FIGURE SUMMARY
Title

A conserved axon type hierarchy governing peripheral nerve assembly

Authors
Wang, L., Mongera, A., Bonanomi, D., Cyganek, L., Pfaff, S.L., Nüsslein-Volhard, C., Marquardt, T.
Source
Full text @ Development

Conserved reliance of sensory afferent axon (SA) extension on pioneer motor efferent axons (MEs). (A) Peripheral axons visualized in 72hpf zebrafish larva. (B-E) Time-lapse sequence of SA, labeled using -8.4neurog1::GFP transgene (yellow), extending peripherally along preformed CaP ME, labeled by NBT::dsRed transgene (magenta). Arrowheads indicate SA growth cone. (F-H) Delayed and aberrant SA extension in the absence of MEs upon islet1E2/E3 morpholino injection. (I) Longitudinal analysis of SA and ME extension and impacts of ME removal (ne7 segments, e3 embryos per stage and condition). (J) Peripheral axons visualized in E4 chick embryo. (K-N) SAs, labeled using the Isl1DRG::mGFP transgene (yellow), extending along preformed MEs, labeled by co-transfected Hb9MN::mCherry. Arrowheads indicate distalmost SA growth cone. Asterisks indicate additional Isl1DRG::mGFP activity in non-DRG neural crest cells. Dotted line indicates trunk/limb boundary. (O-Q) Delayed and aberrant SA extension (visualized using anti-Tuj1 antibody) in the absence of MEs upon Hb9MN::Cre/PGKneolox2DTA co-transfection. (R) Longitudinal analysis of relative SA and ME extension, and impact of ME removal (ne7 sections, e3 embryos per stage and condition). (S) Peripheral axons visualized in E10.5 mouse embryo. (T-W) SAs, labeled using the Brn3atau:lacZ allele, extending along preformed MEs, labeled using the Hb9MN::GFP transgene. (X-Z) Delayed and aberrant SA extension in the absence of MEs in Olig2Cre;Rosa26lxstopDTA embryos. (AA) Longitudinal analysis of relative SA and ME extension, and impact of ME removal (ne8 sections, e4 embryos per stage and condition). hpf, hours post-fertilization; DRG, dorsal root ganglion; MN, motor neurons. Error bars indicate standard error of the mean (s.e.m.). Scale bars: 10 µm in E,H for B-H; 50 µm in K-Q,T-Z.

Effects of partial ME ablation on SA extension. (A) Dorsal whole-mount view of lumbar spinal cord and limbs in E6 chick embryo: peripheral axons visualized by anti-Tuj1 immunodetection (black). Severe reduction, but not loss, of crural (asterisk), peroneal (PN) and tibial nerves (TN) (arrowheads) upon unilateral transfection with a low titer (0.5µg/ml) of Hb9MN::Cre/PGKneolox2DTA plasmids. LFC, lateral femoral cutaneous nerve. (B,C) Transverse section of E6 chick spinal cord: partial ablation of motor neurons (MNs) after unilateral low-titer transfection. Anti-Isl1/2 immunofluorescence (gray) to label DRG neurons and MNs. Anti-TrkA immunofluorescence (yellow) to label DRG neurons. (D) Dorsal whole-mount view of SAs (yellow indicates Brn3atlz) at the sciatic plexus (SP) in E12.5 mouse embryo. DPN, deep peroneal nerve; PN, peroneal nerve; TN, tibial nerve. (E) Reduction, but not loss, of SAs beyond the sciatic plexus after delayed ablation of MEs in Olig2Cre;Isl2lxstopDTA embryo. (F) Transverse section of E12.5 control spinal cord: MNs labeled using Hb9MN::GFP (magenta). Anti-Isl1/2 immunofluorescence (gray) visualizes nuclei of DRG neurons and MNs. (G) Transverse section of E12.5 Olig2Cre;Isl2lxstopDTA spinal cord: severe reduction, but not complete absence, of MNs. Scale bars: 300μm in A; 100μm in B; 200μm in D,F.

SAs influence ME fasciculation but not trajectory or target choice. (A,B) Dorsal whole-mount view: MEs extending into hindlimbs in control (A) and in the absence of SAs in Wnt1Cre;Rosa26lxstopDTA mouse embryos (B). CP, crural plexus; DPN, deep peroneal nerve; PN, peroneal nerve; SP, sciatic plexus; TN, tibial nerve. (C-F) Transverse section of E12.5 thoracic (C,D) and lumbar (E,F) motor columns (magenta indicates Hb9MN::GFP): retrograde DiI tracing does not detect aberrant ME targeting at the level of columnar divisions. (C) Retrograde DiI tracing from epaxial muscle labels medial division of medial motor column (MMC). (D) Retrograde DiI tracing from hypaxial muscle labels lateral MMC. (E) Retrograde DiI tracing from ventral hindlimb labels Hb9::eGFPlow medial division of lateral motor column (LMC). (F) Retrograde DiI tracing from dorsal hindlimb labels Hb9MN::GFPhigh lateral LMC (asterisk indicates a possible Hb9MN::GFPhigh LMCl neuron in the process of lateral migration). Scale bars: 300µm in A,B; 50µm in C-F.

