Segmental identity and cerebellar granule cell induction in rhombomere 1

BACKGROUND: Cerebellar granule cell precursors are specifically generated within the hindbrain segment, rhombomere 1, which is bounded rostrally by the midbrain/hindbrain isthmus and caudally by the boundary of the Hoxa2 expression domain. While graded signals from the isthmus have a demonstrable patterning role within this region, the significance of segmental identity for neuronal specification within rhombomere 1 is unexplored. We examined the response of granule cell precursors to the overexpression of Hoxa2, which normally determines patterns of development specific to the hindbrain. How much does the development of the cerebellum, a midbrain/hindbrain structure, reflect its neuromeric origin as a hindbrain segment? RESULTS: We show that a Gbx2-positive, Otx2-/Hoxa2-negative territory corresponding to rhombomere 1 forms prior to an identifiable isthmic organiser. Early global overexpression of Hoxa2 at embryonic day 0 has no effect on the expression of isthmic signalling molecules or the allocation of rhombomere 1 territory, but selectively results in the loss of granule cell markers at embryonic day 6 and the depletion of cell bodies from the external granule cell layer. By comparison the trochlear nucleus and locus coeruleus form normally in ventral rhombomere 1 under these conditions. Microsurgery, coupled with electroporation, to target Hoxa2 overexpression to rhombic lip precursors, reveals a profound, autonomous respecification of migration. Rhombic lip derivatives, normally destined to occupy the external granule cell layer, violate the cerebellar boundary to form a ventrolateral nucleus in a position comparable to that occupied by rhombic lip derived neurons in rhombomere 2. CONCLUSIONS: Different overexpression strategies reveal that the recognition of migration cues by granule cell precursors is dependent on their identity as rhombomere 1 derivatives. Segmental patterning cues operate autonomously within the rhombic lip precursor pool. By contrast, a subset of coextensive nuclei is refractory to ectopic Hoxa2 and is presumably induced solely by isthmic organiser activity. Thus, graded (isthmic) and segmental mechanisms may operate exclusively of one another in the specification of different neuronal populations within rhombomere 1. The early designation of an Otx2-negative, Hoxa2-negative region, prior to the appearance of the isthmic organiser, is a key initial step in the specification of the cerebellum

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand 10-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultra-structural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes

Ten-fold Robust Expansion Microscopy

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes

Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila

Hunger drives food-seeking behaviour and controls adaptation of organisms to nutrient availability and energy stores. Lipids constitute an essential source of energy in the cell that can be mobilised during fasting by autophagy. Selective degradation of proteins by autophagy is made possible essentially by the presence of LIR and KFERQ-like motifs. Using in silico screening of Drosophila proteins that contain KFERQ-like motifs, we identified and characterized the adaptor protein Arouser, which functions to regulate fat storage and mobilisation and is essential during periods of food deprivation. We show that hypomorphic arouser mutants are not satiated, are more sensitive to food deprivation, and are more aggressive, suggesting an essential role for Arouser in the coordination of metabolism and food-related behaviour. Our analysis shows that Arouser functions in the fat body through nutrient-related signalling pathways and is degraded by endosomal microautophagy. Arouser degradation occurs during feeding conditions, whereas its stabilisation during non-feeding periods is essential for resistance to starvation and survival. In summary, our data describe a novel role for endosomal microautophagy in energy homeostasis, by the degradation of the signalling regulatory protein Arouser

Notch signaling in the development of the inner ear

The sensory patches of the inner ear consist of two types of cell: sensory hair cells and supporting cells. The pattern is such that supporting cells surround each hair cell and no two hair cells touch each other. The aim of this study was to uncover the genetic mechanisms that control the differentiation and patterning of these two cell types. The alternating pattern of hair cells and support cells has led to the suggestion that their differentiation is co-ordinately regulated by cell- cell interactions involving the Notch signaling pathway. The key players in this pathway are Delta, a ligand, and Notch, its receptor, mediating a process known as lateral inhibition - a mechanism which forces neighbouring cells of an initially equivalent group to become different. The findings in this study show that two Notch ligands Deltal and Serrate2 are expressed in the nascent hair cells and are thought to deliver lateral inhibition to their neighbours, which become supporting cells. Intriguingly, the supporting cells also express a Notch ligand, Serratel. To functionally test the role of the Notch signaling pathway in the developing chick inner ear, retroviral vectors were used to misexpress components of the Notch signaling pathway. It is shown that a simple lateral inhibition model based on feedback regulation of the Notch ligands is inadequate to explain the generation and patterning of the sensory hair cells. The Notch ligand Serratel is regulated by lateral induction and not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligand Deltal, Serratel, and Serrate2 in the neighbours of the nascent hair cell. At least one factor. Numb, capable of blocking reception of Notch signaling is concentrated in hair cells

