Number of human embryos in the study, with breakdown by sex and method of pregnancy termination *

Morphology and timing of human posterior neuropore (PNP) closure.

(A, B) Two CS12 embryos viewed from right side, each with 22-23 somites (s), and a looped heart (h). Neural tube closure is complete along most of the body axis, including the forebrain (fb), whereas the PNP remains open caudally. (C, D) Magnified oblique views from upper right side of the caudal region; the open PNP is outlined with dashed lines. (E-H) H&E-stained transverse sections, through the PNP, with section planes as indicated by dashed lines in A, B. The most caudally located sections (E, F) show a relatively flat neural plate (np), although incipient dorsolateral hinge points (DLHPs; arrows) are visible. Note the midline notochord (no) underlying the neural plate, and hindgut (hg) beneath the notochord (in E only). More rostral sections (G, H) show elevated neural folds with DLHPs clearly visible (arrows: unilateral in G, bilateral in H), located where basal contact of the neural plate changes from surface ectoderm (se), to paraxial mesoderm (pm). A median hinge point (MHP; asterisks in G, H) overlies the notochord. (I) PNP length (double headed arrow in C), normalised to somite (s) length (bracketed in C), determined from photographic images of 40 human embryos (24 females; 16 males) at CS10 (n = 4), CS11 (n = 8), CS12 (n = 16) and CS13 (n = 12). Symbol colours indicate the Carnegie Stages assigned at the time of collection. The PNP shows gradual closure, with completion around the 30 somite stage. (J) Somite number of the 40 embryos in I, plotted against days post-conception for each Carnegie Stage. The linear regression equation is shown, with R2 = 0.82, and p < 0.001. Scale bars: 1 mm in A, B; 0.4 mm in C, D; 0.1 mm in E-H.

Development of the tail in human embryos.

(A, B) Whole embryos at CS13 (A) and CS18 (B), showing the range of stages studied (4-6.5 weeks post-conception). The tail bud (arrow) is well formed at CS13 following completion of PNP closure at CS12, whereas, by CS18, tail development and regression are largely complete and only a small tail remnant remains (arrow). (C-H) Higher magnification views of the caudal region at CS13 to CS18. At CS13, the tail bud is relatively massive, tapering gradually and with a rounded end (arrow in C). Somites are visible rostral to the tail bud (arrowheads) with an intervening region of presomitic mesoderm (yellow bracket). At CS14 and CS15 the tail narrows progressively, with distal tapering (arrows in D-E). By CS16, this has yielded a slender structure with narrow pointed end (white arrow in F) in which somites extend almost to the tail tip (yellow arrow in F). Thereafter, the tail shortens progressively (arrows in G, H), develops a marked flexion (asterisk in H), and becomes increasingly transparent (G, H). (I-K) Analysis of embryos in the range CS13-CS16 (Table 2), plotting CS against: (I) days post-conception (p.c.), (J) somite no. and (K) crown-rump (C-R) length in mm. One-way ANOVA on ranks shows all three parameters vary significantly with CS (p-values on graphs). Somite no. reduces significantly between CS16 and CS17/18 (* p < 0.05). Abbreviations: ba, branchial arches; fb, forebrain; fl, forelimb; h, heart; hl, hindlimb; s, somites. Scale bars: 1 mm in A, B; 0.5 mm in C-H.

Measurements of human embryos, CS12-18 *

Programmed cell death during regression of tail structures in mouse and human embryos.

Transverse sections through the tail region of mouse (A-E) and human (F-J) embryos at the stages indicated on the panels. Staining is by TUNEL method (A, D, E) or using an antibody specific for activated caspase 3 (B, C, F-J), with counterstaining by methyl green. Particularly intense programmed cell death (PCD) is observed in the ventral midline mesoderm overlying the ventral ectodermal ridge (ver) of both species (arrows in C, H). PCD can also be detected in the tail-gut (g) of both mouse and human embryos (arrows in A, B, G, I). Significantly, the tail-gut can be clearly identified in a caudal section through an E12.5 mouse tail (arrow in E), whereas it is absent from a more rostral section of the same tail (D). A blood vessel (bv) can be seen where the gut was previously situated in this section. Similarly, in a CS13 human embryo, the tail-gut is absent from a rostral section (F) but present more caudally in the same embryonic tail, with dying cells visible in the distal tail-gut remnant (arrowhead in G). Hence, tail-gut regression proceeds in a rostral to caudal direction in both species. Other abbreviations: no, notochord; nt, neural tube. Scale bar in A represents: 50 µm (A,H,I), 70 µm (B,C,F,G) and 80 µm (D,E,J).

Stage-dependent cell death and multiple neural tube lumens in mouse and human tails.

