The authors have declared that no competing interests exist.
Two theories address the origin of repeating patterns, such as hair follicles, limb digits, and intestinal villi, during development. The Turing reaction–diffusion system posits that interacting diffusible signals produced by static cells first define a prepattern that then induces cell rearrangements to produce an anatomical structure. The second theory, that of mesenchymal self-organisation, proposes that mobile cells can form periodic patterns of cell aggregates directly, without reference to any prepattern. Early hair follicle development is characterised by the rapid appearance of periodic arrangements of altered gene expression in the epidermis and prominent clustering of the adjacent dermal mesenchymal cells. We assess the contributions and interplay between reaction–diffusion and mesenchymal self-organisation processes in hair follicle patterning, identifying a network of fibroblast growth factor (FGF), wingless-related integration site (WNT), and bone morphogenetic protein (BMP) signalling interactions capable of spontaneously producing a periodic pattern. Using time-lapse imaging, we find that mesenchymal cell condensation at hair follicles is locally directed by an epidermal prepattern. However, imposing this prepattern’s condition of high FGF and low BMP activity across the entire skin reveals a latent dermal capacity to undergo spatially patterned self-organisation in the absence of epithelial direction. This mesenchymal self-organisation relies on restricted transforming growth factor (TGF) β signalling, which serves to drive chemotactic mesenchymal patterning when reaction–diffusion patterning is suppressed, but, in normal conditions, facilitates cell movement to locally prepatterned sources of FGF. This work illustrates a hierarchy of periodic patterning modes operating in organogenesis.
Repeating anatomical units, forming a pattern, are present in different parts of the body. This is particularly obvious in the skin, where the many hair follicles become regularly arranged and fixed in place in the embryo as a pattern of evenly spaced spots. The basis for the formation of such repeating patterns has attracted a number of theoretical explanations. One influential theory is that cells first issue chemical signals to one another to produce a map of the pattern, which they then follow to assemble a structure, like a hair follicle. Alternative theories instead suggest that cells cluster together directly, without reference to a pre-existing map, to drive pattern formation. In this study, we find that a set of chemical signals known to be important for early hair follicle formation form a network capable of producing a patterned template to which cells then respond, moving according to its instructions. This supports the signal-based theory for pattern formation. Strikingly, though, by imposing the conditions of the incipient hair follicle evenly across the entire skin, we reveal that skin cells can also form patterns directly by coalescing, without requiring instructions from a pre-existing pattern. However, this ability of cells to pattern directly is normally subservient to their following of a quickly forming pattern template defined by chemical signals. This work highlights that different ways of making biological patterns can coexist in the same embryonic organ but that one is normally subordinated to the other.
Diverse structures, such as the skeletal elements of the limb, rugae of the palate, cartilaginous rings of the trachea, intestinal villi, and feathers, scales, or hair follicles, develop in a periodically patterned manner. Although many specific models to explain the spontaneous emergence of such repeating patterns in embryonic tissues have been proposed [
(A) Schematic illustrating the 2 general theoretical models explaining the emergence of repeating patterns: Turing reaction–diffusion-driven systems and mesenchymal self-organisation. (B) Schematic of the process of hair follicle formation depicting acquisition of epidermal foci of
In the mouse embryo, a periodic pattern of hair follicles first arises at late embryonic day 13 (E13) [
Classical tissue recombination experiments indicated that the dermal mesenchyme is the compartment in which pattern generation occurs, this spatial information subsequently being conveyed to the epidermis through inductive signalling [
Once the spatial pattern has been defined, the cells selected to become a follicle activate expression of other genes to progress their development into construction of the mature organ. These later-acting genes are typically expressed in the ‘de novo’ mode, indicating their activation only after assumption of the new cell fate, as opposed to the ‘restrictive’ mode of expression, which is characteristic of genes involved in the patterning process itself [
Simple signalling interactions have been proposed as the basis of hair follicle periodic patterning, though none represent a complete system capable of de novo pattern formation. WNT signalling coupled with induced expression of Dickkopf (DKK) family members was proposed to contribute to a reaction-diffusion system, though no WNT activator positive feedback loop was identified [
Here, we identify a set of interactions between the BMP, FGF, and WNT pathways capable of breaking symmetry to produce a prepattern that guides local dermal cell condensation. Strikingly, by modulating components of this system to mimic the microenvironment of the hair follicle primordium, we identify a transforming growth factor (TGF) β-driven patterning system in dermal mesenchyme that is independent of epidermal instruction. Whereas TGFβ signalling is capable of driving mesenchymal self-aggregation when reaction–diffusion signalling is suppressed, in normal development, this pathway potentiates cell accumulation at sites of focal FGF production, thereby assuring timely dermal condensate formation according to the epidermal template. Thus, both reaction–diffusion signalling and mesenchymal self-organisation potentials reside in the developing skin, but the former dominates and directs the latter, highlighting the hierarchical nature of patterning mechanisms.
