Xavier Morin

The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives.

The sympathetic, parasympathetic and enteric ganglia are the main components of the peripheral autonomic nervous system, and are all derived from the neural crest. The factors needed for these structures to develop include the transcription factor Mash1 (refs 3,4,5), the glial-derived neurotrophic factor GNDF (refs 6,7,8) and its receptor subunits, and the neuregulin signalling system, each of which is essential for the differentiation and survival of subsets of autonomic neurons. Here we show that all autonomic ganglia fail to form properly and degenerate in mice lacking the homeodomain transcription factor Phox2b, as do the three cranial sensory ganglia that are part of the autonomic reflex circuits. In the anlagen of the enteric nervous system and the sympathetic ganglia, Phox2b is needed for the expression of the GDNF-receptor subunit Ret and for maintaining Mash1 expression. Mutant ganglionic anlagen also fail to switch on the genes that encode two enzymes needed for the biosynthesis of the neurotransmitter noradrenaline, dopamine-β-hydroxylase and tyrosine hydroxylase, demonstrating that Phox2b regulates the noradrenergic phenotype in vertebrates.

A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila

In Drosophila, enhancer trap strategies allow rapid access to expression patterns, molecular data, and mutations in trapped genes. However, they do not give any information at the protein level, e.g., about the protein subcellular localization. Using the green fluorescent protein (GFP) as a mobile artificial exon carried by a transposable P-element, we have developed a protein trap system. We screened for individual flies, in which GFP tags full-length endogenous proteins expressed from their endogenous locus, allowing us to observe their cellular and subcellular distribution. GFP fusions are targeted to virtually any compartment of the cell. In the case of insertions in previously known genes, we observe that the subcellular localization of the fusion protein corresponds to the described distribution of the endogenous protein. The artificial GFP exon does not disturb upstream and downstream splicing events. Many insertions correspond to genes not predicted by the Drosophila Genome Project. Our results show the feasibility of a protein trap in Drosophila. GFP reveals in real time the dynamics of proteins distribution in the whole, live organism and provides useful markers for a number of cellular structures and compartments.

THE BHLH PROTEIN NEUROGENIN 2 IS A DETERMINATION FACTOR FOR EPIBRANCHIAL PLACODE-DERIVED SENSORY NEURONS

neurogenin2 encodes a neural-specific basic helix-loop-helix (bHLH) transcription factor related to the Drosophila proneural factor atonal. We show here that the murine ngn2 gene is essential for development of the epibranchial placode-derived cranial sensory ganglia. An ngn2 null mutation blocks the delamination of neuronal precursors from the placodes, the first morphological sign of differentiation in these lineages. Mutant placodal cells fail to express downstream bHLH differentiation factors and the Notch ligand Delta-like 1. These data suggest that ngn2 functions like the Drosophila proneural genes in the determination of neuronal fate in distal cranial ganglia. Interestingly, the homeobox gene Phox2a is activated independently of ngn2 in epibranchial placodes, suggesting that neuronal fate and neuronal subtype identity may be specified independently in cranial sensory ganglia.

Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2a

Phox2a is a vertebrate homeodomain protein expressed in subsets of differentiating neurons. Here, we show that it is essential for proper development of the locus coeruleus, a subset of sympathetic and parasympathetic ganglia and the VIIth, IXth, and Xth cranial sensory ganglia. In the sensory ganglia, we have identified two differentiation blocks in Phox2a-/- mice. First, the transient expression of dopamine-beta-hydroxylase in neuroblasts is abolished, providing evidence that Phox2a controls noradrenergic traits in vivo. Second, the expression of the GDNF receptor subunit Ret is dramatically reduced, and there is a massive increase in apoptosis of ganglion cells, which are known to depend on GDNF in vivo. Therefore, Phox2a appears to regulate conventional differentiation traits and the ability of neurons to respond to essential survival factors.

Mitotic spindle orientation in asymmetric and symmetric cell divisions during animal development.

