The ability to monitor the proliferative status of live cells both in vitro and in vivo over time has revolutionised our understanding of development, growth and disease. This was first made possible by fluorescent ubiquitination-based cell cycle indicator (Fucci) technology, which distinguishes specific cell cycle phases through the reciprocal degradation of fluorescently tagged, truncated forms of human CDT1 and GMNN proteins. Fucci genetic systems have been successfully implemented in transgenic mice, zebrafish, and axolotls. To date, no viable, stably expressing Fucci line has been developed in an avian species. Although a range of continuously improving Fucci constructs have been developed in recent years, existing in vivo Fucci models remain limited because they rely on older reporter technology that fails to distinguish cells in S, G2, and M phases or to label cells in early G1. As a result, these models can be challenging to interpret and their utility for continuous cell tracking and precise analysis of cell-cycle dynamics is limited. Here, we introduce FuChi, a multicistronic Fucci-expressing chicken line incorporating a newly optimised reporter construct composed of an mCerulean-tagged histone H1.0 linker protein fused via a self-cleaving 2A peptide to the tandem Fucci(CA)2 cell cycle biosensor, with additional epitope tags included for detection in fixed tissues. We show that this system accurately discriminates and permits tracking of cells in G1, S, G2, and M phases both in vitro and in vivo, enabling faithful visualisation of cell cycle status in intact tissues and organs. Using FuChi embryos, we mapped proliferation dynamics across developing tissues, analysed cell cycle states of migrating cells, and performed live imaging of early embryos. These latter experiments revealed that transition from S phase may be a key morphogenetic event during gastrulation as mesendoderm cells egress from the primitive streak to form embryonic structures including the prechordal plate. Pairing this advanced reporter with the intrinsic experimental advantages of the chicken embryo positions FuChi as a premier in vivo system for studying cell-cycle kinetics in development, delivering clear technological improvements over current Fucci models. FuChi chickens provide a powerful new resource for studying embryonic development, organ growth, tissue homeostasis, disease processes, and infection responses.

Fig 1. Basis and design of different Fucci reporters.

A) Schematic of cell cycle stages including the abundance of GMNN and CDT1 proteins, two essential and interacting DNA replication licensing factors that accumulate at distinct phases of the cell cycle. For visualisation purposes, we have chosen to represent the red fluorescence as magenta in all our Fucci reporter system schematics. B) Structure of human CDT1 (hCDT1) and human GMNN (hGMNN) proteins, including key features regulating their proteolysis during the cell cycle. The N-terminus of CDT1 is targeted for degradation by two different E3 ubiquitin ligases, CUL4^Ddb1 and SCF^Skp2, which bind to the PIP box and Cy motif, respectively. Phosphorylation of key amino acids (Thr29 and Ser31) facilitates binding of SCF^Skp2. Ubiquitination and proteolysis of GMNN is regulated through the E3 ligase APC^Cdh1 binding to the destruction box. C) Fucci biosensors utilise fusion proteins of hCDT1 and hGMNN, containing truncated forms of the proteins fused with fluorescent markers. In Fucci(SA) systems, an SCF^Skp2-sensitive hCDT1 probe linked to a fluorescent protein (e.g., mKO2 / mCherry) is used in combination with a truncated APC^Cdh1-sensitive hGMNN fused to a spectrally distinct fluorescent protein (e.g. mAG / mVenus). The expected fluorescent profiles throughout the cell cycle reflect hCDT1 and hGMNN degradation by their respective E3 ligases. Note that double-labelled cells are white and negative cells are grey in the combined schematic. D) Fucci(CA) systems combine a CUL4^Ddb1-sensitive hCDT1 probe and an APC^Cdh1-sensitive hGMNN probe, which create a distinct fluorescent profile from Fucci(SA) based on the degradation of hCDT1. The grey Cy motif denotes the RRL to AAA change in this probe, which renders the Cy motif nonfunctional. As above, double-labeled cells are white and negative cells are grey in the combined schematic. Schematics created in BioRender. Glover, J. (2026) https://BioRender.com/37cu02a.

https://doi.org/10.1371/journal.pbio.3004036.g001

H1.0-Fucci(CA)2 construction

To generate pcDNA5-CAG-H1.0-Cerulean-Fucci(CA), a gene fragment encoding the mCerulean fluorescent protein fused to an HA epitope tag and a porcine teschovirus-1 2A peptide (P2A), flanked by 5′ MluI and 3′ HindIII restriction sites, was synthesised by GeneArt (Thermo Fisher). The mCerulean fragment was cloned into pZERO-1 (Thermo Fisher), and the coding sequence of the mouse H1f0 gene (encoding the H1.0 linker histone) was PCR-amplified from mouse cDNA (Table 1) and cloned into the MluI site.

Subsequently, an mCherry-hCDT1(1/100)PIP(+)Cy(−) gene fragment [11], including a 5′ 6× His tag and HindIII, and 3′ NsiI sites, was synthesised by GeneArt. An mVenus-hGMNN(1/110) fragment [11] was PCR-amplified to incorporate a 5′ Myc tag, NsiI, and 3′ AgeI sites (Table 1). These three components were sequentially cloned into a pcDNA5-Flp backbone modified to contain the CAG promoter in place of CMV [15]. The fidelity of all clones was checked by sequencing.

To generate pB-H1.0-Cerulean-Fucci(CA)2, a polylinker sequence (Table 1) was cloned into pB-H2B-Cerulean-Fucci2a [25] using MluI/PmeI, and the H1.0-Fucci(CA) construct was transferred as an MluI/AgeI fragment.

Transfection into PGCs

To generate H1.0-Fucci(CA)2 PGCs, 2 µg of pB-H1.0-Cerulean-Fucci(CA)2 was co-transfected with 1 µg of piggyBac Hybase vector [26] into a previously derived line of male PGCs (FR2M, Hy-Line birds) using Lipofectamine 2000 (Life Technologies), and grown in FAOT medium [27]. Stably transduced cells were then selected with puromycin (Sigma-Aldrich) and expanded prior to PGC injection.

Generation of FuChi embryos

H1.0-Fucci(CA)2 PGCs were micro-injected into Hamburger–Hamilton [28] (HH) stage 15–16 surrogate iCaspase9 sterile hosts as described previously [29]. Briefly, 1 µl of 25 mM B/B compound (Takara Bio) was added to a 50 µl cell suspension (5,000 PGCs/µl) before 1 µl of this suspension was injected into the dorsal aorta of host embryos within windowed eggs. Eggshells were sealed with medical Leukosilk tape (BSN Medical) and incubated until hatch. Founder F₀ FuChi males were mated with wild-type Hy-Line females to produce heterozygous F₁ FuChi eggs and offspring. Additionally, F₂ embryos were generated from subsequent matings of F₁ adult FuChi males with wild-type Hy-Line females. Both F₁ and F₂ embryos were used for downstream analyses. Chicken experiments were conducted under UK Home Office licence PP9565661 and approved by the Roslin Institute Animal Welfare and Ethical Review Board Committee (study numbers #P305 and #P345). The fertility, viability (of fertile eggs), and transgene transmission rates (determined by fluorescence) were recorded.