To determine the functions and relationships between cell signalling pathways and whether their interactions are sufficient to constitute a complete pattern forming network, we focussed on the BMP, FGF, and WNT/β-catenin pathways [
Having defined conditions to stimulate and repress each of these 3 signalling pathways, we set out to delineate their transcriptional regulatory interactions and assess whether these could comprise a functional pattern forming network. Turnover of activator and inhibitor molecules is required to produce a reaction–diffusion pattern [
We performed a transcriptome-wide screen of mRNA half-lives in E13.5 mouse dermis and epidermis, subsequently applying a data filtering criterion based on half-life (t1/2 ≤ 90 minutes) and focusing upon extracellular molecules (ligands, receptors, and extracellular signal-attenuating molecules) of the BMP, FGF, and WNT pathways (
To identify regulatory relationships between the BMP, FGF, and WNT pathways, we stimulated and repressed each pathway in unpatterned E13.5 skin explants for 6 h and assessed the resulting change in abundance of each candidate transcript (
After excluding candidates unresponsive to BMP, FGF, or WNT signal modulation, 15 genes were found to be regulated within this period (
In this network, some features of classical Turing activator–inhibitor dynamics are present, including activator stimulation of inhibitor production, but we did not detect direct positive feedback for any pathway. Rather, each pathway displays prominent self-inhibition. Thus, in its number of components and its structure, this network does not conform to the classical topology of a reaction–diffusion system. Therefore, we took a mathematical approach (
We grouped the candidates into 5 species: BMP (
Having defined the reciprocal relationships between BMP, FGF, and WNT signalling, including their organisation into a network capable of periodic patterning, we investigated the origin of the dermal condensate and the influences of these pathways on its formation. We performed live cell imaging of TCF/Lef::H2B-GFP E13.5 dorsal skin explants using confocal microscopy (
(A) Single frames from time-lapse sequences of E13.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explant culture captured by confocal microscopy. Dashed circles indicate ultimate condensate location. Scale bar: 50 μm. (B) Analysis of tracked cells showing the probability of joining the dermal condensate based upon initial location relative to its centre. Two hundred and forty individual cells were tracked across 8 condensates from 4 independent skins. (C) Protractor plot showing the distribution of Euclidean angles and Euclidean distances of individual cell movements in 6-h windows for cell tracks that start outside of, but ultimately terminate in, a follicle (condensate = red) and those that remain outside (intercondensate = blue). Tracking was halted on cell entry. (D) Plots showing the mean Euclidean angle (top) and mean level of persistence (bottom) of condensate-entering and intercondensate cells for 6-h windows relative to time of entry into the condensate. Error bars represent SEM (condensate cells
We performed an analysis of the direction of movement of individually tracked cells by dividing each cell track into 6-h windows and determining the Euclidean angle of movement with respect to the future condensate centre for each window, as well as the Euclidean and accumulated distance travelled over this period. To determine whether individual cell movement was directed, we compared the distribution of Euclidean angles and distances for 2 classes of cells: (i) those cells initially outside the condensate area which subsequently entered it (‘condensate’) and (ii) those cells which remained outside the condensate area throughout (‘intercondensate’) (
To investigate the timing of the distinct behaviours of cells entering condensates, we quantified cell behaviour by comparing the three 6-h windows prior to follicle entry (or the end of the track for intercondensate cells). We compared the Euclidean angle and level of persistence (calculated as the Euclidean distance/accumulated distance) of movement by the 2 cell classes (intercondensate and condensate) at 0–6, 6–12, and 12–18 h prior to follicle entry (
Using the TCF/Lef::H2B-GFP line, we set out to determine the relative order of placode specification and cell condensation. We fixed cultured dorsal skin cultures at intermediate stages of development and compared the timing of appearance of
