# Spatiotemporal cellular dynamics of the notochord shape intervertebral disc morphogenesis in the mouse embryo through apoptosis and proliferation

Julie Warin, Estelle Balissat, Pauline Colombier, Lily Paillat, Maeva Dutilleul, Anne Camus

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## ► To cite this version:

Julie Warin, Estelle Balissat, Pauline Colombier, Lily Paillat, Maeva Dutilleul, et al.. Spatiotemporal cellular dynamics of the notochord shape intervertebral disc morphogenesis in the mouse embryo through apoptosis and proliferation. Developmental Dynamics, 2026, pp.1-21. 10.1002/dvdy.70119. hal-05532798

HAL Id: hal-05532798

https://nantes-universite.hal.science/hal-05532798v1

Submitted on 2 Mar 2026

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# Spatiotemporal cellular dynamics of the notochord shape intervertebral disc morphogenesis in the mouse embryo through apoptosis and proliferation

Julie Warin

Estelle Balissat

Pauline Colombier

Lily Paillat

Maeva Dutilleul

Anne Camus

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Nantes Université, Oniris, Inserm, Regenerative Medicine and Skeleton, Nantes, France

**Correspondence**

Anne Camus, Nantes Université, Oniris, Inserm, Regenerative Medicine and Skeleton, RMeS, UMR 1229, 44000 Nantes, France.

Email: anne.camus@univ-nantes.fr

**Present address**

Pauline Colombier, Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA.

**Funding information**

Region Pays de la Loire; French Society of Rheumatology; Institut National de la Santé et de la Recherche Médicale; Centre National de la Recherche Scientifique

**Abstract**

**Background:** The notochord is a midline structure essential for vertebrate embryogenesis, contributing to the development of the nervous system, digestive tract, and vertebral column. In particular, notochord signaling is indispensable for proper patterning and coordinated development of alternating vertebrae and intervertebral discs (IVDs). Later, notochordal cells (NCs) mature and adopt a characteristic vacuolated morphology before giving rise to the core of the forming IVD, the nucleus pulposus (NP). Postnatally, NCs play a pivotal role in maintaining disc integrity through the secretion of specific factors and extracellular matrix (ECM). Despite its importance in disc formation and homeostasis, the morphogenetic mechanisms underlying the notochord's transformation into the NP are insufficiently characterized.

**Results:** We conducted a comprehensive histological and immunohistochemical analysis to investigate the cellular events governing NP formation in the mouse developing spine. Temporal analysis of intracytoplasmic vacuole formation using Lamp-1 marker revealed that vacuolation contributed to NP growth, while cell density progressively decreased. In addition, quantitative analyses demonstrated a notable proliferative capacity within notochordal cells coupled with region-specific apoptotic activity in sclerotome, at future disc sites.

**Conclusions:** This study highlights the intricate balance of cellular proliferation, programmed cell death, matrix remodeling, and vacuolation dynamics as key determinants in shaping the NP along the rostro-caudal axis.

**KEYWORDS**

axial skeleton development, intervertebral disc, morphogenesis, mouse embryos, notochord, nucleus pulposus formation, sclerotome
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# 1 | INTRODUCTION

Notochordal cells (NCs) are a rare cell type present at the midline along the anterior-posterior axis of all chordate embryos. In the early stage, NCs are organized into a mesodermal rod-like structure, the notochord, which, beyond serving a structural function, plays key roles in signaling and in orchestrating the development of surrounding tissues in vertebrates. These include the central nervous system, axial skeleton, and foregut endoderm. Abnormal notochordal development is associated with a variety of birth defects in humans, impacting neural, mesodermal and endoderm derivatives. A substantial number of animal studies have demonstrated the essential role of the morphogen Sonic Hedgehog (SHH), secreted by NCs, in the correct dorso-ventral patterning and differentiation of the neural tube and somites, primarily through its gradient-dependent signaling mechanism that regulates gene expression in target cells.¹,² In somites specifically, once the ventral-medial part differentiates into sclerotome cells, SHH signaling promotes their proliferation and migration around the notochord to form the unsegmented perinotochordal tube of loose mesenchymal sclerotome-derived cells. After their migration is completed, vertebral segmental patterning takes place through sclerotome segmentation, during which cells reorganize to form alternating condensed regions, giving rise to the annulus fibrosus (AF), the fibrous peripheral part of the intervertebral disc (IVD) and loose regions, which develop into the vertebral bodies (VB) of the future vertebral column.²⁻⁶ Genetic studies have demonstrated that NCs contribute to another component of the IVD, the nucleus pulposus (NP). During development NCs regress to finally reside at the center of the disc, forming the highly hydrated part of the IVD where degenerative changes are thought to initiate.⁷,⁸ Once again, SHH signaling is critical for IVD formation, postnatal growth and for maintaining its integrity.⁹⁻¹²

Although the etiology of low back pain is not fully elucidated, it is often associated with aging and degeneration of the disc. NC loss in certain species coincides with the onset of degenerative IVD changes. There is currently no effective treatment for disc degeneration.¹³ This is largely due to a lack of basic knowledge of the molecular and cellular controls of disc differentiation, growth and homeostasis during embryogenesis and at different stages of life. Nevertheless, there is now considerable evidence from in vitro studies that NCs have a significant influence on disc cell homeostasis.¹⁴⁻¹⁶ At present, NCs and their biologically active factors, although promising targets for regenerative or symptom-modifying therapies for IVD disease, involve mechanisms and influencing factors that largely remain to be characterized.

Cell morphology, molecular identity and lineage-tracing experiments have established that the NP develops from the embryonic notochord. However, NCs' cellular changes toward their functional state in the NP, defined by their intrinsic properties and specialized roles throughout developmental stages of skeletal maturation, are insufficiently explored. During early organogenesis, the disc developmental process encompasses the transition from an initial state as notochord primordium (or anlagen) to its mature form of the NP of the adult disc. The transition from notochord to NP involves spatiotemporal changes from a rod-like structure to segmented fibrocartilaginous tissue. Despite descriptive studies, the cellular and molecular mechanisms underlying the transformation of the embryonic notochord into the NP remain poorly understood.¹⁷,¹⁸

To gain insights on the morphogenetic events occurring in the notochord during IVD formation in mice, we performed histological and immunohistochemical analysis to investigate the dynamics of cellular events that orchestrate NP formation, focusing on apoptosis and proliferation. This study also examines in detail cellular vacuolation and extracellular matrix (ECM) formation. These processes were analyzed along the rostro-caudal axis at key developmental stages in IVD formation, where cellular changes and complex tissue rearrangements are particularly pronounced.⁶,¹²,¹⁹⁻²¹

Quantitative analyses reveal a distinct capacity for cellular proliferation in the notochord, alongside evidence of differential apoptotic cell death in the sclerotome at the level of the forming disc. Our results indicate that these morphogenetic events progressively occur following a cranio-caudal pattern during axial development. Altogether, our findings underscore a coordinated spatial and temporal regulation of cell proliferation and apoptosis, along with intracytoplasmic vacuoles formation and ECM remodeling as key cellular processes associated with NP morphogenesis. This study provides a foundation for future investigations into the morphogenesis of the notochord and the NP in genetic mutant contexts, as well as the underlying molecular mechanisms.

# 2 | RESULTS

## 2.1 | Notochord morphological changes during nucleus pulposus development

We first aimed at developing a temporal histological atlas to examine in detail the key cellular events of the notochord developing into the NP found at the core of the adult disc (Figure 1A-O). Both Hematoxylin and eosin (H&E) and periodic acid-Schiff with alcian blue
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<figure><img src="image_6.png" /><figcaption>FIGURE 1 Temporal histological atlas of the notochord and sclerotomes morphological changes during disc development.</figcaption></figure>

(A) Schematic summarizing key steps of notochord transformation into the nucleus pulposus during embryonic and postnatal stages. (B–O). Rostro-caudal oriented sections (top-to-bottom) stained with PAS-Alcian Blue (PAS-AB) and Hematoxylin Eosin Saffron (HES) at E9.5 (B, I), E11.5 (C, J), E12.5 (D, K), E13.5 (E, L), E14.5 (F, M), E17.5 (G, N), and P4 (H, O). Note the distinct fusiform NC enlargement at E13.5. Scale bar = 50 µm. (C'–H'; I'–O') Magnified views of PAS-AB and HES sections. Scale bar = 25 µm. PAS-AB stains nuclei, glycosaminoglycans, and acidophilic structures in blue-purple, blue, and red, respectively. HES stains nuclei, ECM, and cytoplasm in blue-purple, orange, and pink. Stars in D' and K' indicate intercellular space at the notochord midline. (P–U) Immunohistochemistry for collagen VI (COL6) at E11.5–P4 shows dynamic matrix deposition. Note the presence of COL6 in the growth plate at P4 (U). (P', U') Higher magnifications of COL6 staining. Arrows highlight COL6 deposition at the NP periphery. Representative sections of lumbar region have been selected. Scale bar = 25 µm. AF, annulus fibrosus; NP, nucleus pulposus; NTC, notochord; pAF, prospective annulus fibrosus; pNP, prospective nucleus pulposus; pVB, prospective vertebrae; VB, vertebrae.
(PAS-AB) staining were performed on serial histological sections to highlight different features along developmental time from E9.5 to E17.5 and at 4 days post-natal stage (P4). H&E staining, by distinguishing cell morphology and nuclei, helped to reveal the general spatial organization of NCs and previously uncharacterized aspects of their vacuolation, as well as the structural changes of the surrounding tissues, in particular sclerotomes. PAS-AB staining distinguishes the ECM components, and in particular glycosaminoglycan (GAG)-rich regions, which become more prominent as cells differentiate. This staining was useful for highlighting less documented features of the notochord basement membrane, intercellular space formation, and ECM deposition throughout development.