Eggs

The FuChi line is maintained in the National Avian Research Facility (NARF), The Roslin Institute, UK. Eggs were incubated vertically in rocking or non-rocking incubators (Masalles) at 37.8 °C. All post-incubation day 14 embryos were treated in accordance with the ASPA 1986 schedule 1 culling methods. Fluorescent, and therefore transgenic, embryos were identified using a Nightsea SFA-RB fluorescence adapter and/or a Zeiss Axio Zoom.V16 stereo zoom microscope.

Cell culture and transfections

DF1 cells were maintained in DMEM (Life Technologies) containing 10% foetal bovine serum (FBS), 2% chicken serum (CS), 1% penicillin/streptomycin. Cells were transfected with either pB-H1.0-Cerulean-Fucci(CA)2 or pB-H2B-Cerulean-Fucci2A [25], together with a vector expressing piggyBac Hybase using Lipofectamine LTX (Life Technologies) as per the manufacturer’s instructions. The cell lines were termed H1.0-Fucci(CA)2-DF1 and H2B-Fucci(SA)2-DF1 in line with Sakaue-Sawano and colleagues [11].

FuChi primary fibroblasts were derived from HH19 limb bud micromasses. Briefly, HH19 embryos were dissected from the egg into DMEM and all limb buds removed. Limb buds were placed in 10× Trypsin (Life Technologies) on ice for 30–40 min, followed by two washes with PBS and removal of the ectoderm with sharpened tungsten needles. Limb bud mesoderm was collected in 100 μl of micromass culture medium (50:50 DMEM/Ham’s F12, 10% FBS, 1% Antibiotic/Antimycotic, 1% L-Glutamine) and dissociated by trituration. 900 μl of cell culture medium was added and the samples were centrifuged at 300 rcf for 2 min. The cell pellet was resuspended in 10 μl of cell culture medium per limb bud. Ten μl drops were added to individual wells of a 6-well glass-bottom plate and incubated for 30 min to 1 hour at 37 °C. Wells were then flooded with micromass culture medium.

To derive FuChi PGCs, 1–2 µl of blood was extracted from HH16 F₁ FuChi embryos, transferred into FAOT medium and cells expanded as previously described [27].

Cell treatments

Prior to flow cytometry analysis, H2B-Fucci(SA)2-DF1, H1.0-Fucci(CA)2-DF1 cells or FuChi PGCs were grown in their regular medium conditions as described above. Cells were then treated for 16 hours under control conditions (regular medium plus DMSO), basal conditions (regular culture medium without FBS and CS for DF1, or FAOT medium without growth factors for PGCs), or nocodazole-treated conditions (regular culture medium containing 100 ng/ml nocodazole; Sigma-Aldrich). Following treatment, adherent DF1 cells were dissociated from the flasks, while PGCs were collected. Cells were centrifuged, put through a 70 µm cell strainer, and stained with DAPI (Sigma-Aldrich) for 30 min. Cells were analysed for DNA content and mCherry/mVenus fluorescence using a BD Fortessa ×20.

Live cell imaging

Primary FuChi limb micromass cultures were grown on glass-bottom culture plates in micromass culture medium and imaged for 72 hours. H2B-Fucci(SA)2-DF1 and H1.0-Fucci(CA)2-DF1 cultures were grown on glass-bottom plates in cell culture medium overnight then imaged for 48 hours. All live imaging was carried out on a Zeiss LSM880 confocal microscope, with z-stack images taken at 10-min intervals. Cell tracking and generation of individual cell montages was carried out using ImageJ/Fiji [30] and the Fucci Tools plugin [31].

Light sheet imaging and analysis

FuChi eggs were obtained from the NARF and stored at 16 °C until the start of the experiments. The eggs were incubated for 1 hour to equilibrate to 37 °C, after which embryos were isolated on filter paper and mounted in a specially constructed imaging chamber and imaged on a dedicated light sheet microscope as described previously [32]. To image the embryos at 10× magnification, two slightly overlapping scans were performed. During the scanning, the surface of the embryo was kept in focus using a dedicated surface tracking algorithm as previously described [32]. Each scan consisted of 2,500–3,000 successive slices taken at 1.84 µm intervals and imaged at 488 and 562 nm excitation. This procedure allowed us to collect whole embryo images at two wavelengths at 8–10 min intervals.

To process the raw image data, the images were transformed into a rectangular coordinate system and the surface was detected using a surface-finding algorithm. The top 30 z slices were projected onto a plane and the nuclei detected in each channel using a custom MATLAB Laplacian of Gaussian (LoG) blob detector algorithm. Nuclei were classified into three classes, those detected exclusively in either the green or red channel and those detected in both, as determined by their co-localisation. For analysis of variation of mVenus-hGMNN (488 channel) and mCherry-hCDT1 (562 channel) expression during the cell cycle in vivo, selected nuclei were traced manually and their intensities in both channels measured. Individual traces were aligned using the sudden breakdown of mVenus-hGMNN and mCherry-hCDT1 at mitosis. The boundary between the embryonic and extraembryonic area at the start of the experiment was derived from the Lagrangian repeller [33,34] and subsequently evolved for all successive timepoints using calculated tissue velocity [32].

Whole genome sequencing

Sequencing was carried out as in Oh and colleagues [35]. Briefly, embryonic day 9 livers were snap-frozen on dry ice and stored at −80 °C. Genomic DNA was extracted from the frozen samples using a Monarch HMW DNA Extraction Kit (New England Biolabs) according to the manufacturer’s instructions. Diluted genomic DNA quality was assessed using an Agilent TapeStation, and concentrations were quantified with a Qubit fluorometer. gDNA from 5 samples was pooled. Library preparation and sequencing was carried out by Edinburgh Genomics. The SMRTbell library was sequenced on a Pacific Biosystems Revio platform (25M ZMW, HiFi mode) using a single SMRT cell. A total of 58 Gb of DNA sequence was generated, with an average fragment size of 19 kb and a mean Phred quality score of Q30. Data was returned in BAM and FASTQ file formats (ENA accession: PRJEB98847). To detect the genomic location(s) of transgene insertion, HiFi reads in fastq.gz format were first aligned against the corresponding transgene sequence using Minimap2 (v2.24-r1122) [36]. In total, 69 of 2,983,364 HiFi reads were mapped. Transgene-mapped reads were retained by Samtools (v1.18 using htslib v1.18) [37]. The output file was converted from BAM to FASTQ and the transgene-mapped reads were realigned using Minimap2 against the paternal haplotype of GRCg7w (white leghorn layer, GCF_016700215.2) [38].