Based on these findings, we hypothesised that local signalling from the epithelial placode might coordinate the condensation of the underlying dermal cells. We considered FGF a good candidate for the local attractant signal as this induces dermal condensations during feather development [
Having established that FGF attracts mesenchymal cells to form a condensate and knowing that reception of WNT signals by mesenchymal cells is also required for this process [
(A) Epidermis and dermis isolated from E13.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explants cultured with LDN193189 (LDN) (10 μM) or fibroblast growth factor (FGF) 9 (1 μg/ml) for 27 h, counterstained with propidium iodide (PI) and imaged using confocal microscopy. Scale bar: 100 μm. (B) Number of condensates per square mm, (C) mean condensate area, and (D) intercondensate cell density measured in E13.5 TCF/Lef::H2B-GFP skin explants cultured with either LDN or FGF. Error bars represent SEM from 5 independent experiments. Significant difference was calculated using a Student paired
To further define the influence of BMP on dermal cell arrangement, we stimulated this pathway in skin cultures containing existing dermal condensates. BMP treatment led to erosion of condensates, with a profound reduction in their size (
These results indicate roles for FGF and BMP signalling in influencing mesenchymal cell aggregation. We tested whether these influences could be modulated to trigger mesenchymal condensation by mimicking the hair follicle primordium microenvironment—that is, high-FGF and low-BMP signalling (hereafter, FGFHiBMPLo)—across the entire skin. Strikingly, imposing these conditions using FGF9 together with LDN193189 caused dermal condensates to arise in a periodically spaced manner without corresponding expression of the epidermal placode marker
(A) Detection of green fluorescent protein (GFP) signal,
To determine the relative rates of dermal condensate patterning, we cultured skin explants under normal or FGFHiBMPLo conditions and imaged the samples at different time points. Pattern formation under normal conditions is more rapid than in FGFHiBMPLo conditions, with the control pattern appearing and stabilising quickly, while the FGFHiBMPLo pattern is slower to appear (
Time-lapse imaging of the formation of mesenchymal condensates under FGFHiBMPLo conditions reveals the gradual emergence of these focal aggregates across the entire skin (
(A) Time-lapse images showing dermal condensate formation in FGFHiBMPLo conditions. Scale bar: 50 μm. (B) Protractor plot showing the distribution of Euclidean angles and Euclidean distances of individual cell movements in 6-h windows for cell tracks that start outside of, but ultimately terminate in, a follicle (condensate = red) and those that remain outside (intercondensate = blue) under FGFHiBMPLo conditions. Tracking was halted on cell entry. Plots showing (C) the mean Euclidean angle and (D) the mean level of persistence of condensate and intercondensate cells for 360-minute windows relative to time of entry into the condensate. From 6 h before entry, the condensate-bound cells show oriented and persistent movement under FGFHiBMPLo conditions. Error bars represent SEM (condensate cells
The behaviour of these cells entering condensates is remarkably similar to that observed in conditions of normal patterning (
To further investigate cell behaviour between normal (control) and FGFHiBMPLo conditions, we compared summary statistics for individual cell tracks (
As the underlying individual cell behaviour driving condensate formation is the same in control and FGFHiBMPLo conditions, we asked whether these are fundamentally distinct patterning mechanisms or whether they might be differently regulated outputs of a single underlying mechanism. Reaction–diffusion- and cell aggregation-based patterning mechanisms behave differently at tissue boundaries [
As the mesenchymal patterning mechanism is fundamentally distinct from the normal condition, we sought a mechanistic basis for this process. We treated skin cultures with modulators of pathways previously implicated in dermal cell condensation to identify signalling pathways with selective effects on mesenchyme-only patterning. We found that mesenchyme, in both control and FGFHiBMPLo conditions, patterned robustly, despite the presence of inhibitors or activators of the CXCL/CXCR, Notch, or platelet-derived growth factor (PDGF) pathways (
Alteration of TGFβ signalling, however, profoundly suppressed mesenchyme-only patterning while having relatively modest effects on normal patterning. The TGFβ receptor type I and II inhibitor LY2109761 (
