Mib1 prevents Notch Cis-inhibition to defer differentiation and preserve neuroepithelial integrity during neural delamination

<div><p>The vertebrate neuroepithelium is composed of elongated progenitors whose reciprocal attachments ensure the continuity of the ventricular wall. As progenitors commit to differentiation, they translocate their nucleus basally and eventually withdraw their apical endfoot from the ventricular surface. However, the mechanisms allowing this delamination process to take place while preserving the integrity of the neuroepithelial tissue are still unclear. Here, we show that Notch signaling, which is classically associated with an undifferentiated state, remains active in prospective neurons until they delaminate. During this transition period, prospective neurons rapidly reduce their apical surface and only later down-regulate N-Cadherin levels. Upon Notch blockade, nascent neurons disassemble their junctions but fail to reduce their apical surface. This disrupted sequence weakens the junctional network and eventually leads to breaches in the ventricular wall. We also provide evidence that the Notch ligand Delta-like 1 (Dll1) promotes differentiation by reducing Notch signaling through a <i>Cis</i>-inhibition mechanism. However, during the delamination process, the ubiquitin ligase Mindbomb1 (Mib1) transiently blocks this <i>Cis</i>-inhibition and sustains Notch activity to defer differentiation. We propose that the fine-tuned balance between Notch <i>Trans</i>-activation and <i>Cis</i>-inhibition allows neuroepithelial cells to seamlessly delaminate from the ventricular wall as they commit to differentiation.</p></div

Insights into Digit Evolution from a Fate Map Study of the Forearm Using Chameleon, a New Transgenic Chicken Line:Fate Mapping the Chicken Ulna

The cellular and genetic networks that contribute to the development of the zeugopod (radius and ulna of the forearm, tibia and fibula of the leg) are not well understood, although these bones are susceptible to loss in congenital human syndromes and to the action of teratogens such as thalidomide. Using a new fate-mapping approach with the Chameleon transgenic chicken line, we show that there is a small contribution of SHH-expressing cells to the posterior ulna, posterior carpals and digit 3. We establish that although the majority of the ulna develops in response to paracrine SHH signalling in both the chicken and mouse, there are differences in the contribution of SHH-expressing cells between mouse and chicken as well as between the chicken ulna and fibula. This is evidence that, although zeugopod bones are clearly homologous according to the fossil record, the gene regulatory networks that contribute to their development and evolution are not fixed.</p

Loss of cilia causes embryonic lung hypoplasia, liver fibrosis and cholestasis in the talpid3 ciliopathy mutant

Sonic hedgehog plays an essential role in maintaining hepatoblasts in a proliferative non-differentiating state during embryogenesis. Transduction of the Hedgehog signaling pathway is dependent on the presence of functional primary cilia and hepatoblasts, therefore, must require primary cilia for normal function. In congenital syndromes in which cilia are absent or non-functional (ciliopathies) hepatorenal fibrocystic disease is common and primarily characterized by ductal plate malformations which underlie the formation of liver cysts, as well as less commonly, by hepatic fibrosis, although a role for abnormal Hedgehog signal transduction has not been implicated in these phenotypes. We have examined liver, lung and rib development in the talpid(3) chicken mutant, a ciliopathy model in which abnormal Hedgehog signaling is well characterized. We find that the talpid(3) phenotype closely models that of human short-rib polydactyly syndromes which are caused by the loss of cilia, and exhibit hypoplastic lungs and liver failure. Through an analysis of liver and lung development in the talpid(3) chicken, we propose that cilia in the liver are essential for the transduction of Hedgehog signaling during hepatic development. The talpid(3) chicken represents a useful resource in furthering our understanding of the pathology of ciliopathies beyond the treatment of thoracic insufficiency as well as generating insights into the role Hedgehog signaling in hepatic development