PCR

Insertion sites in chromosomes 1 and 2 revealed by long-read sequencing were confirmed by PCR (see Table 1 for primers) using Phusion polymerase (NEB), and the supplied HF or GC buffers. Detection of transgene insertion on chromosome 1 was carried out with HF buffer with the following conditions: 98 °C/30 s, (98 °C/10 s, 60°C/30 s, 72 °C/30 s) for 33 cycles, 72 °C/5 min. Detection of transgene insertion on chromosome 2 was carried out with GC-rich buffer with the following conditions: 98 °C/30 s, (98 °C/10 s, 64 °C/30 s, 72 °C/30 s) for 30 cycles, 72 °C/5 min. PCR samples were run on 2% agarose gels for assessment.

Protein sequence comparison

Cdt1 and Gmnn protein sequences from various species were downloaded from NCBI and aligned in MegAlign Pro 17 (DNAstar) using the MAFFT algorithm.

Cryo-embedding and paraffin processing

For cryo-processing, FuChi embryos were dissected from the egg, briefly washed in PBS, and fixed with 4% PFA or 10% NBF. Following fixation, embryos were washed in PBS then equilibrated in 15% sucrose / PBS at room temperature, then incubated in 15% sucrose / 7.5% gelatin / PBS at 37 °C until fully equilibrated. Blocks of samples embedded in sucrose/gelatin blocks were flash-frozen in isopentane on dry ice, and stored at −80°C. Ten μm cryosections were cut on a Leica CM1950 Cryostat. For imaging, cryosections were incubated in PBS at 37 °C for 15 min to remove gelatin, before mounting in Prolong Gold (Life Technologies) and imaged on a Zeiss LSM880 confocal microscope.

For paraffin embedding, fixed samples (10% NBF or 4% PFA) were washed in PBS, dehydrated through 70%, 90%, and 100% ethanol before clearing in 1:1 ethanol: chloroform, and a final wash in 100% chloroform. Samples were incubated overnight in paraffin wax at 65 °C prior to embedding. 6 μm sections for immunofluorescent analysis were cut using a Thermo Microtome.

Immunofluorescence and fluorescent RNA in situ hybridisation

Immunofluorescent detection of the epitope tags in the H1.0-Fucci(CA)2 biosensor was carried out on formalin-fixed paraffin-embedded (FFPE) sections of FuChi embryos using anti-6× His Tag (Mouse (1:200), Invitrogen #MA1-21315) and anti-Myc Tag (Rabbit (1:100), Cell Signaling Technology #2278) antibodies, or anti-Myc Tag (#2, Mouse (1:500), Cell Signaling Technology #2276). For FFPE sections, after dewaxing, an antigen retrieval step was performed either in EDTA buffer (1 mM EDTA, pH 8.0), Tris-EDTA buffer (10 mM Tris/1 mM EDTA, pH 9.0) or citrate buffer (10 mM sodium citrate, pH 6) using an Antigen Retriever 2100 (Aptum Biologics), or incubation with 5 µg/ml Proteinase K (Invitrogen AM2546) for 20 min at 37 °C. Of note, we tested two different primary antibodies for each epitope tag, across different antigen retrieval conditions. Our results are summarised in Table 2 below. For immunofluorescence on cryosections, samples were equilibrated to room temperature and then incubated for 30 min in PBS at 37 °C to remove gelatin. After these steps, paraffin and cryosections were treated similarly. Sections were permeabilised with TBS containing 0.1%–0.3% Triton X-100 for 10 min, briefly washed in TBS containing 0.1% Tween 20 (TBST), and blocked in 10% goat serum/TBST for one hour at room temperature. Sections were then incubated overnight at 4 °C in primary antibodies (anti-6× His Tag, anti-Myc Tag, or anti-YAP1 (Rabbit (1:200), Proteintech #13584)) diluted in blocking buffer. Samples were washed in TBST before incubation in fluorescent secondary antibodies (Goat anti-rabbit IgG Alexa Fluor 647 (Life Technologies # A-21245), Goat anti-rabbit IgG Alexa Fluor 546 (Life Technologies #A-11035), Goat anti-rabbit IgG Alexa Fluor 488 (Life Technologies #A-11034), Goat anti-mouse IgG Alexa Fluor 647 (Life Technologies #A-21236), or Goat anti-mouse IgG Alexa Fluor 488 (Life Technologies, #A-11029)) diluted 1:500 in blocking buffer for one hour at room temperature. Samples were washed in TBST, counterstained with DAPI, mounted in Prolong Gold and imaged on a Zeiss LSM880 confocal microscope.

For wholemount immunofluorescence, FuChi embryos were dissected and fixed in 10% NBF. Samples were washed 3 × 30–60 min in PBS containing 0.5% Triton X-100 (PBTx) with rocking, before blocking in 10% goat serum / PBTx for 2 hours at room temperature with rocking. Samples were then incubated with anti-DAZL antibody (Rabbit (1:250), Abcam, #AB215718) diluted in blocking buffer for 20 hours at 4 °C with rocking. Following primary antibody incubation, samples were washed 5 × 30–60 min in PBTx, before incubation with secondary antibody (Goat anti-rabbit IgG Alexa Fluor 647; 1:200) for 20 hours at 4 °C with rocking. Samples were then washed 5 × 30–60 min in PBTx, counterstained with DAPI (1:2,000 in PBTx) for 1 hour at room temperature, mounted in Prolong Gold and imaged on a Zeiss LSM880 confocal microscope.

Fluorescent in situ hybridisation was performed by hybridisation chain reaction (HCR; Molecular Instruments). Briefly, FuChi embryos were dissected, and immediately fixed in 4% PFA at room temperature for 1 hour protected from light. Samples were rapidly dehydrated (5 min per dilution series) into 100% methanol, stored at −20 °C for 1–2 hours, before rehydration. HCR was then performed on samples following the manufacturer’s standard protocol using PTCH1 and SHH probes designed and ordered directly from Molecular Instruments. After HCR, embryos were either imaged as wholemounts or processed for cryosectioning; both sample types were imaged using a Zeiss LSM880 confocal microscope.