At E9.5 (20–29 somite pairs) the rod-like notochord structure composed of up to three rows of embryonic notochordal cells (eNCs) in diameter lies directly beneath the ventral side of the neural tube (Figure 1B,I; also described in Götz et al., 1995: PMID: 7781042). The formation of the perinotochordal sheath, an acellular laminin- and collagen-rich basement membrane begins around E10.5, concurrently with the migration of the sclerotome mesenchymal cells, which differentiate from the ventral part of the somite, move medially and form the perinotochordal tube around the notochord (data not shown; also described previously²²,²³). At E11.5 (40–45 somite pairs; Figure 1C,J), eNCs are small, tightly packed within the firm perinotochordal sheath into a rod-like structure approximately 20 µm in diameter. The sclerotomal compartment, initially unsegmented, acquires at this stage a metameric organization with marked regions of different cell density clearly visible (Figure 1C',J'). These phenomena of mesenchymal sclerotome condensation or segmentation result in highly condensed areas corresponding to the disc primordium (or anlagen) that will give rise to the inner and outer annulus fibrosus (AF), and less condensed intervening regions of the vertebral primordium that will undergo cartilaginous differentiation to form the VB.²⁴ At E12.5 (50 to 55 somite pairs), chondrogenesis initiation can be observed at the level of the presumptive vertebrae, marking the early phase of the formation of cartilage before endochondral ossification, during which loose sclerotome cells differentiate into chondrocyte producing ECM (Figure 1D). At E12.5, H&E staining reveals the appearance of numerous small vacuoles or vesicles (Figure 1K,K') while PAS-AB staining highlights the formation of GAG-rich matrix intercellular spaces (Figure 1D'). Remarkably, a distinct acellular channel filled with ECM is observed in the center of the notochord at this stage (Figure 1D,D',K,K'). At E13.5 when somitogenesis ends (up to ~65 somite pairs), the transition from the notochord to the NP is observed with

the accumulation of NCs nuclei at the level of the prospective disc (pAF), while a decrease occurs at the level of prospective VB (pVB; Figure 1E,L). It has been suggested that the developing VB exerts a mechanical stimulus on the embryonic notochord that induces the relocation of NCs toward the IVD area creating a characteristic pattern of swellings and constrictions resembling a string of beads in shape.²²,²⁵,²⁶ This process is commonly referred to as the involution or segmentation of the notochord. At this stage a significant enlargement of the vacuoles is observed in the forming NP which grows to approximately 70 µm in diameter (Figure 1E', L'). At E14.5, the NCs have completely disappeared from the area of the forming VB where only basal lamina remained. In contrast, large vacuolated/physaliferous notochordal cells (vNCs) which are distinct from the eNCs persist and expand at the level of the future disc to shape the newly formed NP (Figure 1F,M,F',M'). As the notochord changes to form the NP, PAS-AB staining reveals regions where GAG-rich ECM accumulates, particularly in the future IVD (Figure 1F,F'). At E17.5 (Figure 1G,N,G',N') and P4 (Figure 1H,O,H',O'), the NP has expanded and adopted a flattened cylindrical shape typical of the adult disc. The outer layers consist of concentric rings of cells and belong to the AF. At these stages, the NP consists primarily of vNCs.

Col6a1 gene encodes a key ECM molecule whose expression progressively increases in the IVD from development through postnatal growth and into adulthood.²⁷,²⁸ Immunodetection was conducted to precisely assess COL6 distribution from E11.5 to E17.5 and P4 (Figure 1P–U). COL6 was detected in the notochord ECM, except in the sheath, from E11.5 onward (Figure 1P’–S’). At E17.5 and P4, a marked positive deposition of COL6 was observed at the periphery of the formed NP (Figure 1T',U'). At E11.5, diffuse COL6 staining was detected in pericellular regions of the sclerotomes (Figure 1P,P'). Remarkably, from E12.5 onward, a stronger COL6 signal marked areas of condensed sclerotome and later, the forming AF (Figure 1Q',R–U,R'–U'). This analysis highlighted that this marker delineates the boundaries of pAF throughout maturation, thus changes in COL6 staining can be used to observe the dynamic evolution of the AF structure over time.

Consistently with previous work of Smits and Lefevre, 2003,²⁹ we observed that cell differentiation and morphogenesis of the notochord proceed in a cranio-caudal sequence. This progression stood out clearly on E14.5 longitudinal section where ongoing morphological changes are apparent (Figure 2). Indeed, the disc-like shape of the NP in the most advanced rostral region differs from that observed in more caudal regions, which are characteristic of newly
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<figure><img src="image_9.png" /><figcaption>**FIGURE 3** Immunohistological characterization of the intracytoplasmic vacuoles during disc development. (A–F) Representative immunohistochemistry of lysosomal-associated membrane protein 1 (LAMP1) on mouse histological sections at E11.5 (A), E12.5 (B), E13.5 (C), E14.5 (D), E17.5 (E), and at P4 (F). Rostro-caudal oriented embryo sections (top-to-bottom). Scale bar = 50 µm. (A'–F') Higher magnifications. Scale bar = 25 µm. Nuclei are counterstained with Mayer's hematoxylin. Note that following chondrogenesis initiation, hypertrophic chondrocytes seen at the level of pVB exhibited small vacuole-like structures at E13.5 (C, C'), that were not further analyzed in this study. (G) Representative maximum intensity projection images of co-immunofluorescence for LAMP1 and Caveolin-1 on mouse histological sections at P4 with the NP oriented from anterior (left) to posterior (right). Nuclei and membranes were labeled using Hoechst and Wheat Germ Aglutinin (WGA) respectively. Overlays of the white box in (G), showing vacuole staining types: ■ LAMP1-predominant, ▲ dual LAMP1/CAV1, and * CAV1-predominant. Scale = 50 µm (G), 10 µm (H–H''). (I) Quantification of LAMP1 positive area from E12.5 to P4, normalized by nucleus number per region (NTC or NP). Each dot represents the normalized value of one ROI (NTC or ≥1NP) and middle lines represent median values. (J) Quantification of cellular density from E12.5 to P4, measured as the number of nuclei per 100 µm² of analyzed area (pNP or NP). Each dot represents the value of one ROI (pNP or ≥1NP). Black line indicates median per stage. Kendall's test was calculated across ROI. (K) Total number of nuclei per pNP or NP region at each developmental stage. (L) NP area in µm² across stages. Each dot represents one ROI. Black line indicates the median per stage. AF, annulus fibrosus; NP, nucleus pulposus; NTC, notochord; pAF, prospective annulus fibrosus; pNP, prospective nucleus pulposus; pVB, prospective vertebrae; VB, vertebrae.</figcaption></figure>
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and provides a biologically relevant estimation of the relative expansion of the vacuolar compartment despite marker heterogeneity. A statistically significant increase was observed from E13.5 to E17.5 (Table 1; Figure 3I). While there was only a slight increase between E12.5 and E13.5, a roughly two-fold increase was observed between E13.5 and E14.5 and again between E14.5 and E17.5. In contrast, little difference has been noted between E17.5 and P4. In parallel, quantitative analysis of NCs' nuclei in NP area confirmed that cellular density decreased steadily over time from E12.5 to P4 (Table 1; Figure 3J). Between E12.5 and E14.5, the mean nuclear density decreased from 1.85 ± 0.10 to 0.97 ± 0.05 nuclei/100 µm² in NP area. The density further reduced by half between E14.5 and E17.5 and between E17.5 and P4, reaching 0.45 ± 0.02 and 0.23 ± 0.01 nuclei/100 µm² in NP area respectively. The calculated coefficient indicated a strong, negative association between developmental stage and nuclei density. To further distinguish between cell loss and tissue expansion, we quantified both the total number of nuclei per NP and the NP area across stages (Figure 3K,L). While the number of nuclei increased progressively from E12.5 to P4 (Figure 3K), the NP area expanded even more markedly (Figure 3L), leading to the apparent sharp decrease in nuclear density (Figure 3J). These analyses confirm that the reduction in density reflects rapid NP expansion rather than a loss of notochordal-derived cells.

Overall, this analysis suggests that notochord expansion and its transformation into the NP are driven by the phenotypic maturation of eNCs into vNCs. The formation

of cytoplasmic vacuoles of increasing volume, along with the deposition of GAG-rich ECM in intercellular space, likely contributes to the observed expansion in tissue volume despite the significant reduction in cellular density as development progresses.