(A) Western blot detection of phospho-SMAD2, total SMAD2 and γ-tubulin in E13.5 skin cultures treated with recombinant transforming growth factor (TGF) β2 (100 ng/ml), the TGFβ receptor inhibitor LY2109761 (25 μM,) or both agents for 8 h. (B) Effects of TGFβ2 supplementation and LY2109761 on normal and mesenchyme-only patterning. Condensates are slow to appear in LY2109761, and expression of the placode marker
Having defined restricted TGFβ as a required self-attractive factor in mesenchyme-only patterning, we sought its role in an interplay between normal patterning and mesenchymal self-organisation. In addition to its function as a direct mesenchymal chemoattractant (
(A, B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of E13.5 or E13.75 (with condensates) skins treated with transforming growth factor (TGF) β2, fibroblast growth factor (FGF) 9, or bone morphogenetic protein (BMP) 4 for 8 or 24 h, respectively, followed by assessment of transcript abundance. TGFβ2 upregulates expression of genes associated with cell movement and the extracellular matrix. Statistical significance from control was calculated using a Student
These gene expression changes related to cell–matrix interactions and movement suggested that TGFβ may play a general role in promoting cell migration. To assess whether TGFβ could promote cell attraction to placodes, as indicated by the prominent condensates it induces during patterning (
Embryonic pattern formation proceeds rapidly; periodic arrangements of limb digits arise within approximately 16 h [
Following definition of the epidermal placode arrangement, dermal cells are locally recruited to assemble a condensate. Previously, a mesenchymal cell-sorting mechanism for hair follicle patterning was proposed based on genetic correlations between hair follicle number and size [
In addition to their contribution to pattern formation through rapid gene regulatory interactions, the BMP and FGF pathways also influence mesenchymal cell behaviour. FGF, normally produced locally at the placode, directs mesenchymal clustering, while BMP suppresses aggregation. By mimicking the hair follicle microenvironment of high-FGF and low-BMP signalling, we identify a latent potential of dermal mesenchyme to self-organise, yielding a stable periodic pattern of cell aggregates in the absence of signalling directives from the epidermis. This demonstrates that dermal condensates do not require spatially restricted signals to form. Rather, the widespread BMP activity in the skin prior to hair follicle patterning, together with the slow rate of dermal self-organisation, subordinates the mesenchyme’s patterning potential to reaction–diffusion patterning in the epidermis. Our identification of a mesenchyme-only patterning condition is, in structural terms, reciprocal to that arising from
Mechanistically, however, these epidermis-only and mesenchyme-only patterns arise through very different mechanisms. Epidermis-only patterning in the
Our findings of dermal condensate formation in the absence of an epidermal prepattern are consistent with modes of tissue patterning in which moving mesenchymal cells themselves are agents of pattern formation. These models do not require interacting diffusible signals and such mechanisms integrate tissue morphogenesis and patterning as part of a single process, rather than occurring in sequence, as in the Turing system [
Together, these results support a model for hair follicle positioning and construction in which a reaction–diffusion system, operating largely in the epidermis [
(A) A signalling network including bone morphogenetic protein–fibroblast growth factor–wingless-related integration site (BMP–FGF–WNT) interactions rapidly produces a periodic prepattern of hair placodes with high WNT/β-catenin activity. (B) FGF20 production by placodes leads to local dermal cell attraction and condensate formation, facilitated by widespread transforming growth factor (TGF) β activity. BMP production inhibits expansion of placode gene expression and further dermal cell attraction. (C) In the absence of epidermal patterning, no localised attractant signals from placodes are present, and dermal BMP signalling is uniform. TGFβ stimulates dermal cell–cell attraction, which is inhibited by BMPs. Suppression of BMP signalling permits TGFβ-driven mesenchymal patterning in the absence of a functioning epidermal reaction–diffusion network. Condensate expansion is restricted by mesenchymal cell depletion from the surrounding dermis.
