Primordial germ cells do not migrate along nerve fibres in marmoset monkey and mouse embryos

in Reproduction
Correspondence should be addressed to R Behr; Email: rbehr@dpz.eu
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Primordial germ cells (PGCs) are the embryonic precursors of spermatozoa and eggs. In mammals, PGCs arise early in embryonic development and migrate from their tissue of specification over a significant distance to reach their destinations, the genital ridges. However, the exact mechanism of translocation is still debated. A study on human embryos demonstrated a very close spatial association between migrating PGCs and developing peripheral nerves. Thus, it was proposed that peripheral nerves act as guiding structures for migrating PGCs. The goal of the present study is to test whether the association between nerves and PGCs may be a human-specific finding or whether this represents a general strategy to guide PGCs in mammals. Therefore, we investigated embryos of different developmental stages from the mouse and a non-human primate, the marmoset monkey (Callithrix jacchus), covering the phase from PGC emergence to their arrival in the gonadal ridge. Embryo sections were immunohistochemically co-stained for tubulin beta-3 chain (TUBB3) to visualise neurons and Octamer-binding protein 4 (OCT4 (POU5F1)) as marker for PGCs. The distance between PGCs and the nearest detectable neuron was measured. We discovered that in all embryos analysed of both species, the majority of PGCs (>94%) was found at a minimum distance of 50 µm to the closest neuron and, more importantly, that the PGCs had reached the gonads before any TUBB3 signal could be detected in the vicinity of the gonads. In conclusion, our data indicate that PGC migration along peripheral nerves is not a general mechanism in mammals.

 

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    PGC locations in the common marmoset monkey (Callithrix jacchus) embryo. Immunohistochemical double staining of OCT4A (brown) and TUBB3 (pink) (A, B, C and D). (A) Representative sagittal overview section of a GD 65 embryo. (B) Higher magnification of area of prospective gonad in (A). (C) Sagittal section of a GD 65 embryo (different than shown in A). Black arrows highlight migrating PGCs in the gut mesenchyme. (D) Developing gonad in a GD 90 embryo containing post-migratory PGCs. Bv, brain vesicle; G(l), gut (lumen); H, heart; M, mesenchyme; Nl, neural lumen; Nt, neural tube; Oe, oesophagus; Pc, plexus choroideus; U, umbilical cord. The asterisks mark the developing genital ridge. Scale bar ≙ 3 mm (A), 300 µm (B, C and D). (E) Graphical representation of tissue locations of PGCs in all investigated slides, separately analysed for each gestational day, n = number of cells available for analysis.

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    PGC distance to closest neuron in the common marmoset monkey (Callithrix jacchus) embryo. (A) Sagittal section of a GD 72 marmoset monkey embryo. Immunohistochemical double staining of OCT4A (brown) and TUBB3 (pink). Inlay shows an exemplary distance measurement of a migratory PGC to the closest neuron. Green arrow highlights a PGC in direct contact with a neuron. Scale bar ≙ 200 µm. (B) Graphical representation of the distance between the observed PGCs and the respectively closest detectable neuron, separately analysed for each gestational day, n = number of cells available for analysis.

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    PGC tissue locations in the mouse embryo. (A, B, C and D) Sections of different embryonic stages immunohistochemically stained for OCT4. (A) Representative transversal section of an E 8.5 embryo. (B) Representative sagittal section of an E 9.5 embryo. (C) Representative transversal section of an E 10.5 embryo. (D) Representative transversal section of an E 11.5 embryo. A, dorsal aorta; Gl, gut lumen; M, mesenchyme; Mes, mesentery; Nt, neural tube. The asterisks mark the developing genital ridge. PGCs are highlighted and colour-coded according to the tissue they were detected in. Scale bar ≙ 90 µm (B), 200 µm (A, C and D). (E) Graphical representation of tissue locations of PGCs in all investigated slides, separately analysed for each embryonic day, n = number of cells available for analysis.

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    PGC distance to closest neuron in the mouse embryo. (A and B) Sagittal and transversal section of E11.5 mouse embryos immunohistochemically double stained for OCT4 (brown) and TUBB3 (pink). Exemplary distance measurements between representative PGCs and the respectively closest detectable neuron. Scale bar ≙ 80 µm (A), 60 µm (B). Asterisk marks the prospective gonad. PGCs are highlighted and colour-coded according to the tissue they were detected in. (C) Graphical representation of the distance between the observed PGCs and the respectively closest detectable neuron, separately analysed for each embryonic day, n = number of cells available for analysis.

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    Neuronal development observed in the mouse embryo. (A) Representative sagittal overview section of an E 10.5 embryo immunohistochemically double stained for OCT4 (brown) and TUBB3 (pink). Scale bar ≙ 2 mm. (B and C) Higher magnification of the area highlighted in (A), immunohistochemically stained for OCT4 (B) or TUBB3 (C). Scale bar ≙ 300 µm. (D, E, F and G) Sections of different embryonic stages immunohistochemically stained for TUBB3. Scale bar ≙ 200 µm. (D) Representative transversal section of an E 8.5 embryo. (E) Representative transversal section of an E 9.5 embryo. (F) Representative transversal section of an E 10.5 embryo. (G) Representative transversal section of an E 11.5 embryo. A, dorsal aorta; Bv, brain vesicle; Gl, gut lumen; H, heart; L, liver; Nl, neural lumen; Nt, neural tube. The asterisks mark the developing genital ridge. Pink circles highlight appearing neurons. (H) Table shows the observed TUBB3 signal intensity in different tissues at different developmental stages in the mouse embryo.

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    Innervation of the common marmoset monkey (Callithrix jacchus) testis. Immunohistochemical staining for OCT4A (brown, A) or TUBB3 (pink, B, C, D, E and F). (A and B) Transversal section of testis and epididymis of a GD 100 marmoset foetus. (C) Transversal section of a GD 117 marmoset foetus. (D) Higher magnification of a GD 117 marmoset foetal testis. Erythrocytes present in the blood vessels unspecifically bind the antibody thereby producing a false-positive signal. (E) Section of a marmoset monkey neonatal testis. (F) Section of a marmoset monkey juvenile testis. Epi, epididymis; Gt, gut; T, testis. The asterisks exemplarily mark seminiferous tubules. Pink arrows highlight neurons. Scale bar ≙ 50 µm.

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