Early embryo culture and imaging

Early embryo cultures were performed as in Chapman and colleagues [39]. Briefly, embryos were collected on filter paper, washed in Ringer’s solution to remove excess yolk, before culturing ventral side up on glass-bottom six-well plate or Petri dish containing a thin layer of albumin/agar (0.3%) mix. Embryos were incubated in a 37 °C imaging chamber, with wet tissue included in the surrounding space and/or unused wells on the plate to ensure humidity, and imaged on a Zeiss LSM880 confocal or Zeiss Axio Zoom.V16 stereo zoom microscope. Drift correction and bleach correction were performed where necessary using Fiji software.

Fluorescence quality assessment

E3 (HH19) embryos were dissected and immediately imaged (0 hours), and at 72 hours. All images were taken with the same fluorescence settings on a Zeiss Axio Zoom.V16 stereo zoom microscope. Samples were fixed in either 4% PFA or 10% NBF for 1 hour to overnight; all fixation durations were included in the analyses, as we observed no differences between fixation durations. Samples were then transferred into either PBS or 70% ethanol, or alternatively stored in PBS or Ringer’s solution throughout.

Image-based determination of cell cycle status in cells and tissues

To parameterise an image-based approach to analyse cell cycle status in cells and tissues, we used data from FuChi-derived PGCs. Briefly, following flow cytometry of FuChi PGC lines under different conditions, a sample of PGCs from each line and treatment group was mounted on slides and imaged using a Zeiss LSM880 confocal microscope. Using the Fucci Tools plugin for ImageJ/Fiji [31], fluorescence intensity values were obtained from imaged cultured PGCs (three independently derived lines; three culture conditions) and benchmarked against flow cytometry results from matched samples. Values for all cells were plotted as scatter plots, using min-max normalised fluorescence values for mCherry and mVenus, calculated for each channel as (𝑥 − 𝑥_min)/(𝑥_max − 𝑥_min), where 𝑥 is the fluorescence value of an individual cell and 𝑥_min and 𝑥_max are the minimum and maximum fluorescence values of the population, respectively. Using the percentages of PGCs in each cell cycle phase as indicated by flow cytometry analysis, we determined empirical boundaries for G1 (mCherry >0.1, mVenus <0.1), S (mCherry <0.1, mVenus >0.1), and G2/M (mCherry >0.1, mVenus >0.1). Cells below both thresholds (mCherry <0.1 and mVenus <0.1) were classified as double-negative and excluded from cell cycle quantification. These boundaries were then used in all subsequent analyses of cell cycle phase in blastodermal cells, presomitic mesoderm (PSM), neural tube, intestine, and migrating germinal crescent PGCs and their neighbouring somatic cells. Images were acquired in 16-bit format, except for the migrating PGC analyses, which were performed using 8-bit images. Imaging settings were selected to avoid pixel saturation prior to fluorescence quantification.

Statistical analysis

All statistical analysis was performed using GraphPad Prism 11. For comparing the cell-cycle populations between apical and basal neuroepithelial cells in the neural tube, multiple paired t-tests with Holm–Šídák correction were used. For comparison of cell-cycle populations between duodenum and colon tissues, unpaired two-way ANOVAs were performed separately for epithelial, mesenchymal and muscle datasets, followed by Šídák’s multiple comparisons tests. Unpaired t-tests, two-tailed and with Welch’s correction, were used to compare E9 and E18 FuChi and wild-type embryo weights. To compare fluorescence levels between fixation and storage conditions, two-way repeated-measures ANOVAs followed by Tukey’s multiple comparisons tests were performed. Details of the specific statistical tests used are also included in the relevant figure legends, along with the number of replicates analysed.

Design of the H1.0-Fucci(CA)2 biosensor

To generate a Fucci-expressing chicken line that overcomes the limitations of previously described Fucci systems, we first aimed to design an optimal Fucci biosensor for chicken. Fucci(SA) sensors (Figs 1B, 1C, 2A, and 2B), the most commonly used Fucci pair, incorporate a truncated form of human CDT1(30–120) that undergoes degradation via the SCF^Skp2 pathway through its Cy motif. While this system functions in mammalian models [12,13,17,40], the Cy motif is not well conserved in non-mammalian species (S1A Fig) and does not function in zebrafish [18], suggesting that it may not function efficiently in chicken. By contrast, Fucci(CA) systems [11] (Figs 1B, 1D, 2A, and 2B), use hCDT1(1–100) with an alternative N-terminal PIP box, which is targeted for degradation via the CUL4^Ddb1 pathway and is well conserved across species (S1A Fig). The regulation of GMNN, the counterpart in both Fucci pairs, is also highly conserved [18] (S1A Fig). This conservation, combined with the additional advantage of S-phase discrimination offered by the PIP box–based CDT1 sensor, suggested that a Fucci(CA) pair would be the best choice for avian species.

Neither Fucci(CA) nor Fucci(SA) discriminate between G2 and M phases and the existing animal models (which are based on Fucci(SA)2) are unable to track cells through early G1 [11]. A tricistronic construct, H2B-Fucci(SA)2, has been described which enabled M-phase discrimination and tracking through early G1 by labelling DNA with an H2B-Cerulean fusion protein [25]. Similarly, the Fucci4 system, although composed of individual constructs and so not useful here, used a fusion with the H1.0 linker histone to achieve the same effect [20]. However, toxicity associated with H2B fusions has also been reported in vivo [41]. Therefore, we selected the H1.0 linker histone fused to mCerulean (optimally spectrally separated from mVenus and mCherry) for use in our chick model. As a final optimisation step, we added HA, 6×His and Myc tags to the H1.0-Cerulean, mCherry-hCDT1(1/100)PIP(+)Cy(−) and mVenus-hGMNN(1/110) components, respectively, to aid in antibody detection in fixed tissues. S1B Fig shows our optimised tricistronic H1.0-Fucci(CA)2 biosensor.

Faithful discrimination of G1, S, G2 and M phases in chicken cells

We compared the utility of our new H1.0-Fucci(CA)2 biosensor against the previously described H2B-Fucci(SA)2 biosensor [25] by using the piggyBac system to stably integrate each of the biosensors (S1B and S1C Fig) into immortalised chicken DF1 fibroblasts. We found that H1.0-Fucci(CA)2 accurately discriminated cell cycle stage, with similar degradation profiles to Fucci(CA)2 mammalian models [11]. This achieved a clear discrimination between G1, S and G2/M populations and a faithful response to cell cycle perturbations (Figs 2C–2F, S1D, and S1F; S1 Video). However, with H2B-Fucci(SA)2 we identified an extended hCDT1 degradation curve, resulting in persistence of mCherry beyond the G1/S transition (Figs 2G–2J, S1E, and S1F; S2 Video), making cell cycle phase difficult to determine accurately. Both the H2B-Cerulean and H1.0-Cerulean fusions resulted in bright nuclear labelling throughout the cell cycle, including early G1, with sharp peaks in fluorescence intensity as the chromosomes condense in M-phase (Figs 2C, 2E, 2G, and 2I). These results establish H1.0-Fucci(CA)2 as a superior cell cycle probe capable of functionally discriminating all cell cycle phases in chicken cells.