Feather arrays are patterned by interacting signalling and cell density waves

Feathers are arranged in a precise pattern in avian skin. They first arise during development in a row along the dorsal midline, with rows of new feather buds added sequentially in a spreading wave. We show that the patterning of feathers relies on coupled fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signalling together with mesenchymal cell movement, acting in a coordinated reaction-diffusion-taxis system. This periodic patterning system is partly mechanochemical, with mechanical-chemical integration occurring through a positive feedback loop centred on FGF20, which induces cell aggregation, mechanically compressing the epidermis to rapidly intensify FGF20 expression. The travelling wave of feather formation is imposed by expanding expression of Ectodysplasin A (EDA), which initiates the expression of FGF20. The EDA wave spreads across a mesenchymal cell density gradient, triggering pattern formation by lowering the threshold of mesenchymal cells required to begin to form a feather bud. These waves, and the precise arrangement of feather primordia, are lost in the flightless emu and ostrich, though via different developmental routes. The ostrich retains the tract arrangement characteristic of birds in general but lays down feather primordia without a wave, akin to the process of hair follicle formation in mammalian embryos. The embryonic emu skin lacks sufficient cells to enact feather formation, causing failure of tract formation, and instead the entire skin gains feather primordia through a later process. This work shows that a reaction-diffusion-taxis system, integrated with mechanical processes, generates the feather array. In flighted birds, the key role of the EDA/Ectodysplasin A receptor (EDAR) pathway in vertebrate skin patterning has been recast to activate this process in a quasi-1-dimensional manner, imposing highly ordered pattern formation

Combination of novel and public RNA-seq datasets to generate an mRNA expression atlas for the domestic chicken

Background: The domestic chicken (Gallus gallus) is widely used as a model in developmental biology and is also an important livestock species. We describe a novel approach to data integration to generate an mRNA expression atlas for the chicken spanning major tissue types and developmental stages, using a diverse range of publicly-archived RNA-seq datasets and new data derived from immune cells and tissues. Results: Randomly down-sampling RNA-seq datasets to a common depth and quantifying expression against a reference transcriptome using the mRNA quantitation tool Kallisto ensured that disparate datasets explored comparable transcriptomic space. The network analysis tool Graphia was used to extract clusters of co-expressed genes from the resulting expression atlas, many of which were tissue or cell-type restricted, contained transcription factors that have previously been implicated in their regulation, or were otherwise associated with biological processes, such as the cell cycle. The atlas provides a resource for the functional annotation of genes that currently have only a locus ID. We cross-referenced the RNA-seq atlas to a publicly available embryonic Cap Analysis of Gene Expression (CAGE) dataset to infer the developmental time course of organ systems, and to identify a signature of the expansion of tissue macrophage populations during development. Conclusion: Expression profiles obtained from public RNA-seq datasets - despite being generated by different laboratories using different methodologies - can be made comparable to each other. This meta-analytic approach to RNA-seq can be extended with new datasets from novel tissues, and is applicable to any species

Mib1 blocks the ability of Dll1 to <i>Cis</i>-inhibit Notch signaling.

