Julian Lewis

Mind Bomb Is a Ubiquitin Ligase that Is Essential for Efficient Activation of Notch Signaling by Delta

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.

Organizing cell renewal in the intestine: stem cells, signals and combinatorial control

The lining of the intestine is renewed at an extraordinary rate, outpacing all other tissues in the vertebrate body. The renewal process is neatly organized in space, so that the whole production line, from the ever-youthful stem cells to their dying, terminally differentiated progeny, is laid out to view in histological sections. A flurry of recent papers has clarified the key regulatory signals and brought us to the point where we can begin to give a coherent account, for at least one tissue, of how these signals collaborate to organize the architecture and behaviour of a stem-cell system.

Autoinhibition with Transcriptional Delay: A Simple Mechanism for the Zebrafish Somitogenesis Oscillator

BACKGROUND The pattern of somites is traced out by a mechanism involving oscillating gene expression at the tail end of the embryo. In zebrafish, two linked oscillating genes, her1 and her7, coding for inhibitory gene regulatory proteins, are especially implicated in genesis of the oscillations, while Notch signaling appears necessary for synchronization of adjacent cells. RESULTS I show by mathematical simulation that direct autorepression of her1 and her7 by their own protein products provides a mechanism for the intracellular oscillator. This mechanism operates robustly even when one allows for the fact that gene regulation is an essentially noisy (stochastic) process. The predicted period is close to the observed period (30 min) and is dictated primarily by the transcriptional delay, the time taken to make an mRNA molecule. Through its coupling to her1/her7 expression, Notch signaling can keep the rapid oscillations in adjacent cells synchronized. When the coupling parameters are varied, however, the model system can switch to oscillations of a much longer period, resembling that of the mouse or chick somitogenesis oscillator and governed by the delays in the Notch pathway. Such Notch-mediated synchronous oscillations are predicted even in the absence of direct her1/her7 autoregulation, through operation of the standard Notch signaling pathway that is usually assumed simply to give lateral inhibition. CONCLUSIONS Direct autorepression of a gene by its own product can generate oscillations, with a period determined by the transcriptional and translational delays. Simple as they are, such systems show surprising behaviors. To understand them, unaided intuition is not enough: we need mathematics.

Maintenance of neuroepithelial progenitor cells by Delta–Notch signalling in the embryonic chick retina

BACKGROUND Neurons of the vertebrate central nervous system (CNS) are generated sequentially over a prolonged period from dividing neuroepithelial progenitor cells. Some cells in the progenitor cell population continue to proliferate while others stop dividing and differentiate as neurons. The mechanism that maintains the balance between these two behaviours is not known, although previous work has implicated Delta-Notch signalling in the process. RESULTS In normal development, the proliferative layer of the neuroepithelium includes both nascent neurons that transiently express Delta-1 (Dl1), and progenitor cells that do not. Using retrovirus-mediated gene misexpression in the embryonic chick retina, we show that where progenitor cells are exposed to Dl1 signalling, they are prevented from embarking on neuronal differentiation. A converse effect is seen in cells expressing a dominant-negative form of Dl1, Dl1(dn), which we show renders expressing cells deaf to inhibitory signals from their neighbours. In a multicellular patch of neuroepithelium expressing Dl1(dn), essentially all progenitors stop dividing and differentiate prematurely as neurons, which can be of diverse types. Thus, Delta-Notch signalling controls a cells choice between remaining as a progenitor and differentiating as a neuron. CONCLUSIONS Nascent retinal neurons, by expressing Dl1, deliver lateral inhibition to neighbouring progenitors; this signal is essential to prevent progenitors from entering the neuronal differentiation pathway. Lateral inhibition serves the key function of maintaining a balanced mixture of dividing progenitors and differentiating progeny. We propose that the same mechanism operates throughout the vertebrate CNS, enabling large numbers of neurons to be produced sequentially and adopt different characters in response to a variety of signals. A similar mechanism of lateral inhibition, mediated by Delta and Notch proteins, may regulate stem-cell function in other tissues.

Neurogenic genes and vertebrate neurogenesis

The neurogenic genes of the Delta-Notch signalling pathway mediate lateral inhibition--a mechanism that controls cell commitment in many tissues and serves in the developing nervous system to single out cells for a neural fate. Recent work has revealed the outlines of the signal transduction pathway from Notch to the nucleus, has clarified the mechanisms by which lateral inhibition causes adjacent cells to become different, and has shown that vertebrates use essentially the same lateral inhibition machinery as flies and worms to regulate neurogenesis.

Early ear development in the embryo of the zebrafish, Danio rerio.

The zebrafish provides an important model for vertebrate inner ear development. The otic placode becomes visible at ∼16 hours (at 28.5°C) and forms a vesicle with a lumen by cavitation at ∼18 hours. Two otoliths appear in the lumen by 19.5 hours, and at about 24 hours the first sensory hair cells are seen, grouped in two small patches, one beneath each otolith, corresponding to future maculae. Staining with fluorescent phalloidin reveals 10–20 hair cells in each macula by 42 hours; between 3 days and 7 days the numbers grow to ∼80 per macula. Neurons of the statoacoustic ganglion are first visible by staining with HNK‐1 antibody at about 24 hours. Serial sections and time‐lapse films show that the neuronal precursors originate by delamination from the ventral face of the otocyst; the peak period of delamination is from 22 hours to 30 hours. The system of semicircular canals forms between 42 hours and 72 hours by outgrowth of protrusions from the walls of the otocyst to form pillars of tissue spanning the lumen. Three further clusters of hair cells also become visible in this period, forming the three cristae. Thus, by the end of the first week, all key components of the ear are present. Subsequent growth produces thousands more hair cells; additional neurons probably derive from proliferation of neuronal precursors within the ganglion. Although the timetable is species‐specific, the principles of inner ear development in the zebrafish seem to be the same as in other vertebrates.