H1.0-Fucci(CA)2 expressing “FuChi” chickens

We used the piggyBac system [42] to generate stably integrated transgenic PGCs expressing the H1.0-Fucci(CA)2 cell-cycle biosensor (Fig 3A). After transplantation of these PGCs into sterile recipient embryos [29], founder F₀ males were bred with wild-type Hy-Line hens to generate heterozygous F₁ (FuChi – “Fucci Chicken”) embryos for analysis (Figs 3A and 3B). FuChi chickens demonstrated fertility, viability and transgene transmission rates of 88%, 92% and 47%, respectively (Fig 3C). FuChi embryos demonstrated strong H1.0-Fucci(CA)2 transgene expression with no observed lethality across all developmental stages (Figs 3B and S2A), were comparable to non-transgenic embryos by visual assessment and by weight as an overall estimate of size (Fig 3D), could be successfully hatched (S2B Fig), and were fertile. Of note, hatched FuChi chicks are initially smaller than wild-type counterparts but do grow at comparable rates (S2C Fig). However, some adult FuChi birds showed poor body condition and required euthanasia, suggesting adverse effects of the H1.0-Fucci(CA)2 transgene and/or consequences of random reporter integration via piggyBac transposition. Mapping H1.0-Fucci(CA)2 integration sites in a pool of FuChi embryos identified insertions on chromosome 1 (NC_052573.1:139,895,885–139,918,586) and chromosome 2 (NC_052574.1:57,700,736–57,701,209). Most embryos analysed carried one of these insertions; however, genotyping of additional embryos indicated that at least one further insertion event was present but was not represented in the sequenced embryo pool (S2D and S2E Fig). We did not observe differences in FuChi reporter expression levels between embryos, regardless of insertion site.

To further investigate the operation of the H1.0-Fucci(CA)2 construct in our FuChi chickens, we derived PGCs and limb micromass cultures from FuChi F₁ embryos for flow cytometry analysis and confocal imaging, respectively. The temporal fluorescent profiles of the H1.0-Fucci(CA)2 biosensor in micromass cells closely matched those of our H1.0-Fucci(CA)2-DF1 cells (Fig 2D) with clear G1, S, G2/M populations identifiable through Fucci(CA)2 biosensor abundance with labelling of nuclei by H1.0-Cerulean throughout the cell cycle and a strong H1.0-Cerulean peak evident during M-phase (Fig 3E and 3F; S3 Video). Flow cytometry analysis of three independent PGC lines (two male, one female) confirmed the fidelity of the reporter in both control and experimental culture conditions in which cell cycle progression was perturbed (Figs 3G and S2F). Taken together, these results establish FuChi chickens as the first viable stably expressing avian cell cycle biosensor model for in vivo studies.

Monitoring cell cycle progression throughout chicken embryonic development

Having validated H1.0-Fucci(CA)2 functionality in cell lines derived from FuChi embryos, we next assessed reporter activity during embryonic development. In HH1 embryos, mVenus-hGMNN-positive proliferating cells were abundant throughout the blastoderm, with many cells co-labelled with mCherry-hCDT1 and mVenus-hGMNN, indicating G2/M phase (Fig 4A and 4B). To quantify these patterns, we applied an image-based cell-cycle classification approach benchmarked against flow cytometry of FuChi-derived PGCs (S3 Fig; see Materials and methods). This analysis showed that the majority of blastodermal cells were in either S phase (29.9%) or G2/M phase (58.5%), consistent with their entry into diapause during lower-temperature storage prior to incubation [43]. During gastrulation, primitive streak cells were strongly enriched for mVenus-hGMNN, indicating high proliferation (Fig 4C and 4D), consistent with previous BrdU labelling studies in similarly staged embryos [44]. At the anterior primitive streak, Hensen’s node showed distinct mCherry-hCDT1/mVenus-hGMNN co-labelling, in line with reports that the node contains G2/M-phase cells (Fig 4C) [45]. In contrast, extraembryonic regions, including the area opaca, contained more G1-positive nuclei (Fig 4C). By HH6, static and live imaging showed that the forming notochord and developing extraembryonic regions were enriched for G1 cells, whereas much of the embryo remained mVenus-hGMNN-positive during axis elongation, with more extensive co-labelling in regions such as the head fold and Hensen’s node (Fig 4E and S4 Video). Live imaging of head fold formation (S5 Video) further showed co-labelled mCherry-hCDT1/mVenus-hGMNN nuclei within the forming head fold, indicating that cell-cycle progression and cell movement occur together during head fold morphogenesis. At later stages, reporter expression was detected across embryonic and extraembryonic tissues, with high mVenus-hGMNN identifying highly proliferative regions including the tailbud, neural tube and developing brain (Fig 4F).

Static and live imaging of somitogenesis showed strong mVenus-hGMNN expression in the PSM before somite formation (Fig 4G and S6 Video). Quantification confirmed that most presomitic mesodermal cells were in G2/M (51.2%) or S (17.1%) phase (Fig 4G). As somites formed, proliferation was evident in both the surrounding epithelial cells and the mesenchymal cells undergoing mesenchymal-to-epithelial transition (Fig 4H and S6 Video). During subsequent somite differentiation, mCherry-hCDT1 expression emerged as a central band within each somite. At HH18, this pattern was restricted to more mature anterior somites (Fig 4F), but was present in all somites at later stages (Fig 4I). A similar switch in Fucci reporter expression has been reported in somites of Fucci2 mouse embryos [46]. At mid-developmental stages, limb buds and regions of the developing brain are proliferative, as indicated by mVenus-hGMNN expression, whereas the lens, condensing cartilage of the limb digits, and feather condensates of the skin were enriched for G1 cells (Fig 4I–4K).