The rationale for the existence of 2 distinct routes to achieve periodic patterning during hair follicle formation may lie in a shifting balance between these 2 potentials in the waves of hair follicles that form at different developmental stages. Thus, it may be that the pattern of secondary or tertiary hair follicles is defined by mesenchymal cell behaviour to a greater extent than the primary follicles focussed upon here. Alternatively, the existence of 2 periodicity generators may be a remnant of the evolutionary history of skin development, as it has been suggested that turnover of patterning mechanisms can occur during the course of evolution [
All animal work was conducted under approval of the Animal Welfare and Ethical Review Body (AWERB) at The Roslin Institute, University of Edinburgh, and by the United Kingdom Home Office in accordance with the Animals (Scientific Procedures) Act 1986. Euthanasia was carried out according to Schedule 1 of the Animals (Scientific Procedures) Act 1986.
Mice were on the FVB/N background, apart from those employed in time-lapse and static imaging of cell rearrangement, which were offspring of a cross between male hemizygous TCF/Lef::H2B-GFP mice [
For the qRT-PCR experiments used in the network derivation, dorsal skin explants were halved along the midline to generate treated and control halves from each embryo. Skin halves were placed onto a cellulose filter (Millipore, pore size 0.45 μm). The tissue was then submerged in DMEM supplemented with 2% FBS (complete DMEM) in a centre-well dish (Falcon) and incubated at 37°C and 5% CO2 for 6 h. For the TGFβ2, FGF9, and BMP4 experiments, whole dissected skins were cultured in complete DMEM containing the required recombinant protein for either 8 h or 24 h.
For longer-term culture, whole skin explants were dissected and mounted on filters as described above, submerged in complete DMEM, and supported by a metal grid in a centre-well dish. E13.5 explants were cultured for 27 h unless stated otherwise. For experiments extending beyond 27 h, medium was replaced every 24 h.
Epidermal–dermal separations for RNA-seq and qRT-PCR experiments were performed by incubating skin samples with 2 mg/mL Dispase II (Gibco) at 37°C for 10 minutes. For imaging, epidermal–dermal separations were performed by incubating skin in 10 mM EDTA/PBS for 25 minutes at 37°C followed by 20 minutes in PBS at RT prior to separation with fine forceps. For counterstaining, samples were fixed in 4% PFA, washed several times in PBS/0.1% Tween 20 (PBST), treated with 20 μg/ml proteinase K for 3 minutes, washed in PBT, incubated with 100 μg/ml RNase for 20 minutes, washed again with PBT and then stained with 1/2000 diluted propidium iodide (PI) solution (Life Technologies) for 5 minutes. Following staining, samples were washed in PBT and mounted in Prolong Gold (Life Technologies).
