Macrophages are essential for tissue repair and regeneration. Yet, the molecular programs, as well as the timing of their activation during and after tissue injury are poorly defined. Using a high spatio-temporal resolution single cell analysis of macrophages coupled with live imaging after sensory hair cell death in zebrafish, we find that the same population of macrophages transitions through a sequence of three major anti-inflammatory activation states. Macrophages first show a signature of glucocorticoid activation, then IL-10 signaling and finally the induction of oxidative phosphorylation by IL-4/Polyamine signaling. Importantly, loss-of-function of glucocorticoid and IL-10 signaling shows that each step of the sequence is independently activated. Lastly, we show that IL-10 and IL-4 signaling act synergistically to promote synaptogenesis between hair cells and efferent neurons during regeneration. Our results show that macrophages, in addition to a switch from M1 to M2, sequentially and independently transition though three anti-inflammatory pathways in vivo during tissue injury in a regenerating organ.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In vertebrates, the total number of vertebrae is precisely defined. Vertebrae derive from embryonic somites that are continuously produced posteriorly from the presomitic mesoderm (PSM) during body formation. We show that in the chicken embryo, activation of posterior Hox genes (paralogs 9–13) in the tail-bud correlates with the slowing down of axis elongation. Our data indicate that a subset of progressively more posterior Hox genes, which are collinearly activated in vertebral precursors, repress Wnt activity with increasing strength. This leads to a graded repression of the Brachyury/T transcription factor, reducing mesoderm ingression and slowing down the elongation process. Due to the continuation of somite formation, this mechanism leads to the progressive reduction of PSM size. This ultimately brings the retinoic acid (RA)-producing segmented region in close vicinity to the tail bud, potentially accounting for the termination of segmentation and axis elongation. https://doi.org/10.7554/eLife.04379.001 eLife digest In humans and other vertebrates, the number of bones (vertebrae) in the spine is determined early in development. The vertebrae form from blocks of tissue called somites that make segments along the body axis—a virtual line running from the head to the tail-end—of the embryo. The somites form as the embryo increases in length, with new somites forming periodically at the back near the embryo's tail-end. A family of genes called the Hox genes are involved in controlling the formation of the somites. However, it is not known whether they directly control the number of somites that form, or whether they control the length of the body of the embryo. Denans et al. studied the Hox genes in chicken embryos. The experiments suggest that the activation of some of the Hox genes in a structure called the tail-bud, which is found at the tail-end of the embryo, slow down the elongation of the body. The Hox genes achieve this by repressing the activity of a signaling pathway called Wnt so that Wnt activity in the tail-bud progressively decreases as the embryo develops. The elongation of the body stops when the levels of a molecule called retinoic acid increase in the tail-bud, which causes the loss of the stem cells that are needed to make the somites. Denans et al.'s findings suggest that Hox genes influence the timing of the halt in elongation, which in turn is important for determining the total number of somites that form. Understanding how Hox genes control the formation of the cells that will make up the somites and influence Wnt signaling is a major challenge for the future. https://doi.org/10.7554/eLife.04379.002 Introduction Body skeletal muscles and vertebrae form from a transient embryonic tissue called paraxial mesoderm (PM). The PM becomes segmented into epithelial structures called somites, which are sequentially produced in a rhythmic fashion from the presomitic mesoderm (PSM). The PSM is formed caudally during gastrulation by ingression of the PM progenitors located initially in the anterior part of the primitive streak (PS) and