We next examined reporter activity across tissue layers and organs, including at later developmental stages. In the neural tube, where cell-cycle-dependent nuclear migration and mitoses at the ventricular surface of the neuroepithelium are well characterised [47], G1 was the largest cell-cycle fraction in both apical and basal neuroepithelial cells, with a higher proportion in basal cells (73.2%). Conversely, apical cells showed relative enrichment for S phase (27.9%) and G2/M (24.9%) compared with basal cells (17.2% S phase; 9.6% G2/M) (Fig 5A). Using H1.0-mCerulean to identify mitotic cells, we observed divisions oriented perpendicular to the dorso-ventral neural tube axis (Fig 5B), as previously described [48,49]. These mitotic cells also showed the expected cytoplasmic mVenus-hGMNN signal during M phase (Figs 1D, 3E and 5B).

In the HH18 eye, the developing lens showed mixed reporter expression, with mVenus-hGMNN-positive cells concentrated centrally and mCherry-hCDT1-positive cells enriched at the periphery (Fig 5C), similar to previous analyses at comparable stages [50]. Cryosections of FuChi retina showed a high proportion of cells in S and G2/M, consistent with the retina being a highly proliferative tissue during vertebrate development [51]. Mitotic figures were often oriented perpendicular to the retinal surface (Fig 5D), consistent with previous data [52]. In other tissues examined at mid-developmental stages, we found the neuroepithelium of developing brain regions displayed high levels of proliferation as previously described (Fig 5E) [53], while the bones of the wing and surrounding muscle contained mostly cells in G1 (Fig 5F).

Finally, we analysed cell cycle profiles in the developing intestine of FuChi embryos across multiple stages (Fig 5G). Before villus formation begins, mVenus-hGMNN-positive proliferating cells are distributed throughout the epithelium and surrounding mesenchyme of the HH29 intestine. As development progresses, luminal ridges transition through a zig-zag phase to form villi, shaped by the surrounding muscle layers [54]. While mVenus-hGMNN-positive cells are present at HH35, by HH38 the muscle layers are predominantly mCherry-hCDT1-positive, indicating enrichment for cells in G1. In the epithelium, proliferative cells become restricted to the villus bases/intervillus regions at later stages, a consistent occurrence across species thought to confine the presumptive stem cell populations to this region [54– 56] (Fig 5G). The mesenchyme contains a mixture of mCherry-hCDT1- and mVenus-hGMNN-labelled nuclei across stages (Fig 5G).

We then compared the duodenum and colon in HH35 FuChi embryos. In chicken, the small intestine will eventually form villi (Fig 5G and 5H), whereas the large intestine forms smaller-scale structures known as cuffs (Fig 5I) [57]. Although the overall reporter profiles were similar, cell-cycle distribution differed significantly between duodenum and colon in the mesenchyme (p = 0.0019) and muscle layers (p < 0.0001), but not in the epithelium. Specifically, the mesenchyme of the duodenum contained fewer cells in G1 than the colonic mesenchyme (47.0% versus 60.5%; p = 0.038) and more cells in G2/M (30.0% versus 14.4%; p = 0.014). In contrast, the muscle layers of the duodenum contained more cells in G1 than the corresponding layers of the colon (41.3% versus 32.4%; p = 0.0038) and fewer cells in G2/M (49.6% versus 60.6%; p = 0.0006). Most notably, epithelial cells at the tips of the forming luminal ridges in the duodenum showed spatially restricted mCherry-hCDT1 expression (Fig 5H; observed in 4/4 intestines analysed), whereas cells at the interridge base were often mVenus-hGMNN-positive. Although similar to the later stage profile (Fig 5G), this pattern suggests that the spatial restriction of proliferation begins several days earlier than previously reported [54].

Together, these results show that the H1.0-Fucci(CA)2 biosensor intensity profiles in FuChi embryos recapitulate known tissue-specific cell cycle patterns and can reveal unrecognised proliferative organisation during chicken embryogenesis.

Robust H1.0-Fucci(CA)2 stability in fixed tissues and immunodetection after paraffin embedding

Next, to assess the stability and usability of H1.0-Fucci(CA)2 in FuChi tissues, we analysed persistence of the fluorescence signal following common methods of fixation and tissue preparation (S4 Fig). H1.0-Fucci(CA)2 signal was stable following a range of fixation methods compared to unfixed specimens (S4A and S4B Fig), with only a slight increase in autofluorescence observed in the fixed samples (S4B Fig).

While H1.0-Fucci(CA)2 signal can be directly observed in cryosections and fixed wholemount tissue (Figs 4 and 5), we wanted to test whether our reporter embryos could be combined with gene and protein expression analysis. We performed fluorescent in situ hybridisation to detect expression of the hedgehog target gene PTCH1 in the developing embryo (Fig 6A). PTCH1 is expressed in the neural tube, limb bud, and somites, with expression closely correlating with proliferating cells in the neural tube (Fig 6A) [58]. Conversely, SHH expression, which induces PTCH1 expression, is restricted to the notochord and floor plate of the neural tube (Fig 6B) [59]. Staining of FuChi embryonic spinal cord cryosections with a YAP1 antibody (Fig 6C) revealed that expression is localised in the progenitor zone, a region associated with high proliferation and regulated by YAP [60]. Additionally, the inclusion of epitope tags in H1.0-Fucci(CA)2 enabled detection of the reporters by immunofluorescence in formalin-fixed paraffin-embedded (FFPE) sections (Figs 6D and S5). The expression profiles of immunostained FFPE sections were comparable to those seen in cryosections across several tissues, including the brain (Figs 5E, 6D, and S5A), intestine (Figs 5G–5I, S5B and S5D) and neural tube (Figs 5A, 6A–6C, S5C, and S5E).

To further explore the utility of FuChi embryos, we tested whether it was possible to assess the proliferation profile of native migrating cells using immunofluorescence on wholemount embryos (Fig 6E). In chickens, PGCs migrate anteriorly from the centre of the blastoderm, ahead of the forming embryo to populate the germinal crescent during early development. Here, they enter the bloodstream and are translocated to the anterior vitelline veins where they exit the bloodstream and migrate into the genital ridges, before finally populating the gonads [61,62]. However, the proliferative status of PGCs during their initial migratory steps, prior to entering the bloodstream, is unknown. Using our imaging-based cell cycle quantification approach, we found that as PGCs migrate towards the germinal crescent the majority of cells reside in G1 (67.3%), compared to those in S (21.7%) and G2/M (11.0%) (Fig 6F). This differs from both neighbouring somatic cells (Fig 6F) and also from PGCs in culture (Figs 3G, S2F, and S3) indicating that, unlike the rapidly proliferating germ cells in the gonads [63], PGCs at these initial migratory stages are not undergoing frequent cell division.