## 2.3 | IVD morphogenesis involves apoptotic events following a cranio-caudal pattern

Changes in cell density and viability have been reported for NC following post-natal growth and during adulthood when they gradually disappear from the NP.³⁹ Apoptotic cell death has been observed during abnormal disc morphogenesis in mouse genetic models, is prevalent in models of disc degeneration, and is also occurring in both aging and degenerated human discs.¹¹,29,31,32,40–47 Yet, the description of apoptosis and its significance in disc morphogenesis during development remain unclear. To bridge this gap of knowledge, we assessed the distribution of programmed cell death along developmental time from E11.5 to E17.5.

First, we performed immunohistochemistry to detect active executioner Caspase-3. Cleaved Caspase-3 positive cells were primarily observed at E11.5 and persisted at E12.5, but were very low or absent at E13.5 and at older E14.5 and E17.5 stages (Figure 4A–C and data not shown). Remarkably, Caspase-3 staining was not found in a localized manner in remnant NC at the level of the pVB at E13.5 or E14.5 (Figure 4C and data not shown). The absence of cell death at these stages indicates that

<table><caption>TABLE 1 Quantification of LAMP1-positive area and cellular density across developmental stages from E12.5 to postnatal P4.</caption><thead><tr><th colspan="6">Normalized LAMP1-positive area</th></tr><tr><th>Embryonic stage (Nb of embryos; Nb of sections)</th><th>E12.5 (1; 1)</th><th>E13.5 (2; 2)</th><th>E14.5 (4; 4)</th><th>E17.5 (3; 3)</th><th>P4 (1; 2)</th></tr><tr><th>Nb of ROI (NTC or NP ≥1) analyzed</th><th>4</th><th>16</th><th>35</th><th>30</th><th>15</th></tr></thead><tbody><tr><th scope="row">Mean ± s.e.m</th><td>15.99 ± 1.19</td><td>24.17 ± 1.60</td><td>50.82 ± 2.72</td><td>96.45 ± 3.45</td><td>103.11 ± 11.06</td></tr><tr><th scope="row">Range (min-max)</th><td>14.56-19.53</td><td>14.67-33.61</td><td>13.75-86.21</td><td>66.11-142.79</td><td>40.97-188.44</td></tr><tr><th scope="row">Pairwise Wilcoxon rank-sum test p-value adjusted</th><td>NA</td><td>NA</td><td>E13.5 vs. E14.5<br>1.66 × 10<sup>-8</sup>****</td><td>E14.5 vs. E17.5<br>1.80 × 10<sup>-14</sup>****</td><td>E17.5 vs. P4<br>7.48 × 10<sup>-1</sup></td></tr></tbody><thead><tr><th colspan="6">Cell density per NP region per 100 µm<sup>2</sup></th></tr><tr><th>Embryonic stage (Nb of embryos; Nb of sections)</th><th>E12.5 (1; 1)</th><th>E13.5 (2; 2)</th><th>E14.5 (4; 4)</th><th>E17.5 (3; 3)</th><th>P4 (1; 2)</th></tr><tr><th>Nb of ROI (NTC or NP ≥1) analyzed</th><th>4</th><th>26</th><th>40</th><th>30</th><th>15</th></tr></thead><tbody><tr><th scope="row">Mean ± s.e.m</th><td>1.85 ± 0.10</td><td>1.24 ± 0.06</td><td>0.97 ± 0.05</td><td>0.45 ± 0.02</td><td>0.23 ± 0.01</td></tr><tr><th scope="row">Range (min-max)</th><td>1.73-2.16</td><td>0.82-1.78</td><td>0.59-2.34</td><td>0.33-0.68</td><td>0.18-0.29</td></tr><tr><th scope="row">Kendall's tau coefficient</th><td colspan="5">τ = -0.78, p = 6.86 × 10<sup>-29</sup></td></tr></tbody></table>

Note: ****p<0.0001
apoptosis does not play a significant role in the elimination of NC at the level of the vertebral disc anlagen during NP formation. In contrast, at earlier stages, apoptotic

cells were predominantly observed in the perinotochordal sclerotomes, exhibiting a distinct spatial distribution (Figure 4A,B). We made the hypothesis that these distinct

<figure><img src="image_13.png" /><figcaption>FIGURE 4 Apoptosis contributes to IVD morphogenetic changes following a cranio-caudal sequence. (A–C) Caspase-3 immunohistochemistry at E11.5, E12.5, and E13.5 on rostro-caudal oriented mouse embryo sections (top-to-bottom). Arrowheads mark active Caspase-3⁺ cells. Scale bars = 50 µm. (D) Quantification of mean apoptotic cells per unit at pVB and pAF levels for E11.5 and E12.5. (E) Quantification of number of apoptotic cells per µm² in perinotochordal regions across cervical, thoracic, lumbar, and caudal levels. At E11.5, lumbar and caudal sclerotomes are not yet condensed, thus combined values are presented in caudal sclerotome histogram. At E12.5, sclerotome cells of the pAF are condensed at all axial levels. (F, G) Representative TUNEL staining at E11.5 (F) and E12.5 (G). White arrowheads indicate TUNEL⁺ cells. Embryo sections are oriented from anterior (left) to posterior (right). Dotted line highlights the notochord. Scale bar = 25 µm. (H, I) Representative immunofluorescence staining of TUNEL and total Caspase-8 at E11.5 and E12.5. White arrowheads indicate TUNEL⁺/Caspase-8⁺ cells. Scale bar = 25 µm. (H', I') Higher magnification of dashed boxes. Scale bar = 10 µm. (J) Schematic illustrating the spatial distribution of apoptotic cells at E11.5 and E12.5 as determined by Caspase-3 quantification. NP, nucleus pulposus; NTC, notochord; pAF, prospective annulus fibrosus; pVB, prospective vertebrae.</figcaption></figure>
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patterns of cell death, observed at E11.5 and E12.5, could mark the initiation of NP morphogenesis. To test this hypothesis, the number and distribution of Caspase-3+ cells were quantified using a defined analysis scheme (See Experimental Procedures and Figure S1A,B). The strategy allowed the measurement of the number of apoptotic cells among notochord or sclerotome across various areas of interest, in particular, distinguishing condensed and non-condensed sclerotomes (alternating formation of pVB and pAF). The quantification also allowed us to compare the distribution in the different rostro-caudal regions, i.e., cervical, thoracic, lumbar and/or caudal which could reflect the progression of axial skeleton morphogenesis. Results of the mean and the range of apoptotic cells per categorization are shown in Table 2 and summarized in Figure 4D at E11.5 and E12.5. No significant difference was found in the mean value of Caspase-3+ cells when comparing their distribution in the notochord at pVB and pAF levels. In contrast, Caspase-3+ cells were detected in the perinotochordal area with greater numbers in the sclerotome of the pAF area compared to pVB (Figure 4D). Moreover, apoptotic cells were significantly higher in the sclerotome of the pAF area “bordering” meaning at the level of analyzed notochord segment versus “anterior” and “posterior” categories. Notably, no apoptotic cell was found in sclerotomes cells in parasagittal sections further away from the notochord segment. In a second phase, we examined in more detail the distribution of Caspase-3+ cells in the sclerotome area “bordering” the notochord as well as in notochord itself along the cervical, thoracic, lumbar and caudal regions (Table 3). The results showed that although not significant (Dunn's test), the number of apoptotic cells per surface area at stage E11.5 was the highest at the caudal region. At E11.5, this value corresponds to the combined presumptive lumbar and caudal regions, as the sclerotomes have not yet condensed enough to allow a clear distinction between these two axial regions (Table 3). In contrast at E12.5, sclerotome cells within the pAF are condensed along all axial levels, enabling clear identification. At stage E12.5, no more apoptotic cells were detected in sclerotomes of the cervical region, while only a few were observed in the thoracic region. However, the comparison of the number of apoptotic cells per µm² using Dunn's test showed that apoptosis was statistically higher in the lumbar and caudal regions when comparing the number of apoptotic events across the different rostro-caudal regions. In summary, apoptotic events were observed at both stages, with the overall incidence of apoptosis decreasing across all regions from E11.5 to E12.5 and with distinct variation in apoptosis levels along the rostro-caudal axis. Thus, the quantification revealed that the degree of apoptosis in