<p><b>(A)</b> Top: Transverse sections of the NT transfected at E2, with the indicated constructs and harvested at E4. Immunostaining for Sox2 (blue) and HuCD (green) labels progenitors and neurons, respectively. Transfection is reported by H2B-Cherry expression. Arrowheads indicate ectopic Sox2⁺ progenitors adjacent to HuCD⁺ transfected neurons. Bottom: Summaries of the effects of Dll1 and Mib1 on neurogenesis. Red and gray cells correspond to electroporated (ep) and non-electroporated cells, respectively. Round and star-shaped cells correspond to progenitors and neurons, respectively. Blue outlines indicate cells changing fate, autonomously or non-autonomously, in each condition. <b>(B, D)</b> Quantification of the Hes5-VNP signal intensity in HuCD⁻ cells either <b>(B)</b> non-transfected (surrounded by at least four transfected cells) or <b>(D)</b> transfected 24 hae with the indicated constructs. Data represent fold change compared to control. <b>(B)</b> <i>n</i> = 54, 35, 35, 54 cells were analyzed for control, Dll1, Dll1+Mib1, and Dll1+mbMib1, respectively. <b>(D)</b> <i>n</i> = 58, 59, 59, 66 cells were analyzed for control, Dll1, Dll1+Mib1, and Dll1+mbMib1, respectively. Data were collected from four to six embryos for each experimental group. ns, <i>p</i> > 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001 (Kruskal-Wallis test). <b>(C, E)</b> Quantification of the differentiation rate in <b>(C)</b> non-transfected neighbors (number of non-transfected HuCD⁺ cells adjacent to a HuCD⁺ transfected cell on the total number of adjacent cells) or <b>(E)</b> transfected cells (number of HuCD⁺ cells on total transfected cells) 48 hae with the indicated constructs. Data represent mean + SEM. <i>n</i> = 14 (6 embryos), 10 (8 embryos), 14 (6 embryos), 18 (6 embryos) sections were analyzed for control, Dll1, Dll1+Mib1, and Dll1+mbMib1, respectively. ns, <i>p</i> > 0.05; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001 (one-way ANOVA). Analyses were performed on the same sections for <b>(B)</b> and <b>(D)</b>, and for <b>(C)</b> and <b>(E)</b>. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004162#pbio.2004162.s007" target="_blank">S1 Data</a>. Scale bar represents 50 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004162#pbio.2004162.s005" target="_blank">S5 Fig</a>. Ct, control; Dll1, Delta-like 1; E, embryonic day; ep, electroporated; hae, hour after electroporation; Hes5, Hairy and Enhancer of Split 5; HuCD, neuron-specific RNA-binding proteins HuC and HuD; H2B-Cherry, Histone 2B fused to Cherry; mbMib1, Mib1 constitutively tethered to the plasma membrane; Mib1, Mindbomb1; ns, nonsignificant; NT, neural tube; Sox2, SRY (sex determining region Y) box 2; VNP, Venus-NLS-PEST.</p

Notch signaling is maintained in prospective neurons.

<p><b>(A)</b> Schematic representation of the Hes5-VNP sequence that was inserted in the Notch reporter transgenic chick line. <b>(B)</b> Left: Transverse sections of the NT of the Hes5-VNP transgenic line at E3 and E4 immunostained for Venus (green) and HuCD (red) to label neurons. Middle: Color coded map of Hes5-VNP intensity. The red line separates HuCD⁻ from HuCD⁺ cells. The black dotted lines delineate the ventral limit of the roof plate and dorsal limit of the motor neuron domain. Right: Distribution of the Hes5-VNP signal intensity in HuCD⁻ and HuCD⁺ cells. Note that cells within the limits of the black dotted lines of the color code panel were labeled in black in the HuCD⁻ population. <b>(C)</b> Top: Time course of the protocol. Bottom: Distribution of the Hes5-VNP signal intensity in FT⁺/HuCD⁻ cells. This population is then divided into EdU⁺ (blue) and EdU⁻ (magenta) cells. A minimum of 58 cells collected from four embryos were analyzed for each group. <b>(D)</b> Left: Transverse sections of the dorsal NT in the Hes5-VNP transgenic line at E4 immunostained for Venus (green), Neurog2 (red), and HuCD (blue). Bottom: Enlarged view of the boxed area showing representative examples of Neurog2+ cells. Right: Distribution of the Hes5-VNP signal intensity in Neurog2⁻ and Neurog2⁺ cells. The latter population was divided based on Neurog2⁺ signal intensity. A minimum of 75 cells collected from six embryos were analyzed for each group. Horizontal bars correspond to medians. ns, <i>p</i> > 0.05; **<i>p</i> < 0.01, ***<i>p</i> < 0.001 (Kruskal-Wallis test). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004162#pbio.2004162.s007" target="_blank">S1 Data</a>. Scale bar represents 25 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004162#pbio.2004162.s001" target="_blank">S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004162#pbio.2004162.s002" target="_blank">S2</a> Figs. E, embryonic day; EdU, 5-ethynyl-2′-deoxyuridine; FT, FlashTag; Hes5, Hairy and Enhancer of Split 5; HuCD, neuron-specific RNA-binding proteins HuC and HuD; Neurog2, Neurogenin 2; ns, nonsignificant; NT, neural tube; VNP, Venus-NLS-PEST.</p

Effects of Notch signaling and apical constriction modulators on apical markers and tissue integrity.