Real-time visualisation of cell cycle dynamics during gastrulation

A major attraction of Fucci systems is that they can be used to image cell cycle progression, not only in isolated live cells, but also in complex tissues and even whole embryos. Control of the cell cycle plays an important role in the embryonic development of all organisms, including chick. However, so far there is only limited information available on cell cycle changes during avian development. To assess the potential of the H1.0-Fucci(CA)2 system for live imaging we used light sheet microscopy to monitor cell cycle changes from the onset of development after egg laying (Eyal-Giladi and Kochav (EG and K) [64] stage XII) to the primitive streak stage (HH4) (Fig 7A and S7 Video). Embryos were imaged at 10x magnification, allowing visualisation and detection of mVenus (hGMNN) and mCherry (hCDT1) in all nuclei of the surface layers of the embryo at 8–10 min time intervals. To quantitatively assess changes in the distribution of cells in different cell cycle phases during the early stages of development, we measured the total number of nuclei detected in G1, S, and G2/M based on mVenus-hGMNN and mCherry-hCDT1 fluorescence as a function of time in both the embryonic and extraembryonic territories (Fig 7B). This revealed that at the onset of development most cells are in S phase and progress into G2 followed by mitosis and G1 in a partially synchronised manner which persists in a further subset of cells during primitive streak formation (Fig 7B). Next, to characterise the temporal cell cycle dynamics in vivo we measured the kinetics of fluorescence changes in individual cells in the embryonic and extraembryonic regions at the onset of development (Fig 7C, S8–S10 Videos). This analysis revealed that the cell cycle is around 10 hours in both territories (Fig 7D; representative of four independent repeats). The synchronised manner of cells progressing from S through G2 followed by mitosis to G1 from the onset of development was also apparent in the global analysis of cells in the specific regions analysed (S6A Fig).

Finally, the epiblast view showed that during primitive streak formation many cells in the deeper layer of the embryo resided in G1 while migrating away from the streak (S6B Fig). To investigate this in more detail, we performed hypoblast side-up experiments (Fig 7E and S11 Video), which allow better viewing of hypoblast cells and cells emerging from the streak after their ingression. Strikingly, these experiments (three independent repeats) revealed that mesendoderm cells emerging from the primitive streak are in S phase, transition through G2/M, and enter G1 during their migration away; this is particularly evident in cells forming the prechordal plate and anterior mesoderm (Fig 7E and S11 Video). These whole embryo live imaging results demonstrate that the H1.0-Fucci(CA)2 system allows tracking of cell cycle progression in vivo for prolonged periods of time. Importantly, it highlights the dynamic cell cycle changes that cells undergo during key morphological events such as gastrulation. The significance of these findings, including the extent to which cell cycle progression influences cell fate, will be elaborated in more detail in future experiments on FuChi embryos.

S1 Fig. Comparison and validation of Fucci reporter systems in chicken cells in vitro.

A) Comparison of the PIP box (targeted by CUL4) and Cy motif (targeted by SCF) degradation domains of CDT1 and the destruction box (targeted by APC) of GMNN across vertebrate species. While the destruction box of GMNN is well conserved in mouse, human and chicken, as are the core residues of the PIP box (Ψ represents any moderately hydrophobic amino acid (L, V, I, or M); Ω is an aromatic residue (Y or F)), the Cy motif of CDT1 is not well conserved across species. B and C) Design of piggyBac Fucci reporter constructs. B) The H1.0-Fucci(CA)2 reporter cassette uses a CAG promoter to drive expression of H1.0-mCerulean, mCherry-hCDT1(1/100)PIP(+)Cy(−), and mVenus-hGMNN(1/110) fusion proteins, each containing an epitope tag (HA, 6× His, or Myc), and which are upstream of the puromycin resistance gene. C) The H2B-Fucci(SA)2 reporter cassette expresses H2B-mCerulean, mCherry-hCDT1(30/120)PIP(−)Cy(+) and mVenus-hGMNN(1/110) fusion proteins, upstream of the puromycin resistance gene. ITR = Inverted terminal repeats. The presence or absence of the PIP domain and Cy motif of human CDT1 proteins are denoted in parentheses. D) Extended time-lapse images showing two full cell cycles of H1.0-Fucci(CA)2 expressing DF1 cells. E) Extended time-lapse images showing two full cell cycles of H2B-Fucci(SA)2 expressing DF1 cells. F) Comparison of Fucci(CA)2 vs. Fucci(SA)2 reporter systems in chicken DF1 fibroblasts. In the absence of serum, an increased proportion of cells are observed in G1 and S phase through DNA content analysis using flow cytometry. This shift is accurately captured in the fluorescent profiles of H1.0-Fucci(CA)2 but not H2B-Fucci(SA)2 cells. For comparison, control conditions are shown in Fig 2F and 2J. Schematics created in BioRender. Glover, J. (2026) https://BioRender.com/37cu02a.

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S2 Fig. Further characterisation of the viability and functionality of FuChi chickens.

A) Table showing fertility, viability and transgene transmission rate of FuChi embryos across various developmental stages. B) FuChi chicks can be hatched, are viable, and reporter expression can be detected. C) Hatched FuChi chicks are smaller than wild-type chicks from the same clutch but gain weight at a similar rate. D) Mapped transgene integration sites in FuChi chickens based on analysis of a pool of five embryos. E) Percentage of chromosome integration sites identified by sequencing present in FuChi embryos. At least one additional transgene insertion is present that was not represented in the embryo pool analysed in (D). In total, 87 embryos were analysed at two developmental stages (HH19; n = 32; HH35; n = 55). F) Validation of H1.0-Fucci(CA)2 reporter system by flow analysis in two independent PGC lines derived from FuChi embryos in both control, growth factor-removed, and nocodazole (Nz)-treated conditions. The reporter profiles accurately reflect DNA content as determined by DAPI staining in both control and experimental conditions. Results from a third PGC line (#2) can be seen in Fig 3G. Schematics created in BioRender. Glover, J. (2026) https://BioRender.com/37cu02a. The underlying numerical data for this figure is detailed in S1 Data.

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S3 Fig. Parameterisation for image-based analysis of the H1.0-Fucci(CA)2 reporter in FuChi cells in vitro and in vivo.

A) Representative confocal images of PGCs cultured under different conditions used for analysis of cell cycle stage. The same experimental cells that were analysed by flow cytometry (Figs 3G and S2F) were used. B) Scatter plot of relative mCherry and mVenus values from image analysis. Gates for each cell cycle are marked. Pink, green and blue dots represent cells from three independently derived FuChi PGC lines. C) Quantification of cell cycle phase in PGCs by image-based and flow cytometry methodologies. Three independently derived FuChi PGC lines were analysed, with 100 PGCs analysed for image-based quantification. Nz = nocodazole. Scale bars = 50 µm. The underlying numerical data for this figure is detailed in S1 Data.