<table><caption>TABLE 2 Distribution of Caspase-3-positive cells in pVB, pAF and notochord along the whole axis.</caption><caption>Caspase 3+ cells in pVB, pAF and notochord along the whole axis</caption><thead><tr><th rowspan="2">Embryonic stage (Nb of embryos; Nb of sections)</th><th colspan="2">E11.5 (4;47)</th><th colspan="2">E12.5 (4;55)</th><th rowspan="2">Notochord cells</th><th rowspan="2">Sclerotome cells</th><th rowspan="2">***pAF (n = 542)</th><th rowspan="2">Notochord cells</th><th rowspan="2">NC</th></tr><tr><th>Sclerotome cells</th><th>pVB (n = 329)</th><th>***pAF (n = 276)</th><th>pVB (n = 619)</th></tr></thead><tbody><tr><td>Ant. (n = 96)</td><td>***Border (n = 137)</td><td>Post. (n = 96)</td><td>Ant. (n = 77)</td><td>***Border (n = 129)</td><td>Post. (n = 70)</td><td>Total# (n = 142)</td><td>Ant. (n = 153)</td><td>***Border (n = 305)</td><td>Post. (n = 161)</td><td>Ant. (n = 127)</td><td>***Border (n = 282)</td><td>Post. (n = 133)</td><td>Total# (n = 382)</td></tr><tr><td>Mean ± s.e.m</td><td>0.09 ± 0.09</td><td>0.55 ± 0.19</td><td>0.08 ± 0.07</td><td>0.77 ± 0.37</td><td>5.49 ± 0.92</td><td>1.47 ± 0.45</td><td>0.08 ± 0.04</td><td>0.03 ± 0.01</td><td>0.16 ± 0.06</td><td>0.03 ± 0.01</td><td>0.90 ± 0.25</td><td>2.61 ± 0.64</td><td>0.78 ± 0.21</td><td>0.09 ± 0.04</td></tr><tr><td>Range (min-max)</td><td>0.00–0.50</td><td>0.00–1.27</td><td>0.00–0.50</td><td>0.21–2.67</td><td>1.33–8.20</td><td>1.11–4.0</td><td>0.00–0.26</td><td>0.00–0.11</td><td>0.00–0.79</td><td>0.00–0.17</td><td>0.00–3.25</td><td>0.00–7.90</td><td>0.00–2.33</td><td>0.00–0.52</td></tr></tbody></table>

Note: Dunn's (1964) test of multiple comparisons following a significant Kruskal-Wallis test with a correction to control the experiment-wise error rate were applied to the quantification data to compare pAF with pVB and NTC and Border, with Ant. and Post. categories. P-values adjusted with the Holm method were ***p < .001. # indicate the total mean value for NTC as no significant difference is observed when comparing Caspase-3+ cells distribution in the notochord between pVB and pAF levels. Ant. = pVB and pAF positioned “anterior” to the notochord segment; Border. = pVB and pAF bordering the notochord; Post. = pVB and pAF positioned “posterior” to the notochord segment.
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<table><caption>TABLE 3 Distribution of Caspase-3-positive cells in sclerotomes bordering the notochord along the rostro-caudal axis: cervical, thoracic, lumbar and caudal regions.</caption><caption>Caspase 3+ cells in notochord and sclerotomes at the level of analyzed notochord segment along the rostro-caudal axis: cervical, thoracic, lumbar and caudal regions</caption><thead><tr><th>Embryonic stage<br>(Nb of embryos; Nb of sections)</th><th colspan="2">E11.5 (4; 47)</th><th colspan="2">E12.5 (4; 55)</th></tr><tr><th>Apoptotic cells/µm²<br>(Nb of area with notochord fragment analyzed)</th><th>Notochord</th><th>Sclerotome</th><th>Notochord</th><th>Sclerotome</th></tr></thead><tbody><tr><td>Cervical</td><td>0.00<br>(12)</td><td>2.46 x 10<sup>-4</sup><br>± 1.37 x 10<sup>-4</sup><br>(12)</td><td>3.92 x 10<sup>-5</sup><br>± 2.26 x 10<sup>-5</sup><br>(11)</td><td>0.00<br>(11)</td></tr><tr><td>Thoracic</td><td>3.90 x 10<sup>-5</sup><br>± 1.85 x 10<sup>-5</sup><br>(40)</td><td>3.54 x 10<sup>-4</sup><br>± 2.03 x 10<sup>-4</sup><br>(40)</td><td>2.40 x 10<sup>-5</sup><br>± 1.40 x 10<sup>-5</sup><br>(30)</td><td>3.14 x 10<sup>-5</sup><br>± 1.22 x 10<sup>-5</sup><br>(30)</td></tr><tr><td>Lumbar</td><td>5.52 x 10<sup>-5</sup><br>± 2.56 x 10<sup>-5</sup><br>(29)</td><td>4.30 x 10<sup>-4</sup><br>± 0.51 x 10<sup>-4</sup><br>(29)</td><td>4.65 x 10<sup>-5</sup><br>± 4.19 x 10<sup>-5</sup><br>(11)</td><td>1.02 x 10<sup>-4</sup><br>± 0.20 x 10<sup>-4</sup><br>(11)</td></tr><tr><td>Caudal</td><td></td><td></td><td>7.29 x 10<sup>-5</sup><br>± 5.44 x 10<sup>-5</sup><br>(36)</td><td>2.25 x 10<sup>-4</sup><br>± 0.42 x 10<sup>-4</sup><br>(36)</td></tr></tbody></table>

Note: Dunn's test (1964) for multiple comparisons was applied to the quantification data following a significant Kruskal-Wallis test with a correction to control the experiment-wise error rate. No statistical difference were found between regions except at E12.5 in sclerotomes cells between cervical and lumbar (¹)(p < .01), thoracic and lumbar (²)(p < .05), thoracic and caudal (³)(p < .001) and cervical and caudal (⁴)(p < .001). P-values adjusted with the Holm method. # indicate the number of apoptotic cells relative to the total area of sclerotomes analyzed at the level of the notochord segment in presumptive lumbar and caudal parts of E11.5 embryos as at this stage the sclerotomes of the pAF are not condensed yet.

sclerotome cells near the notochord fragment increased progressively from head to tail as shown in Figure 4E. This progression of cell death in perinotochordal cells was even more marked at E12.5 with statistical differences between regions. This particular distribution, and the variation between stages E11.5 and E12.5, support the hypothesis that a cranio-caudal wave of apoptotic events occurs in perinotochordal sclerotomes. This dynamic cellular process may contribute to disc morphogenesis, potentially playing a key role in AF formation from the sclerotome and indirectly influencing NP formation from the notochord, both progressing in a parallel head-to-tail sequence (Figure 2).

Apoptotic cell death was also detected by TUNEL staining and was consistent with results described for cleaved Caspase-3 immunostaining (Figure 4F,G, for E11.5 and E12.5, and data not shown for E13.5). Remarkably, confocal microscopy with optical sectioning revealed a consistent occurrence of apoptotic cells specifically at the midline of the notochord at E12.5 along the entire rostro-caudal axis, creating a continuous median intercellular channel in the rod-like structure (Figures 4G and S1C for E12.5 TUNEL assay). Note that, contrasting

with the very low cell death detected in the forming axial skeleton from E13.5, extensive cell death remains visible in the most caudal part of the notochord in E13.5 embryos, where its morphology still retains the rod-like aspect characteristic of the E12.5 stage (Figure S1D for caudal part of E13.5 TUNEL assay). Cell death was also clearly observed on embryonic sections stained with both PAS-AB and H&E where notochord exhibits numerous pyknotic nuclei and cytoplasmic eosinophilia suggestive of apoptosis (Figure S1E,F). Altogether, this analysis reveals specific patterns and a temporal shift of cell death within a short window from E11.5 to E12.5. This occurs in the sclerotome localized in pAF areas adjacent to the notochord. During the same periods, the notochord itself consistently showed a pattern of cell death along the midline (summarized in Figure 4J).

Lastly, cell death was assessed by TUNEL in combination with immunofluorescence for the apical initiator Caspase-8, a key mediator of the extrinsic pathway, to determine whether apoptosis in this developmental context is triggered through autocrine or paracrine interactions. The extrinsic pathway that initiates apoptosis is induced by death ligand binding to a death receptor at
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

the cell surface. Of particular interest, the assessment of total Caspase-8 signal showed elevated expression exclusively in sclerotome cells of the pAF areas adjacent to the notochord segment and was found localized in an area of TUNEL positive cells along the rostro-caudal axis of E11.5 and E12.5 embryos. Although activation status cannot be determined, the spatial distribution and transient high abundance of total caspase-8 suggested modulation of the extrinsic apoptosis pathway (Figure 4H,H',I,I').

## 2.4 | NC proliferation dynamics follows a cranio-caudal pattern during IVD morphogenesis

Although our results show that cell death likely plays a role in IVD morphogenesis, it alone cannot account for segmental expansion and constriction of the NCs. Therefore, we investigated whether differential cell proliferation also contributes to this morphogenetic process. First, we assessed the distribution of mitotic cells by performing anti-phospho-histone H3 antibody (pHH3) immunohistochemistry along developmental time.⁴⁸ Phospho-HH3⁺ nuclei were distributed along the full length of the notochord with no clear evidence of differential proliferation observed in NCs located at the level of PAF or pVB (Figure 5A–D). Indeed, qualitative examination of the sections revealed no distinct pattern of actively dividing cells across the embryonic stages analyzed. These results indicate that pHH3⁺ nuclei do not exhibit clustering beyond what would be expected from a random distribution. In particular, even though at E13.5, the reduced cellularity is occurring in the constricted areas of the notochord, replicating pHH3⁺ NCs were still detectable at pVB level (Figure 5C). Based on the overall distribution of the mitotic cell marker in notochord segments, we concluded that differential cell proliferation could not account for the segmental expansion of the NCs at the pAF levels, which begins to emerge at E13.5.