The orientation of the mitotic spindle has been proposed to control cell fate choices, tissue architecture, and tissue morphogenesis. Here, we review the mechanisms regulating the orientation of the axis of division and cell fate choices in classical models of asymmetric cell division. We then discuss the mechanisms of mitotic spindle orientation in symmetric cell divisions and its possible implications in tissue morphogenesis. Many recent studies show that future advances in the field of mitotic spindle orientation will arise from combinations of physical perturbation and modeling with classical genetics and developmental biology approaches.

Control of planar divisions by the G-protein regulator LGN maintains progenitors in the chick neuroepithelium

The spatio-temporal regulation of symmetrical as opposed to asymmetric cell divisions directs the fate and location of cells in the developing CNS. In invertebrates, G-protein regulators control spindle orientation in asymmetric divisions, which generate progeny with different identities. We investigated the role of the G-protein regulator LGN (also called Gpsm2) in spindle orientation and cell-fate determination in the spinal cord neuroepithelium of the developing chick embryo. We show that LGN is located at the cell cortex and spindle poles of neural progenitors, and that it regulates spindle movements and orientation. LGN promotes planar divisions in the early spinal cord. Interfering with LGN function randomizes the plane of division. Notably, this does not affect cell fate, but frequently leads one daughter of proliferative symmetric divisions to exit the neuroepithelium prematurely and to proliferate aberrantly in the mantle zone. Hence, tight control of planar spindle orientation maintains neural progenitors in the neuroepithelium, and regulates the proper development of the nervous system.

Specification of Neurotransmitter Identity by Phox2 Proteins in Neural Crest Stem Cells

We have investigated the specification of noradrenergic neurotransmitter identity in neural crest stem cells (NCSCs). Retroviral expression of both wild-type and dominant-negative forms of the paired homeodomain transcription factor Phox2a indicates a crucial and direct role for this protein (and/or the closely related Phox2b) in the regulation of endogenous tyrosine hydroxylase (TH) and dopamine-beta hydroxylase (DBH) gene expression in these cells. In collaboration with cAMP, Phox2a can induce expression of TH but not of DBH or of panneuronal genes. Phox2 proteins are, moreover, necessary for the induction of both TH and DBH by bone morphogenetic protein 2 (BMP2) (which induces Phox2a/b) and forskolin. They are also necessary for neuronal differentiation. These data suggest that Phox2a/b coordinates the specification of neurotransmitter identity and neuronal fate by cooperating environmental signals in sympathetic neuroblasts.

A lateral belt of cortical LGN and NuMA guides mitotic spindle movements and planar division in neuroepithelial cells

Knockdown or mislocalization of LGN complex components disrupts the stereotypic biphasic spindle movements regulating planar cell division and neuroepithelial structure in chick embryos.

Regulation of mitotic spindle orientation: an integrated view

Mitotic spindle orientation is essential for cell fate decisions, epithelial maintenance, and tissue morphogenesis. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. Early studies identified the evolutionarily conserved Gαi/LGN/NuMA complex as a key regulator that polarizes cortical force generators. In recent years, a combination of genetics, biochemistry, modeling, and live imaging has contributed to decipher the mechanisms of spindle orientation. Here, we highlight the dynamic nature of the assembly of this complex and discuss the molecular regulation of its localization. Remarkably, a number of LGN‐independent mechanisms were described recently, whereas NuMA remains central in most pathways involved in recruiting force generators at the cell cortex. We also describe the emerging role of the actin cortex in spindle orientation and discuss how dynamic astral microtubule formation is involved. We further give an overview on instructive external signals that control spindle orientation in tissues. Finally, we discuss the influence of cell geometry and mechanical forces on spindle orientation.

Dlg1 controls planar spindle orientation in the neuroepithelium through direct interaction with LGN.

Dlg1 recruits LGN to the cortex of cells in the chick neuroepithelium and may provide instructive cues that drive planar spindle orientation.