Development of the lumbosacral spinal cord is a critical period of embryogenesis. Not only does motor control and sensation in the legs and lower body depend on this event, but proper functioning of bladder, rectum, and genital organs are all critically dependent on nerves arising from the low spinal cord. A major group of congenital malformations termed neural tube defects result when low spinal neurulation fails to be completed or is otherwise abnormal, and these can be open or closed (skin-covered) lesions.

Open spina bifida (also called myelomeningocele) results from defective closure of the primary neural tube, most often at lumbar and upper sacral levels. Subsequent neuroepithelial damage and loss due to prolonged exposure to amniotic fluid (Stiefel et al., 2007) leads to a defect that is disabling in most individuals (Copp et al., 2015). Closed ‘dysraphic’ conditions arise at lower sacral and coccygeal levels of the body axis and result from disturbance of secondary neurulation, in which the neural tube forms without formation of neural folds. Dysraphic conditions involve an abnormal anatomical relationship between the secondary neural tube and surrounding tissues, often with ectopic adipose tissue, as in spinal lipoma and lipomyelomeningocele (Jones et al., 2019). Closed dysraphism may be asymptomatic, but significant disability can occur through tethering of the low spinal cord to non-neural tissues (Agarwalla et al., 2007).

In normal primary and secondary neurulation, the neural tube forms by closure and canalisation respectively (summarised in Figure 1 of Nikolopoulou et al., 2017), with extensive studies of both processes in experimental animals, especially chick, mouse, and rat. Despite the relative inaccessibility of neurulation-stage human embryos (3–7 weeks post-conception), a number of studies have described the anatomical, histological, and ultrastructural features of secondary neurulation (Supplementary file 1). Although limited molecular research has been performed, e.g., to determine the mode of cell death in human tail regression (Vilović et al., 2006), a few studies have begun to address specific gene expression during human secondary body development (Olivera-Martinez et al., 2012; Yang et al., 2014). Increasingly, transcriptomic approaches are being used with organogenesis-stage human embryos (Fang et al., 2010; Yi et al., 2010; Krupp et al., 2012; Xu et al., 2023).

Caudal development comprises not only formation of the secondary neural tube, but also other tissue types within the ‘secondary body’ region. This part of the body axis – beyond the cloacal plate which marks the future anus – includes the secondary notochord, tail somites, caudal vessels, tailgut, and surrounding surface ectoderm (future epidermis). These structures show marked tissue-to-tissue variation in development. For example, tail regression in human embryos involves loss of all tail components, whereas the rodent tail maintains the somites and notochord, but loses the secondary neural tube and tailgut. The regulation of this balance between maintenance and loss of tail structures is not understood.

A related area of interest is the molecular control of axial elongation. A population of self-renewing stem cells, termed neuro-mesodermal progenitors (NMPs), resides in the caudal-most embryonic region (the tailbud), with NMPs giving rise to neural and mesodermal derivatives, including the secondary neural tube and somites (Henrique et al., 2015). NMP maintenance is required for axial elongation, mediated via an interplay between WNT3A and FGF8 expression, which promotes NMP survival in the tailbud. Conversely, endogenous retinoic acid promotes differentiation and regulates body length (Wilson et al., 2009).

In the present study, we examined caudal development in 108 human embryos, at Carnegie stages (CS) 10–18 (3.5–6.5 weeks post-conception). The aim was to gain new information on several unanswered or controversial questions in human neurulation, including: (i) how the embryo transitions from primary to secondary neurulation; (ii) the mode of formation of the secondary neural tube; (iii) the rate of somite formation during low spinal development; (iv) the possible roles of WNT3A and FGF8 in regulating axial elongation; (v) whether a ‘burst’ of apoptosis coincides with termination of axial elongation. A further aim was to gather and present findings on human spinal neurulation that can serve as ‘normative data’ to aid interpretation of research involving multicellular ‘organoid’ structures, that are being increasingly used to model various aspects of human axial development (Denham et al., 2015; Fedorova et al., 2019; Moris et al., 2020; Rifes et al., 2020; Karzbrun et al., 2021; Libby et al., 2021; Amadei et al., 2022).

The study involved 108 human embryos (Table 1), obtained from the Medical Research Council (MRC)/Wellcome Human Developmental Biology Resource (https://www.hdbr.org/), with UK ethics committee approval. Embryos were donated by women undergoing termination of pregnancy for ‘social’ reasons, in most cases by mifepristone/misoprostol-induced (medical) delivery, with a few intact embryos obtained by ultrasound-guided vacuum aspiration (surgical). All embryos in the study were chromosomally and morphologically normal, and were assigned to CS, as described (O’Rahilly and Muller, 1987; Bullen and Wilson, 1997). Comparisons to mouse were with random-bred CD1 embryos, staged by embryonic (E) day, where E0.5 is the day following overnight mating.