later, in the tail-bud (Bénazéraf and Pourquié, 2013). At the end of somitogenesis, the embryonic axis is segmented into a fixed species-specific number of somites which varies tremendously between species ranging from as little as ∼32 in zebrafish to more than 300 in some snakes. The somites subsequently differentiate into their final vertebral and muscular derivatives to establish the various characteristic anatomical regions of the body. Hox genes code for a family of transcription factors involved in specification of regional identity along the body axis (Mallo et al., 2012; Noordermeer and Duboule, 2013). In mouse and chicken, the 39 Hox genes are organized in four clusters containing up to thirteen paralogous genes each. Hox genes exhibit both spatial and temporal collinearity, meaning that they are activated in a sequence reflecting their position along the chromosome and become expressed in domains whose anterior boundaries along the body axis also reflect their position in the clusters. Whether Hox genes control axis length and segment number has been controversial. Mouse mutants in which entire sets of Hox paralogs are inactivated show severe vertebral patterning defects but exhibit normal vertebral counts (Wellik and Capecchi, 2003; McIntyre et al., 2007). In contrast, precocious expression of Hox13 genes in transgenic mice leads to axis truncation with reduced vertebral numbers (Young et al., 2009). Furthermore, mouse null mutations for Hoxb13 or Hoxc13 result in the production of supernumerary vertebrae (Godwin and Capecchi, 1998; Economides et al., 2003). In chicken and fish embryos, the arrest of axis elongation has been linked to the inhibition of FGF and Wnt signaling in the tail-bud which leads to the down-regulation of the transcription factor T/Brachyury and of the Retinoic Acid (RA)-degrading enzyme Cyp26A1 (Young et al., 2009; Martin and Kimelman, 2010; Tenin et al., 2010; Olivera-Martinez et al., 2012). Downregulation of Cyp26A1 in the tail-bud ultimately leads to rising RA levels and to differentiation and death of the PM progenitors which terminates axis elongation. Premature exposure of the tail-bud to high RA levels in chicken or mouse embryos inhibits Wnt and FGF signaling and leads to axis truncation (Tenin et al., 2010; Olivera-Martinez et al., 2012; Iulianella et al., 1999) suggesting that the tail-bud must be protected from the differentiating action of RA. In the Cyp26A1 null mutant mice, RA-signaling reaches the tail-bud, prematurely inducing the downregulation of FGF signaling and the increase of Sox2 expression, resulting in axis truncation posterior to the thoracic level (Abu-Abed et al., 2001; Sakai et al., 2001). In chicken, the tail-bud starts to produce RA when explanted in culture after the 40-somite stage (Tenin et al., 2010). This late RA signaling activity in the tail-bud is involved in the termination of segmentation and axis elongation (Tenin et al., 2010; Olivera-Martinez et al., 2012). At the 40-somite stage, the mRNA for Raldh2, the RA-biosynthetic enzyme becomes expressed in the tail-bud potentially accounting for this late RA activity. What triggers this late expression of Raldh2 in the chicken tail-bud is however unknown. In vertebrates, the termination of axis elongation is accompanied by a progressive reduction in size of the PSM (Gomez et al., 2008; Gomez and Pourquié, 2009). The shrinking of the PSM which brings the segmented region producing RA in the vicinity of the tail-bud might also contribute to the raise in RA levels in the tail-bud and possibly to the late Raldh2 activation in the tail-bud. Thus in the chicken embryo, the timing of elongation arrest (and hence the total number of somites formed) could be in part controlled by the kinetics of PSM shrinking. PSM size depends on the velocity of somite formation which removes cells anteriorly and on the flux of cells from the primitive streak and tail-bud generated during elongation which injects cells posteriorly. How this flux of progenitors ingressing in the PSM is regulated over time, and which genes are regulating this process remain poorly understood. Hoxb1-9 genes have been proposed to control cell ingression of paraxial mesoderm precursors from the epiblast during gastrulation (Iimura and Pourquié, 2006). However, Hoxb1-9 genes are only expressed in anterior regions of the embryo precluding their playing a role in the control of axis termination. Here, we first investigate the relationship between the speed of somite formation and of axis elongation. We show that, in the chicken embryo, activation of Abdominal B-like posterior Hox genes in the tail-bud correlates with the slowing down of axis elongation, while the speed of somitogenesis remains approximately constant. Our data indicate that a subset of progressively more posterior Hox genes, which are collinearly activated in vertebral precursors, repress Wnt and FGF activity with increasing strength, leading to a graded repression of the Brachyury/T transcription factor. This progressively reduces mesoderm ingression and cell motility in the PSM, thus slowing down the elongation process. Due to the continuation of somite formation at a steady pace, this mechanism leads to the progressive reduction of PSM size. Results Activation of posterior Hox genes correlates with axis elongation slowing down We measured the variations of velocities of axis elongation and somite formation in time-lapse videos of developing chicken embryos during the formation of the first 30 somites (Video 1). The velocity of somite formation shows limited variation during this developmental window (Tenin et al., 2010) (Figure 1A, n = 4 embryos for each condition). In contrast, axis elongation velocity increases during the formation of the first 10 somites and then it decreases until the 25-somite stage, when it drops abruptly (Figure 1A and Video 2, n = 41 embryos). The number of PSM cells decreases over time (Figure 1B, n = 5 embryos for each condition) while no significant difference in cell proliferation or apoptosis in the PSM and tail-bud is observed (Figure 1C–F, n = 4 embryos for each condition). Cell motility, which has been implicated in the control of axis elongation (Bénazéraf et al., 2010), also decreased between 15 and 27 somites (Figure 1G, n = 4 embryos for each condition). Thus, a parallel decrease in cell motility and in cell flux to the PSM accompanies axis elongation slow down. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse video of an embryo from Stage 5 HH to 29 somites showing the different phases of axis elongation (Bright-field, ventral view, anterior is up). https://doi.org/10.7554/eLife.04379.003 Figure 1 Download asset Open asset Slowing down of axis elongation correlates with decreasing cell ingression in the PSM. (A) Velocity of axis elongation and of somite formation. (B) PSM cell number. (C–D) Tiling of confocal sections of 20-somite (C) and 25-somite (D) stage embryos. EdU positive cells are labeled in green, phosphorylated histone H3 (pH3) in red, and nuclei in blue (DAPI). (C′, D′) Higher magnification of PSM regions used to quantify the proliferation. (C″, D″) Confocal sections of parasagittal cryosections of tail-bud used to quantify cell proliferation. (E–F) Quantification of cell proliferation (E) and apoptosis (F) in 20–22 and 25–27 somites chicken embryos. (G) Cell motility in the posterior PSM. https://doi.org/10.7554/eLife.04379.004 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse videos showing axis elongation slow-down around the 25-somite stage. Bright-field imaging of chicken embryos at 15–17 somites (left panel), 20–22 somites (middle panel), and 25–27 somites (right panel) (ventral view, anterior is up). https://doi.org/10.7554/eLife.04379.005 Hoxb1-9 genes were shown to regulate cell flux to the PSM by controlling the timing of cell ingression from the epiblast (Iimura and Pourquié, 2006). Activation of Hox genes in the epiblast and tail-bud is collinear and occurs in two phases. First, the paralog groups 1 to 8 (and Hoxb9) are quickly activated within ten hours before the first somite formation (stage 7 HH [Hamburger and Hamilton, 1992]; Figure 2, n = 8 embryos for each condition). This phase is followed by a pause during formation of the first ten somites. Then between the 10 and 40-somite stage, the posterior Hox genes corresponding to the paralog groups 9–13 (and Hoxc8 and Hoxd8) become subsequently activated in a slower phase which takes almost 48 hr (Figure 2, n = 8 embryos for each