Recombinant FGF7 (mouse), FGF9 (mouse and human), BMP4 (mouse), and TGFβ2 (human) were from R&D Systems. LDN193189 (Stemgent), CHIR99021 (Axon biochem), IWR-1 (Tocris), DAPT (Bio-Techne), LY2109761 (Cambridge Biosciences), Pertussis toxin (Tocris), Imatinib Mesylate (Sigma Aldrich), and SU5402 (Sigma Aldrich) were reconstituted according to the manufacturer’s recommendations. We did not detect any significant effect from treatment with recombinant FGF20 protein obtained from 2 commercial suppliers on either placode pattern formation, skin development, or on expression of the direct FGF pathway target gene
Total RNA was isolated from skin explants using Tri Reagent (Sigma) and treated with RQ1 DNAse (Promega) to remove contaminating genomic DNA. cDNA was synthesised from total RNA using random primers and Superscript III reverse transcriptase (Roche) in a 20-μl reaction. Reactions were diluted 20-fold and had 3 μl used as a template for each qRT-PCR. Each reaction was performed in a 20-μl volume using Universal SYBR Green Master Mix (Roche) containing Rox reference dye. Reactions were performed in triplicate, with at least 3 biological replicates used to determine each data point. Relative expression levels were determined from cDNA dilution standard curves and normalised to
See
Skin explants were fixed overnight in 4% PFA at 4°C. Samples were dehydrated into 100% methanol, bleached in 6% H2O2, then rehydrated and treated with 20 μg/mL proteinase K. After postfixing in 4% PFA containing 0.2% glutaraldehyde for 20 minutes, skin explants were hybridised with probe at 60°C overnight in 50% formamide, 5 X saline sodium citrate (SSC), 1% SDS, 50 μg/mL heparin, and 50 μg/mL yeast RNA in diethyl pyrocarbonate (DEPC)-treated H2O. Samples were washed to remove unbound probe and signal detected using an alkaline phosphatase sheep antidigoxigenin antibody (Roche, 1:1,000 dilution) and 5-bromo-4-chloro-3'-indolylphosphate/nitro-blue-tetrazolium (BCIP/NBT) colour reaction (Sigma).
For immunohistochemistry, samples were fixed in 4% PFA, embedded in 0.12 M sodium phosphate buffer/7.5% gelatin/15% sucrose, and cryosectioned. Sections were rehydrated in PBS at 37°C for 30 minutes, briefly washed with tris-buffered saline containing 0.01% Tween 20 (TBST), then incubated in blocking buffer (TBST containing 5% heat-treated sheep serum and 1% BSA) for 1 h at RT before overnight incubation at 4°C with primary rabbit antibodies (1:200 mouse antiactive beta catenin (Millipore #05–665) or rabbit antiphosphorylated SMAD2 (pSMAD2) (Cell Signalling Technologies #3108) diluted in blocking buffer. Samples were washed in TBST, then incubated with fluorescent secondary antibodies (1:500 Life Technologies) in blocking buffer for 1 h at RT. Samples were washed with TBST, counterstained with DAPI (Sigma) and mounted in Prolong Gold (Life Technologies). Sections were imaged using a Zeiss LSM710 confocal microscope (Carl Zeiss).
For immunoblotting, protein was extracted from skin samples using radioimmunoprecipitation assay (RIPA) lysis buffer (Santa Cruz Technologies) and a handheld homogeniser. Protein samples were diluted in NuPage LDS sample buffer (Life Technologies), separated by gel electrophoresis on 4%–12% Bis-Tris NuPage precast gels under denaturing conditions and transferred to nitrocellulose membrane (Amersham). Membranes were blocked in 5% milk/TBST for 1 h at RT, followed by overnight incubation at 4°C in 5% BSA/TBST containing primary antibody (1:1,000 mouse anti-active β-catenin [Millipore #05–665], 1:3000 rabbit anti-β-catenin [BD biosciences #610153], 1:1,000 rabbit anti-phospho SMAD 2/3 [Cell Signalling Technologies #8828], 1:1,000 rabbit anti-SMAD2 [Cell Signalling Technologies #5339], or 1:3,300 mouse anti-γ tubulin [Sigma Aldrich #T6557]). Following primary antibody incubation, membranes were washed in TBST and incubated for 1 h at RT in 5% milk/TBST containing horseradish peroxidase (HRP)-conjugated species-specific secondary antibodies (Dako). Membranes were washed several times with TBST before detection with the Novex ECL chemiluminescent substrate reagent kit (Life Technologies) and developed on ECL Film (Amersham).