Morphology of human PNP closure

Relatively few human embryos with an open posterior neuropore (PNP) have been reported in the literature (Müller and O’Rahilly, 1987; O’Rahilly and Müller, 2002), probably owing to the early stage at which primary neurulation is completed (end of week 4, post-conception). In two intact CS12 embryos (Figure 1A and B; crown-rump length: 3 mm; 22–23 somites), we identified an open PNP by microscopic inspection at collection (Figure 1C and D). Transverse histological sections confirmed an open neural tube in the caudal region, with minimal tissue damage evident, indicating that primary neurulation was not yet complete. The neural plate is relatively flat in the most caudally located sections, although incipient dorsolateral hinge points (DLHPs) are visible (Figure 1E and F). The notochord underlies the neural plate midline, and the caudal end of the hindgut is visible beneath the notochord in one embryo (Figure 1E), but not the other (Figure 1F). In more rostral sections, close to the ‘zippering’ point of PNP closure, elevated neural folds flank a marked ventral midline bend in the neural plate, the median hinge point (MHP), which precisely overlies the notochord (Figure 1G and H). DLHPs are also clearly present, unilaterally in one embryo (Figure 1G) and bilaterally in the other (Figure 1H). As in the mouse (McShane et al., 2015), the DLHPs are situated where the neural plate changes from basal contact with surface ectoderm to basal contact with paraxial mesoderm. We conclude that MHP and DLHPs characterise PNP closure in human embryos at CS12, marking a direct equivalence to Mode 2 spinal neurulation in the mouse embryo (Shum and Copp, 1996).

Figure 1

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Figure 1—source data 1

Measurements from photographic images of individual human embryos, as described in Materials and methods.

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Development and regression of the human embryonic tail

Overall caudal development was studied in 37 human embryos (CS13–18), which covered the period 28–45 days post-conception (Table 2; Figure 2A, B, I). Crown-rump length increased 2.5-fold during this period, from a mean value of 6.4 mm at CS13 to 15.4 mm at CS18 (Table 2; Figure 2J). Observations on the intact embryos showed that the PNP is closed in most embryos by CS13, and a developing tailbud is present which exhibits mild ventral curvature and a thick rounded tip (Figure 2C). Somites are visible proximal to the tailbud (arrowheads in Figure 2C), with an intervening region of presomitic mesoderm at CS13 (yellow bracket in Figure 2C). By CS16, however, the somites extend almost to the tail tip (yellow arrow in Figure 2F). As development progresses, striking changes occur in the tail which continues to lengthen (Table 2) but simultaneously narrows, particularly at the tip, to yield a sharply pointed structure by CS16 (Figure 2D–F). At the same time, the tail straightens and even becomes dorsally bent (Figure 2F). Subsequent to CS16, the tail shortens (Figure 2G; Table 2), and its distal portion becomes increasingly translucent in appearance. By CS18, only a short, curved stump remains (Figure 2H), and the tail is lost completely thereafter. Additional embryonic tails in the CS13–18 range are shown in Figure 2—figure supplement 1.

Figure 2 with 1 supplement see all

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Table 2

Carnegie stage (CS)	Age range (days post-fertilisation)	Number of embryos	Somite number †	Crown-rump length ‡	Tail length (total) ‡	Tail length distal to somites ‡
12	25–27	2	22, 22	3.0, 3.0	N/A	N/A
13	28–30	7	35.4±2.3	6.4±1.3	1.06±0.44	0.49±0.16
14	31–32	5	35.0±2.0	8.4±2.2	0.99±0.21	0.56±0.06
15	33–35	7	35.9±1.8	8.9±0.8	1.18±0.55	0.67±0.15
16	37–39	8	36.6±2.6	11.7±1.2	1.29±0.48	0.46±0.13
17	40–43	4	32.3±2.1	12.2±1.2	1.18±0.20	0.34±0.01
18	44–45	6	32.6±1.1	15.4±2.2	1.14±0.48	N/D

1. N/A: not applicable; N/D: not determined.

2. *

   Somite numbers: mean ± SD (except CS12, where actual somite numbers are shown). Somite number was available for all embryos except n=5 at CS18.

3. †

   Summary of embryos that underpin Figure 1A–H (CS12) and Figures 2 and 3 (CS13–18). For full data set, see Supplementary file 2.

4. ‡

   Lengths (mm): mean ± SD (except CS12, where actual lengths are shown). Length measuements were available only for a subset of embryos. See Supplementary file 2 for full details.