To further investigate the overall proliferative potential of the NCs during IVD morphogenesis, we performed immunohistochemistry for Ki-67 to label proliferative cells (Figure 5E–H). Interestingly, we observed that as early as E11.5, only a subset of the notochord population was stained, indicating the remaining cells had entered a quiescent (G0) state. Moreover, from E13.5 stage, variations in the distribution of the Ki-67⁺ proliferative cell subpopulation became evident. We hypothesized that these distinct patterns of proliferation activity may contribute to NP morphogenesis. To test this hypothesis, the number and distribution of Ki-67⁺ cells were quantified using a defined analysis scheme. The strategy allowed us to count proliferative NCs while distinguishing them into

two categories-“at the level of pVB” or “at the level of pAF”- and to compare their distribution across the four different rostro-caudal regions (Figure S1G). In the early stages, the distribution of Ki-67⁺ cell in the notochord was homogeneous between pVB and pAF categories, with above 50% and 40% of Ki-67⁺ NCs detected at E11.5 and E12.5 respectively, regardless of the rostro-caudal regions (Figure 5I; Table 4). In contrast, during the transition from notochord to NP at E13.5 and E14.5 stages, the percentage of Ki-67⁺ cells progressively increased in notochord segments at the level of pAF compared to the pVB. A difference of 25% and 38% of proliferative cells was observed in pAF areas at E13.5 and E14.5, respectively (Figure 5I; Table 4). At E14.5 stage, the Ki-67⁺ count in pAF was twice higher than in pVB across the entire rostro-caudal axis, suggesting that NCs in the forming disc preferentially retain their proliferative capacity. From E14.5 onward, no remnant NC in the developing VB is observed as the NP is formed. Within the NP structure, the overall proportion of proliferating NCs progressively decreased across the E14.5, E17.5, and P4 stages, with a sharp decline from approximately 80% to 35% after birth (Figure 5I; Table 4). We then examined in greater detail the proliferative capacity of NCs across distinct axial regions, with a specific focus on the transition from notochord to NP at E13.5 and E14.5 (Figure 5G,H,J,K; Table 5). This analysis revealed that the proliferative capacity of NCs at the pAF level was higher than at the pVB level, particularly at the cervical, thoracic and lumbar regions. At E13.5, proliferation was statistically more pronounced in the cervical and thoracic regions whereas at E14.5, it became statistically more prominent in the thoracic and lumbar regions. No statistical difference was observed in the caudal regions, where the NP has not yet formed at these stages, and the notochord retains a comparable morphology to E12.5 (Figure 2).

Our results provide evidence that Ki-67 expression distinguishes patterns of proliferating NCs that collectively contribute to NP formation. They indicate a dynamic shift in Ki-67⁺ cells distribution, aligning with the cranio-caudal morphogenesis process. They also reveal differences in NCs' proliferative capacities between regions of the forming NP and vertebrae. Altogether, our findings support the hypothesis that the spatial and temporal patterns of proliferation within the notochord contribute to the morphogenetic process of NP formation.

## 3 | DISCUSSION

Morphogenesis involves diverse processes that shape embryonic tissues and structures at the origins of the
<figure><img src="image_20.png" /><figcaption>FIGURE 5 Proliferative capacity of notochordal cells contributes to IVD morphogenetic changes following a cranio-caudal sequence. (A-D) Representative immunohistochemistry of phosphohistone 3 (pHH3) on mouse histological sections at E11.5 (A), (N = 3 embryos; n = 6 sections), E12.5 (B), (N = 2 embryos; n = 4 sections), E13.5 (C), (N = 7 embryos; n = 14 sections), and E14.5 (D), (N = 3 embryos; n = 9 sections). (E-H) Representative immunohistochemistry of Ki-67 on mouse histological sections at E11.5 (E), E12.5 (F), E13.5 (G) and E14.5 (H). (I) Quantification of Ki-67⁺ notochordal cells according to their location at pAF or pVB levels along developmental time. After E14.5 the intervertebral disc is formed and the notochord is absent from the area where the vertebrae is formed. (J-K) Quantification of Ki-67⁺ notochordal cells at E13.5 (J) and E14.5 (K) across rostro-caudal levels (cervical, thoracic, lumbar, caudal), and at the pAF or pVB levels. The x-axis indicates the number of sections analyzed. Statistical analysis: Kruskal-Wallis test followed by Dunn's post hoc test (1964) with Holm correction for multiple comparisons. Asterisks indicate significance (*p* < .05; **p** < .001; ***p** < .0001). Embryo sections are displayed in a rostro-caudal orientation (top-to-bottom). Scale bar = 50 µm. NP, nucleus pulposus; ns, non-significant; NTC, notochord; pAF, prospective annulus fibrosus; pVB, prospective vertebrae.</figcaption></figure>

organs. It can involve cell proliferation, apoptosis, migration, rearrangement, cellular orientation, adhesion, and mechanical forces. Here, we highlight tightly linked events of controlled proliferation and apoptosis coordinated during mouse spine development that contribute to notochord expansion and NP tissue specialization in the forming IVD. We showed that significant differences in

proliferation and apoptosis at specific developmental stages contribute to the normal progression of NP morphogenesis and are associated with changes in notochord and sclerotome cell arrangements. Additionally, we showed that these processes are accompanied by the phenotypic maturation of eNCs into mature vNC, marked by prominent cytoplasmic vacuoles and ECM deposition,
<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<table><caption>TABLE 4 Quantification of Ki-67-positive notochordal cells according to the proximity with the prospective annulus fibrosus or prospective vertebrae.</caption><thead><tr><th colspan="8">Percentage of Ki67+ cells in notochord and nucleus pulposus localized at the pVB and pAF levels from E11.5 to P4</th></tr><tr><th rowspan="3">Embryonic stage (Nb of embryos; Nb of sections)</th><th colspan="2">E11.5 (3; 20)</th><th colspan="2">E12.5 (3; 7)</th><th colspan="2">E13.5 (10; 97)</th><th colspan="2">E14.5 (11; 88)</th><th colspan="2">E17.5 (3; 13)</th><th colspan="2">P4 (3; 6)</th></tr><tr><th colspan="2">Notochord</th><th colspan="2">Notochord</th><th colspan="2">Notochord</th><th colspan="2">Notochord</th><th colspan="2">Nucleus pulposus</th><th colspan="2">Nucleus pulposus</th></tr><tr><th>pVB</th><th>pAF</th><th>pVB</th><th>pAF</th><th>pVB</th><th>pAF</th><th>pVB</th><th>pAF</th><th>pVB</th><th>pAF</th><th>pVB</th><th>pAF</th></tr></thead><tbody><tr><td>Nb of unit with notochord fragment analyzed</td><td>7</td><td>8</td><td>9</td><td>10</td><td>24</td><td>27</td><td>21</td><td>24</td><td>NA</td><td>3</td><td>NA</td><td>49</td></tr><tr><td>% Ki67+ cells ± s.e.m</td><td>57.43 ± 5.69</td><td>55.90 ± 4.14</td><td>46.35 ± 3.32</td><td>44.91 ± 4.25</td><td>43.03 ± 3.23</td><td>67.83 ± 3.67</td><td>37.76 ± 3.25</td><td>75.38 ± 3.05</td><td>NA</td><td>79.04 ± 5.44</td><td>NA</td><td>34.54 ± 1.72</td></tr></tbody></table>

therefore sustaining NP morphogenesis despite a progressive decline in cellular density. Our work provides a precise description and quantitative assessments of these specific morphogenetic events, establishing a reference resource for phenotypic analysis in IVD development, in particular in NP morphogenesis, and enabling cross-study comparisons in mice.