condition). Hoxa13 is the first Hox13 activated at the 25-somite stage, when axis elongation slows down abruptly. Thus, there is a striking correlation between the timing of posterior Hox genes activation and the beginning of axis elongation slow down (Figures 1A and 2). Figure 2 Download asset Open asset Collinear activation of Hox genes in paraxial mesoderm precursors. (A) Table showing the collinear onset of Hox genes expression in the epiblast/tail-bud generated from (B) Chicken embryos hybridized in whole-mount with Hoxa (blue), Hoxb (yellow), Hoxc (red), and Hoxd (green) probes. Each panel shows the beginning of activation of each Hox gene in paraxial mesoderm precursors in the epiblast of the anterior primitive streak or in the tail-bud. Hox probe used is indicated on the top of each panel. Anterior is up. Dorsal view. https://doi.org/10.7554/eLife.04379.006 A subset of posterior Hox genes can regulate cell ingression and axis elongation In order to test the role of posterior Hox genes on the control of cell ingression and cell motility in the developing chicken embryo, we used an in vivo electroporation technique, allowing to precisely target the paraxial mesoderm precursors in the epiblast of the anterior primitive streak (Bénazéraf et al., 2010) (Video 3). We developed a strategy allowing to overexpress two different sets of constructs in largely different populations of paraxial mesoderm cells by performing two consecutive electroporations of the paraxial mesoderm (PM) precursors of the epiblast of stage 4–5 HH embryos. Embryos are first electroporated on the left side of the primitive streak with a control Cherry construct, and then on the right side of the streak with a second vector expressing the yellow fluorescent protein Venus and a Hox construct (Figure 3A). This strategy results in essentially different PM cells expressing the two sets of constructs with the Cherry expressing cells enriched on the left side whereas Hox expressing cells are mostly found on the right side. When no Hox construct is present in the Venus vector, the Cherry and Venus-expressing populations of cells were observed to extend from the tail-bud to the same antero-posterior level of the paraxial mesoderm indicating that they began ingressing at the same time (Figure 3B, n = 8 embryos). In contrast, cells expressing Cherry were always extending more anteriorly than cells expressing Venus and one of the following posterior Hox gene: Hoxa9, Hoxc9, Hoxd10, Hoxd11, Hoxc11, Hoxa13, Hoxb13, or Hoxc13, indicating that these Hox genes can delay cell ingression of the PSM progenitors (Figure 3B–C n > 6 for each condition and not shown). This simply reflects the fact that cells ingressing later become located more posteriorly. Strikingly, the effect on ingression was progressively stronger when overexpressing more 5′ genes suggesting a collinear trend (Figure 3C). Inverting the order in which the constructs are electroporated did not affect the final phenotype. The distance between the anterior boundaries of the two domains was found to progressively increase with more posterior Hox genes as shown by measuring the ratio between Venus and Cherry posterior domains (Figure 3A–C). Over-expression of Hoxa10, Hoxc10, Hoxa11, Hoxc12, Hoxd12 and Hoxd13 showed no difference with the control Cherry vector (Figure 3A–C, n > 6 for each condition and data not shown). Using consecutive electroporation of Hoxd10 and Hoxc11 constructs labeled with Cherry and Venus, respectively, we observed that Hoxc11 has a stronger effect on ingression than Hoxd10 (Figure 3—figure supplement 1, n = 12 embryos). A similar result was observed when Hoxa13 was compared to Hoxc11 in the same assay (Figure 3—figure supplement 1, n = 6 embryos). Thus, a subset of posterior Hox genes is able to delay PSM cell ingression in a collinear manner. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse video showing the precise targeting of PSM progenitors and the ingression of the epiblast cells to form the PSM. Bright-field (purple) merged with fluorescent images of PSM cell progenitors electroporated with a control H2B-Venus (ventral view, anterior is up) from stage 6 HH onwards. https://doi.org/10.7554/eLife.04379.007 Figure 3 with 1 supplement see all Download asset Open asset Posterior Hox genes can regulate cell ingression in a collinear fashion. (A) Consecutive electroporation protocol. The ratio of the green domain (green bar, Hox expressing) over the red domain (red bar, control vector) measures the ingression delay. (B) Embryos consecutively electroporated first with Cherry and then with Venus together with control, Hoxa9, Hoxc11, or Hoxb13 vectors. Arrowheads: anterior boundary of Cherry (red) and Venus (green) domains. (C) Ratio of Venus over Cherry domains for posterior Hox genes. Dots: electroporated embryos. Bar indicates the mean. Stars: p-value of two-tailed Student's t-test applied between the different conditions. *p < 0.05; **p < 0.01; ***p < 0.005. Error bars: standard error to the mean (SEM). https://doi.org/10.7554/eLife.04379.008 To analyze the effect of posterior Hox genes on ingression, PM precursors were electroporated with Venus and a Hoxa13 or a control construct and harvested after 5 hr when the electroporated cells start to ingress. No ectopic expression of laminin (Figure 4A,B–C″), acetylated tubulin (Figure 4A,D–E″), or E-cadherin (data not shown) was observed after Hoxa13 over-expression. We compared the number of Venus-positive cells in epiblast vs primitive streak and mesoderm in embryo sections. The majority of Hoxa13-expressing cells were still found in the epiblast while control cells have ingressed into the primitive streak and mesoderm indicating that Hoxa13 delays ingression by retaining cells in the epiblast (Figure 4F–H, n = 4 embryos for each condition). No up-regulation of the neural marker Sox2 was observed in the tail-bud of embryos electroporated with Hoxa13 (Figure 4I–J, n = 8 embryos for each condition) and very few cells were observed in the neural tube of embryos electroporated with Hox constructs (see Figure 3B, Figure 4I–J, Figure 6A–B, Figure 7D–J and Figure 9A and Videos 4–8). This indicates that the effect on ingression is not caused by the recruitment of PM precursor cells to a neural fate. Ingression of cells from the epiblast to the primitive streak occurs via an epithelium to mesenchyme transition which involves first destabilization and then complete loss of basal microtubules in these cells. This process has been shown to be regulated by a basally localized activity of the small GTPase RhoA (Nakaya et al., 2008). In order to test if the effect of the posterior Hox genes on delaying PSM progenitors ingression could involve regulation of microtubule stability, we used a dominant negative form of RhoA (DN-RhoA) as a tool to destabilize basal microtubules in the epiblast (Nakaya et al., 2008). We performed consecutive electroporations at stage 5 HH to overexpress a control Cherry vector in one population of PSM progenitors and Hoxa13 with DN-RhoA vectors in another population and allowed the embryos to develop for 20 hr. We observed that the two populations of cells reach the same anterior level (Figure 4K, n = 5/5 embryos) indicating that these cells ingressed at the same time. Altogether, these results suggest that Hox genes control PSM progenitors ingression through the regulation of basal microtubule stability in the epiblast. Figure 4 Download asset Open asset Epiblast cells overexpressing Hox genes do not convert to a neural fate. (A) Transverse section of a stage 7 HH chicken embryo labeled with phalloidin (white) to highlight the actin network and with laminin (red) to identify the epiblast basal membrane. Colored boxes indicate the different phases of differentiation of the mesoderm: epiblast (purple), ingressing cells (yellow), and mesoderm (blue). (B–E″) Transverse sections at the PSM progenitors level 5 hr after electroporation of a control Venus or of Hoxa13. (B-C”) Laminin immunolabeling (red) after Venus (B–B″) or Hoxa13 over-expression (C–C″). (D–E″) Acetylated α-tubulin immunolabeling (red) after Venus (D–D″) or Hoxa13 (E–E″) over-expression. (F–G) Transverse cryosections of the anterior primitive streak of an embryo electroporated with Venus (F) or with Venus and Hoxa13 (G). White arrow: cells ingressed in the primitive streak (F) and non-ingressed epiblast cells (G). Green: Venus; red: laminin; blue: nuclei. (H) Quantification of ingression in embryos electroporated with control