Control of segment number in vertebrate embryos

The vertebrate body axis is subdivided into repeated segments, best exemplified by the vertebrae that derive from embryonic somites. The number of somites is precisely defined for any given species but varies widely from one species to another. To determine the mechanism controlling somite number, we have compared somitogenesis in zebrafish, chicken, mouse and corn snake embryos. Here we present evidence that in all of these species a similar ‘clock-and-wavefront’ mechanism operates to control somitogenesis; in all of them, somitogenesis is brought to an end through a process in which the presomitic mesoderm, having first increased in size, gradually shrinks until it is exhausted, terminating somite formation. In snake embryos, however, the segmentation clock rate is much faster relative to developmental rate than in other amniotes, leading to a greatly increased number of smaller-sized somites.

Dll1- and Dll4-Mediated Notch Signaling Are Required for Homeostasis of Intestinal Stem Cells

BACKGROUND & AIMS Ablation of Notch signaling within the intestinal epithelium results in loss of proliferating crypt progenitors due to their conversion into postmitotic secretory cells. We aimed to confirm that Notch was active in stem cells (SCs), investigate consequences of loss of Notch signaling within the intestinal SC compartment, and identify the physiologic ligands of Notch in mouse intestine. Furthermore, we investigated whether the induction of goblet cell differentiation that results from loss of Notch requires the transcription factor Krüppel-like factor 4 (Klf4). METHODS Transgenic mice that carried a reporter of Notch1 activation were used for lineage tracing experiments. The in vivo functions of the Notch ligands Jagged1 (Jag1), Delta-like1 (Dll1), Delta-like4 (Dll4), and the transcription factor Klf4 were assessed in mice with inducible, gut-specific gene targeting (Vil-Cre-ER(T2)). RESULTS Notch1 signaling was found to be activated in intestinal SCs. Although deletion of Jag1 or Dll4 did not perturb the intestinal epithelium, inactivation of Dll1 resulted in a moderate increase in number of goblet cells without noticeable effects of progenitor proliferation. However, simultaneous inactivation of Dll1 and Dll4 resulted in the complete conversion of proliferating progenitors into postmitotic goblet cells, concomitant with loss of SCs (Olfm4(+), Lgr5(+), and Ascl2(+)). Klf4 inactivation did not interfere with goblet cell differentiation in adult wild-type or in Notch pathway-deficient gut. CONCLUSIONS Notch signaling in SCs and progenitors is activated by Dll1 and Dll4 ligands and is required for maintenance of intestinal progenitor and SCs. Klf4 is dispensable for goblet cell differentiation in intestines of adult Notch-deficient mice.

Delta-Notch signalling controls commitment to a secretory fate in the zebrafish intestine

The transparency of the juvenile zebrafish and its genetic advantages make it an attractive model for study of cell turnover in the gut. BrdU labelling shows that the gut epithelium is renewed in essentially the same way as in mammals: the villi are lined with non-dividing differentiated cells, while cell division is confined to the intervillus pockets. New cells produced in the pockets take about 4 days to migrate out to the tips of the villi, where they die. We have generated monoclonal antibodies to identify the absorptive and secretory cells in the epithelium, and we have used these antibodies to examine the part that Delta-Notch signalling plays in producing the diversity of intestinal cell types. Several Notch receptors and ligands are expressed in the gut. In particular, the Notch ligand DeltaD (Delta1 in the mouse) is expressed in cells of the secretory lineage. In an aei mutant, where DeltaD is defective, secretory cells are overproduced. In mind bomb (mib), where all Delta-Notch signalling is believed to be blocked, almost all the cells in the 3-day gut epithelium adopt a secretory character. Thus, secretory differentiation appears to be the default in the absence of Notch activation, and lateral inhibition mediated by Delta-Notch signalling is required to generate a balanced mixture of absorptive and secretory cells. These findings demonstrate the central role of Notch signalling in the gut stem-cell system and establish the zebrafish as a model for study of the mechanisms controlling renewal of gut epithelium.

Instability of Hes7 protein is crucial for the somite segmentation clock

During somitogenesis, a pair of somites buds off from the presomitic mesoderm every 2 hours in mouse embryos, suggesting that somite segmentation is controlled by a biological clock with a 2-hour cycle. Expression of the basic helix-loop-helix factor Hes7, an effector of Notch signaling, follows a 2-hour oscillatory cycle controlled by negative feedback; this is proposed to be the molecular basis for the somite segmentation clock. If the proposal is correct, this clock should depend crucially on the short lifetime of Hes7. To address the biological importance of Hes7 instability, we generated mice expressing mutant Hes7 with a longer half-life (∼30 min compared with ∼22 min for wild-type Hes7) but normal repressor activity. In these mice, somite segmentation and oscillatory expression became severely disorganized after a few normal cycles of segmentation. We simulated this effect mathematically using a direct autorepression model. Thus, instability of Hes7 is essential for sustained oscillation and for its function as a segmentation clock.