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S4 Fig. Reporter fluorescence quality post-fixation.

A) Example images of FuChi or wild-type embryos which were fixed and/or stored in each medium condition as annotated and imaged at 0 and 72 hours to detect H1.0-mCerulean (mCerulean), mCherry-hCDT1 (mCherry) or mVenus-hGMNN (mVenus) expression. B) Fluorescent reporter levels at 0 and 72 hours in FuChi and wild-type embryos following fixation and/or storage. Fixation in 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA) stabilises H1.0-Fucci(CA)2 reporter expression while reducing the increase in autofluorescence levels observed without fixation. Error bars represent S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001; two-way repeated-measures ANOVAs followed by Tukey’s multiple comparisons tests. Scale bars = 500 µm. The underlying numerical data for this figure is detailed in S1 Data.

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S5 Fig. Detection of cell-cycle reporter profiles by antibody staining in paraffin-embedded tissues.

A) Immunofluorescent staining of formalin-fixed paraffin-embedded (FFPE) brain sections detecting the His-tag, fused to mCherry-hCDT1, and the Myc tag, fused to mVenus-hGMNN. EDTA-based antigen retrieval enabled specific detection of both tags, revealing the proliferative profile of the brain tissue. Proliferative cells (green) were largely restricted to the neuroepithelium (Ne) and choroid plexus (Cp), consistent with native reporter expression observed in cryosections of FuChi embryos. B) Antibody staining of FFPE intestinal sections from FuChi embryos revealed an expression profile similar to that observed in FuChi intestinal cryosections, with proliferative cells mainly localised to the epithelial layers. Asterisks indicate non-specific staining. C) Antibody staining of FFPE neural tube sections detected the characteristic proliferative profile of the neural tube: apical cells were predominantly in S/G2/M phase and appeared green, whereas basal cells were predominantly in G1 phase. D) Proteinase K treatment provided an alternative antigen retrieval approach for detecting His-tag expression in FFPE sections; however, co-staining with the Myc antibody used in panels A–C was not compatible due to loss of Myc signal with proteinase K treatment. E) Although not compatible with His-tag co-staining, a second primary antibody was identified for detection of the Myc tag in FFPE FuChi sections, producing strong staining across antigen retrieval conditions. Further details of staining conditions are provided in Table 2. Scale bars, 50 µm.

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S6 Fig. Cell cycle analysis in developing FuChi embryos using light sheet microscopy.

A) Cell counts of selected embryonic and extraembryonic regions of the FuChi embryo visualised by light sheet microscopy across time in Fig 7C. All cells detected in the analysed regions were categorised based on their fluorescence profile at the time interval; mCherry-hCDT1-positive (G1; magenta), mVenus-hGMNN-positive (S; green), or positive in both channels (Late S, G2/M; black). These results are representative of four independent repeats. B) 3D epiblast (Ep) view of a HH5 FuChi embryo at two successive time points 6 hours apart. Four optical sections (i–iv) taken from anterior (Ant.) to posterior (Pos.) are also shown at positions indicated by white lines. Note the difference in cell cycle status (G1) of cells egressing from the primitive streak (Ps) (arrows). Scale bars as annotated. The underlying numerical data for this figure is detailed in S1 Data.

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S1 Video. Imaging of DF1 cells expressing the H1.0-Fucci(CA)2 biosensor.

These cells express H1.0-mCerulean (cyan), mCherry-hCDT1(1/100)PIP(+)Cy(−) (magenta), and mVenus-hGMNN(1/110) (green) fusion proteins. Images were captured every 10 min by confocal microscopy. Scale bar = 50 µm.

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S2 Video. Imaging of DF1 cells expressing the H2B-Fucci(SA)2 biosensor.

These cells express H2B-mCerulean (cyan), mCherry-hCDT1(30/120)PIP(−)Cy(+) (magenta), and mVenus-hGMNN(1/110) (green) fusion proteins. Images were captured every 10 min by confocal microscopy. Scale bar = 50 µm.

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S3 Video. Visualisation of H1.0-Fucci(CA)2 biosensor expression in limb micromass cell cultures derived from a FuChi embryo. mCherry-hCDT1 (magenta), mVenus-hGMNN (green), and H1.0-mCerulean (cyan) show dynamic expression through cell division.

Images were captured every 10 min by confocal microscopy. Scale bar = 50 µm.

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S4 Video. Live imaging of a developing FuChi embryo from HH4.

Left panel: mCherry-hCDT1 (magenta) and mVenus-hGMNN (green) expression. Hensen’s node (Hn) displays co-expression of both reporters (G2/M), whereas the forming notochord (Nc) is enriched for mCherry-hCDT1 labelling (G1). Right panel: H1.0-mCerulean (cyan) reporter expression. Images were captured every 15 min using an Axio Zoom.V16 stereo zoom microscope. Scale bar = 500 µm.

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S5 Video. Time-lapse imaging of head fold formation in a FuChi embryo.

Left panel: mCherry-hCDT1 (magenta) and mVenus-hGMNN (green) expression. Right panel: H1.0-mCerulean (cyan) reporter expression. Images were captured every 10 min by confocal microscopy. Scale bar = 100 µm.

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S6 Video. Visualisation of somite formation in FuChi embryos.

The initial part of the video shows the sequential formation of somites in a FuChi embryo; left panel: mCherry-hCDT1 (magenta) and mVenus-hGMNN (green) expression; right panel: H1.0-mCerulean (cyan) reporter expression. Scale bar, 100 µm. The second part of the video shows magnified views of individual somites visualised using the mCherry-hCDT1 and mVenus-hGMNN reporters. Images were captured every 15 min by confocal microscopy. Scale bar, 15 µm.

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S8 Video. The dynamic position of selected 330 x 330 µm regions in the embryonic and extraembryonic areas selected for analysis.

The regions (yellow squares) were tracked over time using the velocity field calculated using particle image velocimetry. The time interval between images is 10 min. Scale bar = 500 µm.

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S9 Video. Cutout of a small epiblast region from the embryonic area of a developing FuChi embryo.

Coloured circles indicate detection of mVenus-hGMNN (green) or mCherry-hCDT1 (magenta) fluorescence. Manually tracked cells are outlined by blue squares. Scale bar = 50 µm.

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S10 Video. Cutout of a small epiblast region from the extraembryonic tissue of a developing FuChi embryo.

Coloured circles indicate detection of mVenus-hGMNN (green) or mCherry-hCDT1 (magenta) fluorescence. Manually tracked cells are outlined by blue squares. Scale bar = 50 µm.

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