or Hoxa13-expressing constructs. (I–J) In situ hybridization of 2-day old chicken embryos electroporated with Venus (I) or Hoxa13-Venus (J) expressing vectors. Left panel shows Sox2 expression in the neural tube and tail-bud, and right panels show GFP immunohistochemistry. (K) Chicken embryo consecutively electroporated with a control (Cherry, red) and a mix of Hoxa13+DN-RhoA (Venus, green). Arrowheads: anterior boundary of Cherry (red) and Venus (green) domains. Stars: p-value of two-tailed Student's t-test applied between the different conditions. ***p < 0.005. Error bars: standard error to the mean (SEM). https://doi.org/10.7554/eLife.04379.010 We next tested the effect of over-expressing posterior Hox genes on axis elongation (Figure 5A–C, Video 4, n = 47 embryos). Over-expression of either Hoxa9, Hoxc9, Hoxd10, Hoxd11, Hoxc11, Hoxa13, Hoxb13 or Hoxc13 but not of Hoxa10, Hoxc10, Hoxa11, Hoxc12, Hoxd12 and Hoxd13 in PM precursors caused a significant decrease of elongation velocity (Figure 5A–C and not shown). The effect of Hox genes becomes progressively stronger for more posterior genes (Figure 5C and not shown, Video 4). Therefore, the same posterior Hox genes can alter cell ingression and axis elongation with a similar collinear trend (Figures 3C and 5C). The cell-autonomous control of ingression by posterior Hox genes (Figure 4F–H) is expected to reduce the supply of motile cells in the posterior PSM. This should slow down elongation movements and could explain why such a non-cell autonomous effect on axis elongation is observed while only 30–50% PM cells express the Hox constructs. These data suggest that a subset of posterior Hox genes controls the slowing down of axis elongation by regulating ingression of PM precursors. Figure 5 Download asset Open asset Posterior Hox genes control the axis elongation velocity in a collinear fashion. (A–B) Time-lapse series of chicken embryos electroporated either with control (A) or Hoxa13 (B). Red line: position of Hensen's node. ss = somite-stage. (C) Velocity of axis elongation of embryos electroporated with either a control, Hoxa9, Hoxc9, Hoxd10, Hoxd11, Hoxc11, Hoxa13, Hoxb13, or Hoxc13 expressing constructs. Stars: p-value of two-tailed Student's t-test applied between the different conditions. *p < 0.05. Error bars: standard error to the mean (SEM). https://doi.org/10.7554/eLife.04379.020 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Effect of Hoxa9, Hoxc11, and Hoxa13 electroporation on axis elongation and ingression. Bright-field (purple) merged with fluorescent images of PSM cell progenitors electroporated with either a control H2B-Venus (first panel from the left), Hoxa9-ires2-H2B-Venus (second panel from the left), Hoxc11-ires2-H2B-Venus (third panel) or a Hoxa13-ires2-H2B-Venus (right panel) constructs (green) (ventral view, anterior is up) from Stage 6 HH onwards. Over-expression of Hoxa9, c11, and a13 affects ingression and axis elongation in a collinear fashion. https://doi.org/10.7554/eLife.04379.015 In Drosophila and vertebrates, Hox genes expressed posteriorly can suppress the function of more anterior ones, a property termed phenotypic suppression or posterior prevalence (Duboule and Morata, 1994). We previously showed that posterior prevalence applies for the control of cell ingression by Hoxb1-9 genes (Iimura and Pourquié, 2006). To test whether this property also applies to the posterior Hox genes with an effect on axis elongation, we performed consecutive electroporations first with a mix of Hoxd10 and Hoxc11 constructs (leading to expression in the same cells, in green Figure 6A) and then with a mix of Hoxc11 and a control construct (a mutated Hoxc11 unable to bind DNA (Hoxc11mutH), in red Figure 6A). We observed that cells over-expressing the two functional Hox genes reach the same anterior position as cells over-expressing Hoxc11 and control (Figure 6A,C, n = 10 embryos). Thus Hoxc11 function is dominant over Hoxd10. Similarly, we observed dominance of Hoxa13 over Hoxc11 in the same assay (Figure 6B,C, n = 8 embryos). Therefore, posterior prevalence appears to generally apply for Hox control of cell ingression in the mesoderm (Iimura and Pourquié, 2006). As a result, the effect of Hox genes on cell retention in the epiblast