Mode of cell death during tail regression

In an initial study, transverse histological sections through human and mouse embryonic tails were processed for immuno-peroxidase staining using anti-activated caspase 3. Positive cells were readily identified in the tails of both species (Figure 3A–N), arguing for a role of caspase-dependent apoptosis during tail regression in human and mouse. Principal sites of apoptotic cell death include the regressing tailgut (Figure 3D and F) and, most abundantly, the ventral mesoderm overlying the epithelial ventral ectodermal ridge (Figure 3C and E). We also detected terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells in both mouse and human tail sections (data not shown), further confirming the presence of apoptotic cells during tail development/regression.

Figure 3

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Evidence for regression of the tailgut from rostral to caudal

While the human embryonic tail appears to regress from caudal to rostral (Figure 2), the proximal (rostral) part of the tailgut is reported to degenerate before the more distal (caudal) part in both rat (Butcher, 1929; Qi et al., 2000) and mouse (Nievelstein et al., 1993). To examine this question in human embryos, we performed immunofluorescence for anti-activated caspase 3, which confirmed the presence of apoptosis in both tailgut and ventral mesoderm (Figure 4A’ and B’). Using DAPI (4′,6-diamidino-2-phenylindole)-stained sections along the secondary body axis (Figure 4A and B), we determined the cross-sectional area of neural tube, notochord, and tailgut in two CS14 and one CS15 embryos. Total tail area served as a measure of axial position. The neural tube showed a progressive increase in area towards the proximal (rostral) end of the tail, while the notochord showed no change in area along the body axis (Figure 4C). Strikingly, the tailgut showed the reverse trend, with a marked caudal-to-rostral reduction in cross-sectional area (Figure 4A, B, and D). Tailgut nuclear number also diminished from caudal to rostral (Figure 4E). Hence, although the rostral tailgut has not disappeared by CS15, it appears to be diminishing in size proximally, at the same time as it is being formed, as a prominent tail structure, caudally. We conclude that rostral-to-caudal loss of the tailgut may be a general phenomenon among mammalian embryos.

Figure 4

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Figure 4—source data 1

Quantification of total tail, neural tube (NT), notochord, and tailgut areas (in square microns), and tailgut nuclear counts in three human embryos at CS14 (x2) and CS15.

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Expression of FGF8 and WNT3A during human tailbud elongation

To begin an assessment of the mechanisms that may regulate elongation of the human embryonic tail, and its cessation, we performed whole-mount in situ hybridisation for FGF8 and WNT3A (n=2 embryos minimum for each gene at each stage). These genes are developmentally regulated during axial elongation in chick and mouse embryos, with strong expression in the tailbud during elongation, and down-regulation before axial growth ceases. Direct inactivation or indirect down-regulation of the genes leads to premature axial truncation (Wilson et al., 2009).

In accordance with these findings, we observed strong expression of FGF8 in the tailbud at CS12 and CS13, as revealed in whole embryos (Figure 5A and B) and longitudinal sections through hybridised caudal regions (Figure 5E and F). At CS14, FGF8 expression reduced dramatically so that only a small ‘dot’ of expression was detected in the tailbud (Figure 5C and G), and by CS15 expression of FGF8 was no longer detectable in the tail (Figure 5D and H). Expression of WNT3A followed a similar pattern with strong expression in the tailbud at CS12 (Figure 5I and M), reduced expression intensity at CS13 (Figure 5J and N), a remaining ‘dot’ of tailbud expression at CS14 (Figure 5K and O), and no detectable WNT3A expression in the tail at CS15 (Figure 5L and P). We conclude that expression of FGF8 and WNT3A mirrors the relationship seen in mouse and chick, with strong tailbud expression during active axial extension, and dramatic down-regulation of both genes before the onset of axial growth cessation. It is striking that down-regulation appears complete by CS15, even though the embryonic tail does not reach its maximum length until some days later, at CS16 (Table 2). Down-regulation of Fgf8 and Wnt3a, well in advance of cessation of axial elongation, has also been observed in mouse embryos (Cambray and Wilson, 2007).

Figure 5

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Mode of secondary neural tube formation in human embryos

In our initial study of tail morphology, 9 out of 15 human embryonic tails showed multiple lumens in some transverse sections (Figure 3G and H), whereas the other 6 exhibited only a single neural tube lumen. In a second group of serially sectioned tails (Figures 4 and 6), 6 out of 10 tails had regions of duplicated neural tube. Hence, we find a 60% (15/25) frequency of neural tube duplication, confirming previous findings of multiple neural tube lumens in many human embryonic tails (Bolli, 1966; Lemire, 1969; Saitsu et al., 2004). We most often identified two neural tube profiles in a single transverse section, but in some cases more were observed (e.g. at CS15; Figure 3G).