Transverse sections through the tails of mouse (A-C) and human (D-I) embryos stained with antibodies to activated caspase 3 antibody (A-H; Casp) or phospho-histone H3 (I; PH3). Developmental stages are indicated on the panels. (A-C) In mouse embryos, the tail bud displays a stage-dependent burst of PCD at E13.5 (arrows in B), with absence of caspase 3-positive cells 12 hours earlier, at E13.0 (A), and only occasional dying cells 12 hours later, at E14.0 (arrow in C). Note the absence of a neural tube in the mouse tail bud tip, and the sparse nature of the tail bud mesenchyme at E14.0. (D-F) Human embryonic tail buds show a similar developmental sequence to the mouse, with absence of PCD at CS13 (D), abundant dying cells at CS15 (arrows in E) and cessation of PCD by CS18 (F). Unlike the mouse, the secondary neural tube extends to the tail bud tip (nt in D-F), with this terminal neural tube portion having a single lumen in all three embryos. (G-I). Multiple neural tube profiles were observed in 9/15 human tails, with variability in the number of lumens: four lumens are visible in one CS15 embryo (arrowheads in G) but only two in sections of a second embryo of the same stage (H,I). Adjacent sections through this second human embryonic tail demonstrate both PCD (arrows in H) and mitotic activity (arrow in I) occurring simultaneously in the secondary neural tube, whereas the mesenchyme overlying the ventral ectodermal ridge (ver) exhibits plentiful PCD (red arrow in H), but no mitotic activity (I). Scale bar in A represents: 50 µm in A-C, F-I and 30 µm in D,E.

FGF8 and WNT3A expression in the elongating caudal region of human embryos.

Whole mount in situ hybridisation for FGF8 (A-D) and WNT3A (E-H) in embryos at CS12 (A, E), CS13 (B, F), CS14 (C, G) and CS15 (D, H). Both genes show prominent expression domains in the tail bud at CS12 when axial elongation is underway and the posterior neuropore (PNP) is closing (arrows in A, E). At CS13, following PNP closure, expression of FGF8 and WNT3A remains prominent although less intense and more localised to the terminal tail bud than at CS12 (arrows in B, F). By CS14, both genes exhibit much smaller, high localised expression domains that each appears as a ‘dot’ within the tail bud region (arrows in C, G). By CS15, axial elongation has ceased, the tail tip has narrowed and is increasingly transparent. At this stage, expression of neither gene can be detected (asterisks in D, H). Note that whole embryos are shown in B, F, G while isolated trunk/caudal regions are shown in A, C, D, E, H. No. embryos analysed: FGF8, n = 2 for each stage; WNT3A, n = 2 for each stage except n = 3 for CS13. Scale bars: 500 μm.

FGF8 and WNT3A expression in sections through the elongating caudal region of human embryos.

Vibratome sections with sagittal orientation of the whole mount in situ hybridisation embryos shown in Fig 5. Expression of FGF8 (A-D) and WNT3A (E-H) is shown in embryos at CS12 (A, E), CS13 (B, F), CS14 (C, G) and CS15 (D, H). Tail bud region is outlined by dotted lines. Extensive expression domains are visible for both FGF8 and WNT3A at CS12 (A, E) whereas at CS13 (B, F) both genes are less intensely expressed, with transcripts for WNT3A particularly reduced compared with CS12. At CS14, only a small ‘dot’ of remaining expression is visible close to the tail bud tip for each gene. At CS15, no FGF8 or WNT3A expression is detectable in the caudal region. No. embryos analysed: FGF8, n = 2 for each stage; WNT3A, n = 2 for each stage except n = 3 for CS13. Scale bars: 100 μm.

Mode of formation of the human secondary neural tube.

(A,B) Two different hypotheses on the mode of human secondary neural tube (nt) formation. As in chick, it has been suggested that multiple lumens form initially in the tail bud (tb), and then coalesce to form the secondary neural tube (A). Alternatively, the finding of multiple neural tube lumens in sections of some human tails may reflect splitting of the secondary neural tube at more rostral levels (B). (C-K) Representative serial transverse sections (haematoxylin and eosin) through three different human embryonic tails at CS17 (C-E, F-H, I-K). Sections close to the tail bud tip (left side: C,F,I) show a broad, dorsoventrally flattened neural tube with a single lumen. There is no evidence of multiple lumens coalescing at such caudal levels in any embryos. Further rostrally (middle: D,G,J), sections show a neural tube with round profile and single lumen. Sections further rostrally in two embryos (right side: E,H) show evidence of secondary neural tube splitting, with two lumens (green arrows in E; nt1, nt2) and three lumens (green arrows in H; nt1, nt2, nt3). The third embryo shows a folded neural tube with ventral midline extension (yellow arrow in K) but only one lumen. Asterisk in K: dorsal gap in neural tube is a post-mortem artefact (also in J). Red arrows: secondary notochord (not). Scale bars: 50 µm.

Publications on human secondary neural tube and body formation, showing topics covered