 and Phox2a/b but not Hand2 and Gata2/3 are induced in parasympathetic precurosors by BMPs. This implies that the only transient expression of TH and DBH is due to the absence of Hand2 and Gata2/3. Signals controlling cholinergic differentiation of ciliary neurons are unknown.

Fig. 2: This figure illustrates the effect of inhibiting the function of BMPs on the development of the chick ciliary ganglion. The unilateral application of the BMP antagonist noggin in the vicinity of the optic vesicle in 2-day-old chick embryos results at embryonic day 5 in a unilateral block of ciliary ganglion development (left, white arrowheads), which is reflected by the lack of generic neuronal differentiation (SCG-10) (A), Mash1 expression (B) and Phox2b expression (C). The ciliary ganglion on the contralateral side develops normally, as well as the sensory trigeminal (TG) which does not express the autonomic markers Mash1 and Phox2b. BMP inhibition does not affect the migration and aggregation of Sox-10-positive neural crest cells to form a normal size ciliary ganglion (D; black arrowhead). This illustrates that BMPs are essential for the initiation of autonomic neuron development. CG, ciliary ganglion, TG, trigeminal ganglion.


Perspectives

While these data begin to reveal a genetic network that controls noradrenergic neuronal fates and demonstrates the potential of these factors in neural crest stem cells, the way in which different types of autonomic neurons are generated is poorly understood. Transcription factors that are differentially expressed in sympathetic versus parasympathetic ganglia and may control the specific development of neuronal subtypes have been identified by gene profiling and will now be functionally analysed.

As most of the transcription factors involved in the development of noradrenergic neurons are also expressed in mature neurons, a role in the maintenance of differentiated functions is implicated, which is currently investigated. Further interesting questions are how glial cells are specified in autonomic ganglia, how the machineries that direct generic and subtype-specific aspects of neuronal differentiation are coordinated, and how neuronal differentiation is linked to cell cycle exit.
admin, 13-02-2008