A Genetic Screen for Olfactory Habituation Mutations in <em>Drosophila</em>: Analysis of Novel <em>Foraging</em> Alleles and an Underlying Neural Circuit

<div><p>Habituation is a form of non-associative learning that enables animals to reduce their reaction to repeated harmless stimuli. When exposed to ethanol vapor, <em>Drosophila</em> show an olfactory-mediated startle response characterized by a transient increase in locomotor activity. Upon repeated exposures, this olfactory startle attenuates with the characteristics of habituation. Here we describe the results of a genetic screen to identify olfactory startle habituation (OSH) mutants. One mutation is a transcript specific allele of <em>foraging</em> (<em>for</em>) encoding a cGMP-dependent kinase. We show this allele of <em>for</em> reduces expression of a <em>for-T1</em> isoform expressed in the head and functions normally to inhibit OSH. We localize <em>for-T1</em> function to a limited set of neurons that include olfactory receptor neurons (ORNs) and the mushroom body (MB). Overexpression of <em>for-T1</em> in ORNs inhibits OSH, an effect also seen upon synaptic silencing of the ORNs; <em>for-T1</em> may therefore function in ORNs to decrease synaptic release upon repeated exposure to ethanol vapor. Overall, this work contributes to our understanding of the genes and neurons underlying olfactory habituation in <em>Drosophila</em>.</p> </div

Flatmounts of brains up to embryonic day (E) 6 were prepared by opening the neural tube along the dorsal midline: cb – cerebellum, mb – midbrain, hb – hindbrain, rl – rhombic lip, ml – ventral midline

<p><b>Copyright information:</b></p><p>Taken from "Segmental identity and cerebellar granule cell induction in rhombomere 1"</p><p>BMC Biology 2004;2():14-14.</p><p>Published online 15 Jun 2004</p><p>PMCID:PMC446226.</p><p>Copyright © 2004 Eddison et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> The expression of (blue) at E6 in one half of the anterior hindbrain viewed as a flatmount (rostral top, mediolateral axis runs left to right). Granule cell precursors born at the rhombic lip express as they migrate over dorsal rhombomere 1 (r1) to condense as the external granule cell layer (EGL [egl]). is also expressed in a ventral wedge of r1 precursors (arrow) abutting the anterior r2 boundary and ventral midline. The relationship of this positive precursor pool (blue) to the anterior boundary of (red) is shown inset. Overexpression of (red) leads to a loss of in the EGL and prominent ventral wedge (arrow). This is replaced by a weaker, broadened, ventral domain similar to that in the hindbrain. At E9, in transverse section through a control embryo, the neuregulin receptor labels the EGL. Red label dorsally reflects high levels of expression of the control virus, RCANBP(B)(red), within the ventricular zone of the cerebellum (cb). Injection of the active RCASBP(B)virus leads to a reduction in . Patches of expression (arrows) correspond to residual EGL overlying a locally thickened cerebellum. Cresyl violet stains in control and RCASBP(B)-infected cerebella reveal an absence of cell bodies within a superficial, subpial layer (arrows), normally occupied by the EGL, following overexpression. Scale bar (D,E) = 500 μm

<i>for</i> alleles enhance olfactory startle habituation.

<p><b>A)</b><i>for^11.247</i> and <i>for²⁶¹⁴</i> show enhanced OSH. A reduction of distance traveled (compared to <i>Ctrl</i>) was seen in both alleles at pulse 2 (p<0.01), 3 and 4 (p<0.001;(n = 12). <b>B)</b><i>for^11.247</i> and <i>for²⁶¹⁴</i> have an enhanced HI (indicating more habituation). Significant difference was seen between <i>Ctrl</i> and <i>for^11.247</i> or <i>for²⁶¹⁴</i> (p<0.001; n = 12). <b>C)</b> Compared to <i>Ctrl,</i> the precise excision, <i>for^Δ11.247,</i> had a normal HI (p>0.05; n = 6). Unless indicated, significance was established by a One-Way-ANOVA with post-hoc Newmans-Keuls tests in all figures (*p<0.05, **p<0.01, ***p<0.001).</p