should become progressively stronger as more posterior genes become activated. Figure 6 Download asset Open asset Posterior prevalence of posterior Hox genes. (A) Embryos consecutively electroporated first with Hoxc11-Cherry + Hoxc11mutH-Cherry and with Hoxd10-Venus + Hoxc11-Venus shown 24 hr after reincubation. (B) Embryos consecutively electroporated first with Hoxa13-Cherry + Hoxa13mutH-Cherry and then with Hoxa13-Venus + Hoxc11-Venus shown 24 hr after reincubation. Red arrowheads: anterior boundary of Cherry-expressing cells. Green arrowheads: anterior boundary of Venus-expressing cells. (C) Quantification of the ratio of Venus over Cherry expressing domains for the experiments shown in A and B. Each dot corresponds to one electroporated embryo and bar indicates the mean. (D–E) Luciferase assay measuring Wnt/βcatenin pathway activity after over-expression of the BATLuc construct together with a Renilla-expressing vector and either (D) control, Hoxa9, Hoxa13 or the combination of Hoxa9 and Hoxa13 expressing vectors. (E) Blow-up of the samples shown in (D). (F) BATLuc assay with serial dilutions of the Hoxa13 plasmid (in μg/μl on the x axis). (G) Western blot labeled with an anti-HA antibody showing embryos electroporated with Hoxa13 under the control of a doxycycline-responsive promoter activated with different doses of doxycycline (in μg/ml). (H) BATLuc assay after Hoxa13 over-expression under the control of a doxycycline-responsive promoter activated with different doses of doxycycline (in μg/ml on the x axis). Stars represent the p value of the two-tailed Student's t-test applied between the different conditions. **p < 0.01; ***p < 0.005. Error bars represent the standard error to the mean (SEM). https://doi.org/10.7554/eLife.04379.011 Pbx1 acts as a cofactor regulating cell ingression controlled by anterior Hox genes Expression of anterior Hox genes in the primitive streak is maintained during the fast axis elongation phase occurring during the formation of the first ten somites, suggesting that there must be a mechanism blocking their effect on ingression during this time window (Figure 1A). TALE (Three Amino-acid Loop Extension) family members have been shown to differentially interact with anterior and posterior Hox genes (Chang et al., 1995; Moens and Selleri, 2006). In chicken, the only TALE gene expressed in PM precursors is Pbx1 which is detected in the primitive streak from stage 4 to 7 HH (Figure 7A, n = 8 embryos for each condition [Coy and Borycki, 2010]). Electroporation of a siRNA targeting Pbx1 in the epiblast resulted in strong down-regulation of Pbx1 (Figure 7B–C, n = 4 embryos for each condition). In consecutive electroporations performed first with Cherry and a control siRNA and then with Venus and a siRNA targeting Pbx1, cells electroporated with the Pbx1 siRNA were found extending more anteriorly than control cells (Figure 7D,K, n = 19 embryos). The effect of Pbx1 siRNA on ingression could be rescued by co-expressing Pbx1 (Figure 7E,K, n = 16 embryos). We compared in consecutive electroporations the effect of expressing first a control siRNA with either Hoxb7, Hoxb9, Hoxa9, Hoxc9, Hoxd10, Hoxd11, Hoxc11, Hoxa13, Hoxb13 or Hoxc13, and then the Pbx1 siRNA with the same Hox gene. Cells co-expressing Hoxb7 or Hoxb9 and the Pbx1 siRNA reached more anterior levels than cells co-expressing these Hox genes and the control siRNA (Figure 7F–G, K, n = 10 and 15 embryos respectively). In contrast, cells co-expressing either a control or the Pbx1 siRNA together with a posterior Hox gene were found to extend up to the same anterior level (Figure 7H–K and not shown, n > 8 embryos for each condition). Over-expression of Pbx1 in PM precursors after the 3-somite stage slowed down axis elongation (Figure 7L and Video 5, n = 12 embryos), suggesting that Pbx1
Significance The vertebral column provides essential structural and protective functions. The total number of vertebral elements and their specific morphologies are remarkably reproducible within a given species, yet can be tailored to the requirements of separate vertebrate species. Major genetic determinants driving formation of the vertebral column are known, but how they are regulated to achieve a highly reproducible structure remains to be fully elucidated. In this report, we show that the miR-196 family of microRNAs are essential in defining correct vertebral number and vertebral identity in mouse. We reveal the molecular landscape controlled, either directly or indirectly, by miR-196 activity, to demonstrate that miR-196 impacts many key developmental signalling pathways and reinforces a timely trunk-to-tail Hox code transition.