Affi-Gel Blue Gel beads (Bio-Rad) were washed twice in PBS and incubated in either 100 μg/ml recombinant human FGF9, 100 μg/ml recombinant human TGFβ2, or 100 μg/ml BSA diluted in PBS for at least 2 h at RT or overnight at 4°C. Beads were placed onto a nitrocellulose Millipore filter (pore size 0.45 μm), and dissected E12.5 TCF/Lef::H2B-GFP dorsal skin explants were manoeuvred and placed on top of the beads, dermis side down. Skins were then imaged, as described in
E13.5 TCF/Lef::H2B-GFP dorsal skin explants were dissected and imaged as described previously using a custom imaging chamber [
All image analysis tasks were performed using custom written macros for the Fiji [
A cell was deemed to acquire a ‘condensate identity’ if, at the end of the tracking period, it was incorporated within this structure; for analysis purposes, tracking was halted on follicle entry. Cells that did not enter the condensate were termed ‘intercondensate.’ Visual analysis of these cells up to a later time point was also performed to ensure that these cells did not become part of the condensate at a later stage.
(A) E13.5 (prior to hair patterning) dorsal back skin from TCF/Lef::H2B-GFP mice demonstrates GFP expression specifically in dermal cell nuclei thereby allowing mesenchymal cells to be visualised during hair follicle morphogenesis. (B) E13.5 TCF/Lef::H2B-GFP dorsal skin explant cultured for 27 hrs and imaged for GFP (left). Epidermis and dermis separated and counterstained with propidium iodide (PI) (right). In the epidermis, signal has appeared in the placodes, while the dermis shows widespread nuclear GFP, which reveals the aggregated cells comprising the dermal condensates. (C) Dose effects of modulators of BMP, FGF and WNT signalling on periodic patterning assessed by
(TIF)
E13.5 TCF/Lef::H2B-GFP dorsal skin explants were treated with increasing concentrations of FGF7 for 27 hrs, GFP expression analysed and
(TIF)
(A) Schematic of experimental approach to define mRNA half-lives and derive a network of interactions between BMP, FGF and WNT pathways. Briefly, paired skin halves were either processed for RNA isolation immediately or else cultured in Actinomycin D to inhibit RNA synthesis for a set period, then processed for RNA isolation. Epidermal and dermal layers were isolated and RNA-sequencing performed on each layer separately. The ratio of RNA species abundance between the paired skin samples was used to calculate t1/2. Candidate genes were identified based on half-life and a gene regulatory network was derived through systematic experimental and mathematical approaches. (B) [3H] cytidine incorporation assay to determine Actinomycin D dose sufficient to block transcription in cultured embryonic skin. Each point represents the [3H] cytidine incorporation per A260 unit for a single skin sample. (C) Transcripts from RNA-seq experiments expressed above threshold level in epidermis or dermis, grouped according to half-life. Of the 37,315 transcripts detectable by RNA-seq, 11,970 were detected in the epidermis and 12,597 in the dermis. (D) qRT-PCR validation of selected mRNA half-lives from RNA-seq data. E13.5 dorsal skin explants were treated with Actinomycin D for 30, 60 or 120 minutes. Relative expression was calculated by normalising the expression at each experimental time to the expression of that gene at time 0. The raw numerical values for (B-D) can be found in
(TIF)
(A) qRT-PCR detecting
(TIF)
(A) E12.5 TCF/Lef::H2B-GFP dorsal skin explants cultured with either FGF9 (1 μg/ml), LDN193189 (LDN) (10 μM) or both for 48 hrs show a periodic pattern of cellular aggregates and lack
(TIF)
E12.5 TCF/Lef::H2B-GFP skin explants were cultured in the presence of FGF9 (1 μg/ml), LDN193189 (LDN) (10 μM) or both for 27 hours, then imaged and this condition was either reapplied (A) or removed (B) and culture continued for a further 24 hours. Mesenchymal condensations formed in the FGFHiBMPLo conditions are stable and continue to resolve after removal of FGF9 and LDN. Scale bar: 250 μm.