Figure 6

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These initial findings of neural tube duplication prompted a more detailed examination of the suggestion that multiple neural tube lumens represent a mode of human secondary neurulation similar to that seen in the avian embryo (Lemire, 1969; Saitsu et al., 2004; Pang, 2020). In chick, multiple lumens form distally in the tailbud, and coalesce more rostrally to form the secondary neural tube (Criley, 1969; Schoenwolf and Delongo, 1980; Yang et al., 2003; Gonzalez-Gobartt et al., 2021). An alternative view would be that multiple lumens arise only later in human secondary body formation (Catala, 2021), perhaps representing splitting of the already formed secondary neural tube (Figure 6A).

To help resolve this issue, we asked two questions: (i) what is the status of the secondary neural tube where it is first formed, close to the tailbud tip? (ii) at what level of the tail axis is neural tube duplication most often seen? In our initial series of human tails, a single lumen was invariably noted in the distal-most portion of the neural tube, just rostral to the tailbud tip (CS13–18; Figure 3L–N). This was confirmed in the second series of 10 serially sectioned tails (CS14–15; Figure 6B, C, and D), thus demonstrating that secondary neurulation in human produces a neural tube with a single lumen. When multiple secondary neural tube lumens were visible in the embryonic tail, this was invariably located at more rostral levels of the tail (Figure 3G and H; Figure 4C; Figure 6B’, C’’, and D’’), with variation in location between embryos. We conclude that multiple secondary neural tube lumens in human embryonic tails likely represent splitting, which tends to occur rostrally and does not represent a secondary neurulation mechanism involving coalescence of multiple lumens, as in chick. Humans therefore resemble other mammals, including rat and mouse, in initially forming a single secondary neural tube lumen in the tailbud.

The development and later disappearance of the human tail has been of interest to embryologists for more than a century (Catala, 2021). To better appreciate the knowledge base for human embryonic caudal development, we conducted a systematic literature review using several search terms (see Materials and methods). This generated a long list of publications that was filtered to include only those with primary data relating to caudal development in human embryos. The final list comprises 28 papers (Supplementary file 1) that span over 100 years of research, from the early 1900s (Kunitomo, 1918; Streeter, 1919) to recent times (Xu et al., 2023). This work was based on at least 925 human embryos obtained from varying sources (Supplementary file 1) including induced termination of pregnancy for ‘social’ reasons (where most embryos are expected to be normal), as well as spontaneous abortion (miscarriage) and ectopic pregnancy, where embryonic abnormalities are likely to be frequent. Hence, interpretation of embryo morphology in these studies needs to take into account the mode of procurement of the human specimens.

Several aspects of caudal development are addressed by the studies in Supplementary file 1. These include: completion of primary neurulation with PNP closure, the transition into secondary neurulation, the mode of formation and regression of the secondary neural tube, the observation of multiple neural tube lumens, the formation and regression of somites, notochord and gut in the tail region, the role of programmed cell death in tail regression, and initial studies of gene expression in human embryos. Taken together with the findings of the present study, this accumulated literature provides a strong morphological evidence base for in vivo human caudal development, against which in vitro studies can be judged in the emerging field of stem cell-derived organoid differentiation. The latter is producing multicellular structures that may ultimately provide experimentally tractable models for some aspects of human axial development (Denham et al., 2015; Fedorova et al., 2019; Moris et al., 2020; Rifes et al., 2020; Karzbrun et al., 2021; Libby et al., 2021; Amadei et al., 2022).

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Chloe Santos

   Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Contributed equally with

   Abigail R Marshall and Ailish Murray

   Competing interests

   No competing interests declared

2. Abigail R Marshall

   Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Contributed equally with

   Chloe Santos and Ailish Murray

   Competing interests

   No competing interests declared

3. Ailish Murray

   Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

   Present address

   Moorfields Eye Charity, London, United Kingdom

   Contribution

   Investigation, Writing – review and editing

   Contributed equally with

   Chloe Santos and Abigail R Marshall

   Competing interests

   No competing interests declared

4. Kate Metcalfe

   Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Priyanka Narayan

   Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

6. Sandra CP de Castro

   Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Eirini Maniou

   Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

   Present address

   Department of Industrial Engineering, University of Padua and Veneto Institute of Molecular Medicine, Padua, Italy

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

8. Nicholas DE Greene

   Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

   Contribution

   Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4170-5248

9. Gabriel L Galea

   Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2515-1342

10. Andrew J Copp

    Developmental Biology & Cancer, UCL Great Ormond Street Institute of Child Health, London, United Kingdom

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing – review and editing

    For correspondence

    a.copp@ucl.ac.uk

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2544-9117

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