Le corps des vertébrés est allongé selon un axe antéro-postérieur. Cette forme spécifique se met en place durant l’embryogenèse par des phénomènes morphogénétiques d’élongation. Les mécanismes d’élongation qui mènent à la formation des parties antérieures du corps sont très bien décrits mais, par contre, ceux qui concernent les parties les plus postérieures ont été moins bien étudiés. Nous avons choisi l’embryon de poulet comme modèle d’étude pour aborder cette problématique. Avec des expériences d’ablation par microchirurgie, nous avons d’abord montré que la partie caudale du mésoderme présomitique (PSM) était primordiale dans le phénomène d’allongement postérieur. Grâce à des techniques de vidéo-microscopie, nous avons par la suite mis en évidence un gradient caudo-rostral de motilité directionnellement postérieure au sein du PSM. En soustrayant le mouvement du tissu grâce à un marquage de la matrice extracellulaire, nous avons démontré que ce gradient correspond à un gradient de motilité cellulaire non directionnel, indiquant que les mouvements postérieurs sont dus à la déformation tissulaire et non aux déplacements propres des cellules. Par des expériences de perte et de gain de fonction de la voie de signalisation FGF (Fibroblast Growth Factor), nous avons montré que cette voie de signalisation régule le gradient de motilité non directionnelle et l’allongement postérieur de l’embryon. Enfin, nous avons effectué des expériences suggérant que l’effet du FGF sur l’allongement de l’embryon ne passe pas par la régulation de la prolifération cellulaire mais bien par un effet sur la motilité cellulaire. Nous proposons donc un nouveau modèle d’élongation dans lequel le gradient de motilité non directionnelle présent dans le PSM contrôle l’allongement postérieur de l’axe embryonnaire.
Summary Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the anteroposterior axis remains largely unknown. Hox genes have long been suspected to control limb position, however supporting evidences are mostly correlative and their role in this process remains unclear. Here we show that Hox genes determine the avian forelimb position in a two-step process: first, their sequential collinear activation during gastrulation controls the relative position of their own successive expression domains along the body axis. Then, within these collinear domains, Hox genes differentially activate or repress the genetic cascade responsible for forelimb initiation. Furthermore, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether our results which characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb position in avians, show a direct and early role for Hox genes in this process.
Abstract The degree and dynamics of translational control during mammalian development remain poorly understood. Here we monitored translation of the mammalian genome as cells become specified and organize into tissues in vivo . This identified unexpected and pervasive translational regulation of most of the core signalling circuitry including Shh, Wnt, Hippo, PI3K and MAPK pathways. We further identify and functionally characterize a complex landscape of upstream open reading frames (uORFs) across 5′-untranslated regions (UTRs) of key signalling components. Focusing on the Shh pathway, we demonstrate the importance of uORFs within the major SHH receptor, Ptch1 , in control of cell signalling and neuronal differentiation. Finally, we show that the expression of hundreds of mRNAs underlying critical tissue-specific developmental processes is largely regulated at the translation but not transcript levels. Altogether, this work reveals a new layer of translational control to major signalling components and gene regulatory networks that diversifies gene expression spatially across developing tissues.
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