During the early developmental phase (E11.5-E12.5; summarized in Figure 6), our analyses revealed a transient activation of apoptosis via the death receptor pathway in the perinotochordal sclerotome, preferentially localized at the level of the forming disc. Quantitative analysis confirmed that cell death follows a temporal cranio-caudal shift. Apoptosis is a highly regulated form of programmed cell death that classically involves the intrinsic (mitochondrial-dependent) and extrinsic (death receptor or Fas-dependent) pathways. Various works have shown that caspases play key roles in controlling cellular shape and tissue remodeling.⁴⁹ The apical Caspase-8 which initiates programmed cell death induced by the extrinsic pathway was detected exclusively in sclerotome cells of the pAF at the proximity of the notochord, following a cranio-caudal sequence at E11.5 and E12.5. One plausible explanation for the localized occurrence of apoptosis in the condensed perinotochordal sclerotome is that it creates space potentially alleviating physical constraints, thereby facilitating the accumulation of proliferative vacuolated NCs which collectively contribute to the formation of the NP. Apoptosis within the condensed sclerotome may be determined by signaling cues involving SHH, WNT, or TGF-β pathways known to be involved in the apoptotic process. Although the origin of the apoptotic stimulus, whether from the notochord or the sclerotomes, remains to be determined, the results suggest that specific spatial cues, such as changes in the extracellular environment, play a critical role in determining where and when a cell undergoes apoptosis. Alternatively, apoptosis between E11.5 and E12.5 may be part of the morphogenetic events promoting cartilaginous inner AF development and possibly eliminating cells with an inappropriate VB or fibrous annulus developmental fate. Our study also highlights that extensive cell death also occurs simultaneously at the midline of the notochord along the entire rostro-caudal axis at E12.5. These apoptotic events appear to lead to the formation of an acellular median channel with no apparent epithelial lining. Concomitantly, with the initiation of vacuolation within the notochord starting from E11.5 along the entire axis, the earliest signs of ECM deposition appear within the newly formed intercellular channel (Figure 6). While apoptosis is likely to play a role in the transformation of the rod-like notochord into a hollowed structure, the precise function of
<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<table><caption>TABLE 5 Quantification of Ki-67-positive notochordal cells localized at the prospective vertebrae or prospective annulus fibrosus level along the cervical, thoracic, lumbar, and caudal regions.</caption><caption>Number of Ki67+ cells in notochord localized at the pVB or pAF level along the cervical, thoracic, lumbar and caudal regions</caption><thead><tr><th rowspan="2">Embryonic stage (Nb of embryos; Nb of sections)</th><th colspan="2">E11.5 (3; 35)</th><th colspan="2">E12.5 (3; 14)</th><th colspan="2">E13.5 (10; 97)</th><th colspan="2">E14.5 (11; 88)</th></tr><tr><th>Notochord</th><th>pVB</th><th>Notochord</th><th>pVB</th><th>Notochord</th><th>pVB</th><th>Notochord</th><th>pVB</th><th>pAF</th></tr></thead><tbody><tr><td>Cervical (Nb of unit with NTC fragment analyzed)</td><td>(20)</td><td>(21)</td><td>(11)</td><td>(12)</td><td>(46)</td><td>(99)</td><td>(11)</td><td>(83)</td></tr><tr><td>% Ki67+ cells ± s.e.m.</td><td>55.53 ± 9.67</td><td>54.15 ± 7.82</td><td>42.36 ± 7.97</td><td>41.76 ± 11.26</td><td>48.60 ± 2.63</td><td>75.33 ± 1.25***</td><td>49.12 ± 2.56</td><td>73.93 ± 1.66</td></tr><tr><td>Total nuclei per unit, Mean ± s.e.m.</td><td>25.21 ± 1.79</td><td>31.00 ± 1.67</td><td>27.64 ± 6.56</td><td>35.42 ± 3.83</td><td>9.88 ± 1.66</td><td>27.21 ± 3.73</td><td>6.03 ± 0.98</td><td>30.59 ± 1.84</td></tr><tr><td>Thoracic (Nb of unit with NTC fragment analyzed)</td><td>(33)</td><td>(36)</td><td>(18)</td><td>(20)</td><td>(49)</td><td>(83)</td><td>(25)</td><td>(156)</td></tr><tr><td>% Ki67+ cells ± s.e.m.</td><td>52.40 ± 7.21</td><td>48.24 ± 11.28</td><td>47.49 ± 6.02</td><td>51.91 ± 5.76</td><td>42.57 ± 1.25</td><td>71.11 ± 1.63****</td><td>27.54 ± 0.44</td><td>73.14 ± 3.53***</td></tr><tr><td>Total nuclei per unit, Mean ± s.e.m.</td><td>16.20 ± 11.70</td><td>24.03 ± 3.36</td><td>46.58 ± 10.51</td><td>39.76 ± 6.54</td><td>13.80 ± 2.20</td><td>24.55 ± 1.29</td><td>5.96 ± 0.80</td><td>33.20 ± 3.53</td></tr><tr><td>Lumbar (Nb of unit with NTC fragment analyzed)</td><td>(13)</td><td>(14)</td><td>(3)</td><td>(5)</td><td>(16)</td><td>(27)</td><td>(25)</td><td>(77)</td></tr><tr><td>% Ki67+ cells ± s.e.m.</td><td>77.57 ± 11.83</td><td>72.31 ± 3.43</td><td>48.67 ± 0.20</td><td>45.65 ± 4.82</td><td>30.16 ± 3.28</td><td>57.97 ± 4.35*</td><td>40.65 ± 1.08</td><td>84.95 ± 2.94****</td></tr><tr><td>Total nuclei per unit, Mean ± s.e.m.</td><td>17.51 ± 5.41</td><td>30.54 ± 3.82</td><td>53.50 ± 10.50</td><td>52.00 ± 16.00</td><td>31.38 ± 5.81</td><td>31.74 ± 2.44</td><td>11.80 ± 2.21</td><td>44.95 ± 2.66</td></tr><tr><td>Caudal (Nb of unit with NTC fragment analyzed)</td><td>NA</td><td>NA</td><td>(2)</td><td>(7)</td><td>(16)</td><td>(22)</td><td>(19)</td><td>(28)</td></tr><tr><td>% Ki67+ cells ± s.e.m.</td><td>NA</td><td>NA</td><td>50.00</td><td>34.66 ± 11.14</td><td>52.03 ± 1.16</td><td>43.58 ± 2.19</td><td>37.13 ± 3.80</td><td>52.86 ± 3.18</td></tr><tr><td>Total nuclei per unit, Mean ± s.e.m.</td><td>NA</td><td>NA</td><td>24.00 ± 0.00</td><td>51.04 ± 5.29</td><td>47.33 ± 3.81</td><td>40.79 ± 1.61</td><td>20.22 ± 3.99</td><td>24.03 ± 3.13</td></tr><tr><td>% Ki67+ cells all regions ± s.e.m</td><td>57.43 ± 5.69</td><td>55.90 ± 4.14</td><td>46.35 ± 3.32</td><td>44.91 ± 4.25</td><td>43.03 ± 3.23</td><td>67.83 ± 3.67</td><td>37.76 ± 3.25</td><td>75.38 ± 3.05</td></tr></tbody></table>

Note: Only E13.5 and E14.5 embryonic stages show statistically significant differences. Dunn's test (1964) for multiple comparisons was applied to the quantification data following a significant Kruskal-Wallis test with a correction to control the experiment-wise error rate. No statistic difference was found between pAF and pVB at E11.5 and E12.5. At E13.5, a higher number of Ki-67+ cells is found at the pAF level in the thoracic (***p < .0001) and lumbar (*p < .05) regions. At E14.5, a higher number of Ki-67+ cells is found at the pAF level in the thoracic (***p < .0001) and lumbar (***p < .0001) regions. P-values adjusted with the Holm method.
<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /><figcaption>FIGURE 6 Disc development model. Schematic overview of IVD morphogenesis from E11.5 to E17.5-P4. Major structural changes occur as the NCs accumulate at the future disc sites to form the NP, but are not maintained where the VBs form. Our findings provide evidence for two distinct developmental patterns of morphogenetic events, as illustrated here by thoracic and lumbar levels (see Figure S2 for full rostro-caudal axis). The first pattern (E11.5–12.5) involves a synchronous decrease in cell density, an increase in cytoplasmic vacuole number and size, and ECM deposition over time along the axis. Note the early NC apoptosis that uniformly creates a midline channel (black dash lines) most likely facilitating ECM deposition (yellow). The second pattern (E13.5–14.5) reflects a rostro-caudal progression similar to the one observed in NP morphology with differential apoptosis in sclerotomes (E11.5–12.5) and differential proliferative capacities in NCs located at pAF and pVB levels. Arrows indicate proposed physical cues from pAF apoptosis (straight) and pressure constraint from forming pVB (curved), both guiding NC expansion into pAF, along with the increased ability for NCs to divide in this area. By P4, proliferative NCs remained localized at the NP periphery.37,68,69 ECM, extracellular matrix; pAF and pVB, prospective annulus fibrosus and vertebrae. Created in https://BioRender.com.</figcaption></figure>

this process in normal morphogenesis remains to be elucidated. One possibility is that the physical stress associated with vacuole formation activates the intrinsic apoptosis pathway, leading to inner channel formation. This might be essential for creating space for ECM deposition and remodeling, thereby facilitating cellular rearrangement and phenotypic changes during the notochord-to-NP transition.⁵⁰ Moreover, apoptosis observed in the notochord at E11.5 and E12.5 may also allow intercellular space formation at the level of pVB, facilitating subsequent vertebrae morphogenesis.