(TIF)
E12.5 TCF/Lef::H2B-GFP skin explants were cultured over a period of 48 hrs and imaged at time intervals as labelled. FGFHiBMPLo pattern formation is slower to resolve than control condensate formation. Scale bar: 1 mm.
(TIF)
FGFHiBMPLo condensations are formed in the presence of pertussis toxin (PTX, a G-protein inhibitor), imatanib mesylate (IM, a PDGFR inhibitor) and DAPT (a NOTCH inhibitor). Scale bars: 250 μm.
(TIF)
(A) Images of E13.5 TCF/Lef::H2B-GFP skin cultured for 48 hours with or without TGFβ inhibition by LY2109761 (25 μM). Hair follicle primordia in control skin have formed a dermal papilla and polarised from anterior to posterior (inset), while TGFβ inhibited samples have not progressed to this stage. Scale bar: 250 μm. (B) Haematoxylin stained sections of skin samples in panel (A). Placode downgrowth is delayed in the LY2109761 condition, consistent with the reported phenotype of the
(TIF)
Transcript names are in coloured text according to the pathway in which they operate: BMP (green), WNT (red), FGF (blue).
(XLSX)
Results from experiment outlined in
(XLSX)
(XLSX)
(XLSX)
(XLSX)
(XLSX)
(XLSX)
(XLSX)
(DOCX)
(DOCX)
Confocal time-lapse video of cultured E13.5 TCF/Lef::H2B-GFP dorsal skin explant undergoing pattern formation. Scale bar: 100 μm.
(AVI)
Time-lapse video of cultured E13.5 TCF/Lef::H2B-GFP dorsal skin explant undergoing pattern formation. Randomly selected cells were tracked for each individual follicle analysed. The boundary of the final condensate is indicated by the white circle. Cells which ultimately enter and form the condensate are indicated as yellow tracks while those that remain outside as intercondensate cells appear as red tracks. Scale bars as annotated.
(MP4)
Time-lapse imaging of E12.75 TCF/Lef::H2B-GFP dorsal skin explants cultured in the presence of either FGF9 loaded or control BSA beads. Scale bars as annotated.
(M4V)
Live cell imaging of E13.5 TCF/Lef::H2B-GFP dorsal skin explants cultured in FGF9 (1 μg/ml) and LDN193189 (LDN) (10 μM) either alone or in combination. Treatment with FGF9 and LDN results in labyrinthine patterning of dermal condensates. Scale bars as annotated.
(M4V)
Confocal time-lapse video of cultured E13.5 TCF/Lef::H2B-GFP dorsal skin explant treated with FGF9 (1 μg/ml) and LDN 193189 (LDN) (10 μM) and imaged undergoing pattern formation.
(AVI)
5-bromo-4-chloro-3'-indolylphosphate
bone morphogenetic protein
bovine serum albumin
4’6-diamidino-2-phenylindole dihydrochloride
diethyl pyrocarbonate
Dickkopf
Dulbecco’s modified Eagle’s medium
ectodysplasin A receptor
fibroblast growth factor
green fluorescent protein
horseradish peroxidase
nitro-blue-tetrazolium
neural cell adhesion molecule
platelet-derived growth factor
propidium iodide
radioimmunoprecipitation assay
saline sodium citrate
tris-buffered saline containing Tween
transforming growth factor
wingless-related integration site