<p><b>(A)</b> Transverse sections of the NT transfected at E2 with the indicated constructs, harvested at E3 and immunostained for N-Cadherin (red). <b>(B)</b> Transverse sections of the NT transfected at E2 with the indicated constructs, harvested at E4 and immunostained for N-Cadherin (red); and for Sox2 (red) and HuCD (blue) to label progenitors and neurons, respectively. Transfection is reported by GFP expression. Summary: Schematic of the effects observed on tissue integrity. Gray cells correspond to electroporated cells. Scale bar represents 50 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004162#pbio.2004162.s004" target="_blank">S4 Fig</a>. ΔMaml1, dominant-negative Mastermind-like 1; E, embryonic day; EP, electroporation; GFP, green fluorescent protein; HuCD, neuron-specific RNA-binding proteins HuC and HuD; N, neuron; <i>Neurog2</i>, <i>Neurogenin 2</i>; NT, neural tube; P, progenitor; <i>RII-C1</i>, <i>Shroom3 binding site on ROCK2</i>; <i>Shroom3</i>, <i>Shroom family member 3</i>; Sox2, SRY (sex determining region Y) box 2.</p

Model for the role of Mib1-dependent Notch activity in the regulation of neuronal delamination.

<p>Top: Prospective neurons maintain a high level of Notch activity until they fully differentiate. Mib1 is required during that transition phase to keep Dll1 from <i>Cis</i>-inhibiting the Notch receptor. This allows Notch to be <i>Trans</i>-activated by Dll1 present on neighboring cells (not represented here), resulting in the release of the NICD. When the Dll1/Mib1 ratio is sufficiently high, <i>Cis</i>-inhibition takes place and Notch activity is rapidly turned off. Bottom: Sustained Notch activity allows prospective neurons to shrink their apical area and keeps them from differentiating. As Notch activity is decreased, N-Cadherin levels are down-regulated and neuronal differentiation markers start being expressed. Dll1, Delta-like 1; Mib1, Mindbomb1; NICD, Notch intracellular domain; <i>T</i>, time.</p

Sequence of events leading to neuron delamination.

<p><b>(A)</b> Left: Apical view of the NT electroporated at E2 with ZO1-GFP/iRFP (green), along with the constructs indicated on the left, and harvested at different hae, followed by an immunostaining for N-Cadherin. The boxed area indicates the cell of interest. Right: Quantification of the apical area ratio (ratio of the area of one transfected cell versus the mean area of four of its close non-transfected neighbors) and N-Cadherin level ratio (ratio of the average pixel intensity within the apical circumference of one transfected cell corrected by the background versus the mean of average pixel intensity of four of its close non-transfected neighbors) at different hae. Data represent mean + SEM. <b>(B)</b> N-Cadherin intensity ratio as a function of apical area ratio at 24 hae. Data were taken from <b>(A)</b>. The “median” used as a threshold to discriminate between small and large apical areas corresponds to the median of the control (0.62). **<i>p</i> < 0.01; ***<i>p</i> < 0.001 (one-way ANOVA). <b>(C)</b> Top: Apical view of the NT transfected with ZO1-iRFP (green) along with the indicated constructs and immunostained for Tuj1 (red) and Par3 (blue). Bottom: Three-dimensional view of the cell represented above but showing only the ZO1-iRFP and Tuj1 stainings. Right: Scatterplot of the mean apical area ratio for Tuj1⁺ cells. Each point represents one apical area ratio calculated as in <b>(A)</b>. <i>n</i> = 49, 66, 51 cells collected from five embryos were analyzed for control, Neurog2, and ΔMaml1, respectively. ns, <i>p</i> > 0.05; ***<i>p</i> < 0.001 (Kruskal-Wallis test). Horizontal bars correspond to means. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004162#pbio.2004162.s007" target="_blank">S1 Data</a>. Scale bar represents 2 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004162#pbio.2004162.s003" target="_blank">S3 Fig</a>. ΔMaml1, dominant-negative Mastermind-like 1; E, embryonic day; EP, electroporation; GFP, green fluorescent protein; hae, hour after electroporation; iRFP, infrared fluorescent protein; Neurog2, Neurogenin 2; ns, nonsignificant; NT, neural tube; Par3, Partition defective protein 3; ZO1, Zonula Occludens 1.</p