Over the late developmental phase (E13.5-E14.5), results indicate that following apoptosis, distinct spatial and temporal patterns of proliferation contribute to localized tissue expansion and differentiation. Quantitative analyses revealed that proliferative cell distribution

within the notochord shifts dynamically, supporting cranio-caudal NP morphogenesis. We hypothesize that an earlier cranio-caudal apoptotic wave in sclerotomes could facilitate NP expansion from the notochord which proceeds in a similar head-to-tail sequence (Figures 6 and S2). Our findings suggest that the dynamic interplay between these cellular processes together with a progressive increase in ECM deposition and intracytoplasmic vacuoles formation and expansion, as well as cell rearrangement, plays a crucial role in continuously shaping the NP along the rostro-caudal axis of the embryo (Figure 6). Consistent with previous reports on the proliferative characteristics of cells of the ventral node and the derived notochordal plate using BrdU labelling from E7.5 to E14.5,25,51–53 we showed here that a significant portion of NCs are quiescent from E9.5 onward. A recently
published study using EdU injection to map proliferation
showed that the enlargement of the NP involves localized
changes in notochord proliferation, prior to hypertrophic
differentiation of the VB region as assessed by Collagen
10 marker.⁵⁴ Supporting this result, our work reveals that
NCs' proliferative capacity appeared not uniform and
suggests regulatory influences on proliferation at later
phases during disc formation (E13.5-E14.5), when the
notochord transitions into the NP. In addition, quantita-
tive analyses revealed that the distribution of proliferative
NCs shifts dynamically in a cranio-caudal direction,
likely contributing to notochord-to-NP morphogenesis
through progressive cell expansion and rearrangement.
Thus, we hypothesize that distinct proliferative dynamics
driven by local signaling occur in specific areas to shape
the NP along the rostro-caudal axis (Figures 6 and S2).
Across developmental stages, we observed that mitotic
activity marked by pHH3 was low and appeared ran-
domly distributed, in contrast to the broader and more
localized proliferative domains marked by Ki-67. This
pattern may reflect that some NCs transiently pause in
G1–G2, or alternatively, that the sparse pHH3 signal sim-
ply reflect the overall rare but ongoing mitotic events,
consistent with the gradual increase in total NCs number
observed during NP development in this study. There-
fore, Ki-67⁺ cells provide a more comprehensive repre-
sentation of the proliferative activity during NP
development and reveal regional differences that pHH3
staining alone does not capture.

Both biochemical and biomechanical signals could
be influencing these distinct proliferative behaviors;
this remains to be elucidated. Extensive crosstalk
between biochemical and mechanical stimuli during
tissue morphogenesis and growth is now well estab-
lished.⁵⁵,⁵⁶ Changes in mechanical force distribution
may create differences that influence cell proliferation
during subsequent development and morphogenesis of
the disc. These could explain that cells located in areas
of forming VBs sense forces, in contrast to cells in areas
of forming NPs, leading to distinct proliferation behav-
iors. A specific phenotype described as “rod-like noto-
chord persistence” has been reported by several
studies, supporting the idea that physical mechanisms
may be responsible for the notochord to NP transition,
forming the basis of the “pressure” model.⁹,²¹,²⁵,⁵⁷,⁵⁸
Mutations affecting *Pax-1* gene expression in the scler-
otome result in a higher proliferation rate of NCs at
E12.5 and “notochord persistence” at the level of the
vertebrae.⁵,⁵⁹ Although the mechanism has not been
elucidated, this phenotype suggests that sclerotomes
signal back to the notochord to control its expansion
strictly at the level of the forming disc. Alternatively,
the Repulsion/Attraction model has been suggested,

where locally expressed attractant/repulsive signals yet
to be determined could explain notochord cell move-
ment toward the forming NP and/or away from the
forming VB.²¹ Evidence that NCs migrate or are rear-
ranged under any type of pressure or discrete biochem-
ical signal remains to be determined during disc
formation. Further investigations are needed to dem-
onstrate that part of these signals or mechanisms exert
influence on the proliferative behavior of the noto-
chord cell as well as on apoptosis patterning within the
perinotochordal sclerotome. In addition, in-depth
mechanistic knowledge on the intricate and bidirec-
tional interactions between the notochord and the
sclerotome is needed to further understand disc
morphogenesis.

So far, mechanisms regulating the balance between
proliferation and death during IVD development
remain largely unknown⁴³,⁶⁰ Previous studies in mouse
have shown the importance of SHH signaling but also
of the integrity of the notochordal sheath for NCs' sur-
vival and NP growth.⁹,²⁹ Noteworthy, the *Jun* gene has
been reported to be involved in the regulation of NCs'
survival at the time of NP formation. Notochord-
specific conditional knockout mutant embryos exhib-
ited increased apoptosis in the notochord and not in
the sclerotome, reduced cellularity of the IVD, and sub-
sequent fusion of the vertebrae resulting in severe mal-
formations of the axial skeleton.⁴⁵ Hippo pathway
activity during spine development also warrants fur-
ther investigation in mice.⁶¹ Genetic analyses have
shown that *Tead1* and *Tead2* transcription factor and
co-activator YAP protein are essential for maintaining
the notochord at E8.5, potentially through the regula-
tion of proliferation and apoptosis.⁴⁶ Furthermore, the
linked timing of apoptosis and vacuolation in verte-
brates' notochords may reflect apoptosis induction
through mechanical stress response to the apparition
of vacuoles as discussed in the “turgor pressure-sheath
strength” model.¹⁹ This later mechanism provides the
notochord with the necessary rigidity for its role as a
skeletal element for lower-vertebrate embryos' develop-
ment.⁶² Previous studies in Zebrafish and Xenopus
have shown a correlation between a disorganized
extracellular sheath, an impairment in vacuolation,
and the regulation of apoptosis during notochord
development and axis elongation.⁶³⁻⁶⁵ More in-depth
functional evaluations are required to elucidate the
role of proapoptotic and antiapoptotic members of the
Bcl-2 family in the notochord along developmental
time in order to demonstrate that inhibition of apopto-
sis would delay or prevent the transformation of the
notochord into NP. Further insights into the regulation
in NC survival will also help our understanding of the
<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

origin and the cause of rare malignant tumors called chordoma.⁶⁶ These have been proposed to develop in the axial skeleton from rare intraosseous remnants and dormant notochord cells with prominent vacuoles.⁶⁷

Molecular and genetics studies should be conducted to investigate changes in cell behavior using in vivo approaches to assess the impact of genetic alterations in animal models on distinct phenotypic traits in spine development. Although research efforts have improved our understanding of the mechanisms that control NC proliferation in IVD at postnatal stages,⁶⁸,⁶⁹ further studies are required during IVD development to determine cell-autonomous and non-cell-autonomous effects. It remains unclear whether notochord constriction at the pVB results from notochord-intrinsic processes or forces imposed by differentiating sclerotomes, ECM remodeling and proliferation within the vertebral primordium. The relative contributions of apoptosis, mechanical compression, cell rearrangements or migration, and cell fate changes warrant further investigations for a complete understanding of axial skeleton development as an integrated system.

Developmental events examined in this study align with conserved features of IVD formation across mammals. Although rodents uniquely retain NCs into adulthood, the early notochord-to-NP transition and the contribution of sclerotome-derived cells follow processes comparable to those described in larger animal models, including human embryo.¹⁴ These similarities support the relevance of the mouse model for understanding the fundamental developmental processes in the window examined. In addition, recent advanced 3D in vitro pluripotent stem cell-based systems modeling axial development in mammals⁷⁰,⁷¹ will allow us to dissect the effects of signaling pathway activity perturbation, particularly in relation to cellular defects affecting notochord and disc formation. Both in vivo and in vitro approaches will enhance our understanding of the molecular pathways regulating NC survival, which is also essential to gain deeper insights into both disease mechanisms and regenerative medicine.

# 4 | EXPERIMENTAL PROCEDURES

## 4.1 | Mice

Experiments on mice were conducted according to the French and 138 European regulations on care and protection of laboratory animals (EC Directive 86/609, French Law 2001-486 issued on June 6, 2001). Embryos and newborns (4 days postnatal, P4) for histology and immunohistochemistry were collected from spontaneous pregnant mice and newborns respectively from CD1-Swiss background.

## 4.2 | Histological staining and immunohistochemistry

Embryos from embryonic day E9.5 to E17.5 and postnatal P4 Newborns were collected and were fixed in 4% paraformaldehyde (PFA, Sigma, P6148) at 4°C for 24 h and embedded in paraffin (VWR, 10048502) for sectioning in the coronal plane. Spine samples of E17.5 and postnatal P4 were decalcified (EDTA, Euromedex, EU0007, 0.5 M pH = 7.5 at 4°C) for 7 days before paraffin embedding. Five µm sections were stained following standard protocol with Hematoxylin Eosin Saffron (HES) and Periodic Acid Schiff/Alcian Blue (PAS-AB).

For immunohistochemistry detections, after antigen retrieval, sections were permeabilized (with Triton X-100, Merck T8787, 0.5% for 20 min) and then the endogenous peroxidase were saturated for 20 min in H₂O₂ 3% (Merck, H1009) in PBS 1X (Vwr, 733-1645). The sections were incubated with the primary antibodies overnight at 4°C after incubation with blocking solution. The next day, the sections were incubated with secondary antibodies conjugated with biotin. Revelation was performed after 45 min incubation in streptavidin-peroxidase solution and of DAB substrate (3,3'-Diaminobenzidine). Nuclei were stained with Mayer Hematoxylin (Diapath, C0305), mounted with Eukitt (Kindler GmbH; B0914) and scanned using a Hamamatsu Nanozoomer HT (Hamamatsu Photonics KK) digital scanner at 40X magnification. TABLE S1 summarizes the dilution and specific buffers for retrieval for each antibody.

## 4.3 | Immunofluorescence and TUNEL assay

Following collection, embryos and mice spines were directly embedded in Super Cryo-embedding Medium (SCEM) (Section Lab) and frozen in isopentane/dry ice, without decalcification and fixation. A specially prepared adhesive film is fastened to the cut surface of the sample in order to support the section while allowing a slow cut. We adapted Kawamoto's Film Method⁷² for the use of a Carbone steel blade. Samples were cut along the coronal plane and 7 µm sections were mounted on slides (CryoStar NX70, Thermo Fisher Scientific) and post-fixed in 2% PFA (2 min) before permeabilization in 0.2% Triton X-100 (in PBS for 10 min and in a blocking solution; 10% Foetal Calf Serum, Sigma, G9023; 4% BSA, Sigma, A9647; 0.1% Triton X-100 in PBS) for 1 h. Next, sections were incubated overnight at 4°C with primary antibodies followed by 1 h incubation in secondary antibody (Table S1). Nuclei were stained by Hoechst 33258 pentahydrate (bis-benzimide) (2ug/mL, Thermofisher; H3569) and sections were mounted with ProLong™ (Thermofisher,
P36934). Frozen sections for TUNEL staining were post-fixed in 4% PFA for 5 min and cellular membranes were permeabilized with Proteinase K (10 µg/mL, Merck P6556) for 5 min. Apoptotic cells were detected using the Dead-EndTM Fluorometric TUNEL system (Promega, G3250). Confocal immunofluorescence images were acquired with LFOV FLIM Nikon confocal microscope.

## 4.4 | Quantification of LAMP1 normalized positive area and cellular density

Quantifications regarding LAMP1-positive area and cellular density were performed on LAMP1 H-DAB images within the open source software QuPath.⁷³ Images with excessively high or weak DAB signal intensity, as well as NP that were partially out of focus, were excluded from analysis. All measurements were confined to Notochord or NP regions of interest (ROIs), generated using the extension “Segment Anything Model (SAM),”⁷⁴ and manually corrected when necessary. For stages E13.5 and E14.5, note that when the forming NP were connected together with remnant NCs at the pVB level, with no visible separation, they were manually segmented to only quantify cells in the NP area (see Figure S1H,H'). At these stages, regions where the notochord remained rod-shaped were excluded from the quantification. Color deconvolution was adjusted manually and applied to all images to separate DAB from hematoxylin staining. Nuclei were automatically segmented on the original RGB image using Stardist⁷⁵ and the “he_heavy_augment” model, with prediction threshold set at 0.5. Detections with median hematoxylin intensity <0.1 or >1.0 were excluded from nuclei count. LAMP1-positive area in µm² was calculated by applying a pixel threshold on the DAB channel. Exported QuPath annotations (ROI and DAB-positive area) and detection counts (nuclei) were analyzed using R (version 4.4.3). For each ROI, cell density was calculated as the total number of detected nuclei divided by the measured ROI area. For normalization, LAMP1-positive area was divided by the number of detected nuclei within the ROI.

## 4.5 | Quantification and distribution of Caspase-3 signal for apoptosis

Following immunohistochemistry using anti-Caspase-3 antibody to detect active Caspase-3 apoptotic cells, high-resolution images were acquired to visualize notochordal segments along the rostro-caudal axis for the quantification. Despite careful sample orientation during paraffin embedding, embryonic histological sections often contain

interrupted fragments of notochord when the cut is slightly off the strict sagittal plane. Specific regions containing fragments of notochord (NTC) or sclerotome of prospective vertebrae (pVB) or prospective annulus fibrosus (pAF) were manually delineated (see Figure S1A). The quantification strategy allowed the measurement of the number of apoptotic cells among notochord or sclerotome across various areas of interest, enabling distinctions between pVB and pAF. The schematic shown in Figure S1A summarizes the distinct areas counted and classified as (i) “Anterior”: including up to two pVBs positioned rostrally relative to the anterior extremity of the notochord fragment, (ii) “Posterior”: including up to two pVBs positioned posteriorly relative to the posterior extremity of the imaged notochord fragment, and (iii) “Bordering the notochord”: defining the pVB and pAF units flanking both extremities of the imaged notochord fragment. Images were analyzed using ImageJ software for region segmentation. For clarity and consistency in spatial classification, colored lines indicated each region of interest on schematic overlays (see Figure S1A). Caspase-3 positive cells were quantified within each region using the CellCounter plugin of ImageJ. A standardized classification of axial levels was applied across the entire rostro-caudal axis of the mouse embryo, allowing the cervical (posterior to the occipital somites; lack of rib; somites 6–12), thoracic (rib-bearing; somites 13–25; level of the heart, lungs, and thymus), lumbar (absence of ribs; somites 26–31; level of the kidneys, pancreas, stomach, and liver), and caudal regions (sacral + tail; somites 32 onward; level of the large intestine and bladder) to be consistently identified and compared on serial sections (Figure S1B). For reproducibility, quantification was repeated across multiple embryo sections and notochord fragments along the rostro-caudal axis (see Tables 2 and 3). Data were compiled and statistical analysis performed to assess spatial distribution of apoptotic cells in these defined embryonic domains.

## 4.6 | Quantification and distribution of Ki-67 signal for proliferation

Following proliferative cell detection via immunohistochemistry using Ki-67 staining, high-resolution images were acquired to visualize notochordal fragments along the rostro-caudal axis. Images were analyzed using ImageJ software for region segmentation. Specific regions containing fragments of notochord seen on the histological section were manually drawn with a dotted line (see Figure S1G). For clarity and consistency in spatial classification, areas of notochord were delineated and colored distinctively according to their location: in blue
<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

notochord located at the pVB level and in orange noto-
chord located at the pAF level = future disc area. Ki-67
positive cells were quantified within each area using the
CellCounter plugin of ImageJ. A consistent classification
was applied across the rostro-caudal axis (Figure S1B).
For reproducibility, quantification was repeated across
multiple E13.5 and E14.5 embryo sections and notochord
fragments along the rostro-caudal axis (see Tables 4 and
5). Data were compiled and statistical analysis performed
to assess spatial distribution of proliferative cells in these
defined embryonic domains.

**4.7** | Statistical analysis

Statistical analyses were performed using R (version 4.4.3) and GraphPad Prism (version 8.0.1). For the distribution of Caspase-3⁺ and Ki-67⁺ cells, multiple groups were compared, a Kruskal-Wallis test was conducted, followed by Dunn's post hoc test (Dunn, 1964) for pairwise comparisons. To control for the experiment-wise error rate, *p*-values were adjusted using the Holm method. Pairwise comparisons of normalized LAMP1-positive area were assessed using the Wilcoxon rank-sum test. To correct for multiple comparisons, *p*-values were adjusted using the Benjamini-Hochberg procedure to control the false discovery rate (FDR). To evaluate the relationship between developmental stage and nuclei density, Kendall's rank correlation test was calculated using individual ROI. Kendall's tau coefficient assesses the strength and direction of the monotonic association, with values >0.8 indicating strong, 0.4-0.8 moderate, and <0.4 weak correlation.

ACKNOWLEDGMENTS

The authors would like to thank A. Henry for the initia-
tion of the project, N. Wyckens for technical advice and
S. Schneider-Maunoury as well as C. Vesque for critical
discussion on the manuscript. We acknowledge
M. Ambroset from the MicroPICell core facility (SFR
Bonamy, BioCore, Inserm UMS 016, CNRS UAR 3556,
Nantes, France), member of the Scientific Interest Group
(GIS) Biogenouest, IBISA, and the national infrastructure
France-Bioimaging supported by the French national
research agency (ANR-24-INBS-0005 FBI BIOGEN),
the SC3M facility from Inserm/Nantes Université/ONIRIS,
UMR 1229 RMeS Laboratory and SFR Francois Bonamy,
and the UTE-IRS-UN Animal facilities staff for animal
care, breeding, and for their investment in the project.
This research was funded by INSERM, CNRS, and by
grants from the Region Pays de la Loire (BIODIV and
RFI BIOREGATE-CAVEODISC) and the French Society
of Rheumatology (SPHERODISC II). The funders had no

role in the design of the study, analyses, or interpretation
of data; in the writing of the manuscript, or in the deci-
sion to publish the results.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

All data supporting the findings of this study are avail-
able within the article and its Supporting Information.

ORCID

Anne Camus https://orcid.org/0000-0002-8820-0407

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Warin J, Balissat E, Colombier P, Paillat L, Dutilleul M, Camus A. Spatiotemporal cellular dynamics of the notochord shape intervertebral disc morphogenesis in the mouse embryo through apoptosis and proliferation. *Developmental Dynamics*. 2026;1-21. doi:10.1002/dvdy.70119