Conserved late extension of sympathetic efferent axons (SEs). (A) Transverse section of 32dpf zebrafish: SEs [blue, anti-tyrosine hydroxylase (TH) immunofluorescence] extending from sympathetic chain ganglion (SCG) along preformed peripheral nerves (red, SA/ME: anti-Tuj1 immunofluorescence). (B,C) TH+ SCG neurons prior to initiation SE axon extension at 18dpf. (D,E) Higher magnifications of A: TH+ SEs (arrowheads) extending along preformed peripheral axons. (F) Transverse sections of E7 chick embryo at trunk levels: TH+ SCG neurons (blue) around initiation SE extension relative to preformed PNs (red). (G,H) Magnified view of SCG neurons relative to preformed peripheral nerves. (I,J) TH+ SE axons begin extending from SCG along PNs at E8 (arrowheads). (K,L) SE axon advancing further peripherally along peripheral nerves at E9 (arrowheads). (M) Transverse section of E12.5 mouse embryo: TH+ SEs (arrowhead) beginning to extend from SCGs along pre-extending MEs and SAs in the ramus communicans (RC). (N,O) SCG and rc just prior to initiation of SE extension (arrowhead). (P,Q) Detailed view of SEs (arrowheads) extending along SAs and MEs of RC at E12.5. nc, notochord; DR/VR, dorsal/ventral ramus; vi, intersegmental blood vessel; DNR/VNR, dorsal/ventral nerve roots; RC, ramus communicans. Scale bars: 20µm in A,C,E; 100µm in F,H,J,L,M; 50µm in O,Q.

SEs extend along MEs and SAs to innervate dermis. (A-C) Transverse section of E14.5 mouse embryo at trunk levels: the majority of trunk-innervating SEs (arrowheads) extend along pre-extending axons (magenta indicates Hb9::eGFP) (A,C), rather than intersegmental blood vessels (vi) (gray indicates anti-Pecam1 immunofluorescence) (B,C). Subsets of SEs directly extend medially (m) and dorsally (d) along vasculature (B,C). (D-F) Dorsal whole-mount view of E18.5 mouse: dorsal cutaneous nerve (dCN) axons fanning out in trunk dermis and longitudinal projections by MEs into subdermal cutaneous maximus (cm) muscle (D,F). (E) SE projections (blue indicates anti-TH immunofluorescence) through dCN into dermis. (F) ME (magenta indicates Hb9::eGFP) innervation of subdermal cm muscle. (G-I) Longitudinal section through dCN at E18.5: separately labeled SEs (blue) and SAs (yellow indicates anti-TrkA immunofluorescence) can be seen. (J) Three axon types in dorsal ramus (ventral ramus, visceral or vascular trajectories are not depicted for simplicity). Magenta dots indicate cross-sections of cm MEs. DR, dorsal ramus; vi, intersegmental blood vessel; RC, ramus communicans; VR, ventral ramus. Scale bars: 150µm in A; 300µm in D; 50µm in G.

SE trajectories are configured by pre-extending SAs and MEs. (A,B) Transverse section of E14.5 mouse embryo: SE (blue), SA (yellow) and ME (magenta) axons extending into dorsal (DR) and ventral (VR) nerve rami. (C) Whole-mount view of dorsal cutaneous nerve (dCN) axons fanning out into trunk dermis. (D) Visualization of SE axons only in same specimen. (E) Summary: three axon types in DR (VR, visceral or vascular trajectories are not depicted for simplicity). Magenta dots indicate cross-sectioned longitudinally projecting cutaneous maximus (cm) MEs. (F,G) Loss of dorsal (asterisk) and ventral misrouting of SA projections in the absence of MEs (OligCre;Rosa26fxstopDTA) (F) mirrored by SE (G) (asterisk). The converse dorsal misrouting of ventral SAs observed upon ME removal is not shown for simplicity. (H,I) Intermittent loss of dCNs (asterisks) and aberrant pattern of SA projections in the absence of MEs is mirrored by SEs (I). (J) Summary of F-I. (K-N) Initial peripheral extension of SEs along MEs in the absence of SAs (AdvCre;Isl2fxstopDTA) (note the higher degree of SE fasciculation, compared with control), but failure of SEs to innervate dermis (M,N) (remaining axons in M are subdermal cm MEs). (O) Summary of K-N. (P-Q) Normal appearance of ME, SA trajectories in the absence of SEs (DbhCre;Rosa26fxstopDTA). (R,S) Normal appearance of dorsal cutaneous nerves in the absence of SEs. (T) Summary of appearance of P-S. All images are representative of at least five embryos per condition. Scale bars: 100µm in B,G,L,Q; 300µm in D,I,N,S. sg, sympathetic ganglion.

Visualizing and manipulating peripheral MEs and SAs in zebrafish. (A) Lateral wholemount view of peripheral MEs (magenta: TgNBT:dsRed) and SAs (yellow: Tg- 8.4neurog1:GFP) in 72 hpf zebrafish larva. (B-E) Detailed view of trunk PN segments. (B) Monochromatic rendering of all axons. (C) Overlay of ME and SA signals. (D) Separate visualization of MEs. (E) Separate visualization of SAs. (F-H) Example: control SAs and MEs extending from dorsal root ganglion (DRG) neuron and neural tube, respectively. (I-K) Example: aberrant SA extension at segment lacking MEs upon motor neuron ablation by islet1E2/3-MO injection. (L-Q) Time-lapse sequence of highly aberrant SA extension in the absence of MEs. Scale bars: 300 µm in A; 50 µm in E; 20 µm in H,K,Q.

Visualizing and manipulating peripheral MEs and SAs in chick. (A) Dorsal wholemount view of MEs (magenta: Hb9MN::mCherry) in E5 chick embryo (-/+: untransfected/transfected hemitubes). (B) Overlay of MEs with pan-axon label (grey: anti- Tuj-1 immunofluorescence). (C) Dorsal wholemount view of SAs (yellow: Isl1DRG::mGFP) in E5 chick embryo. (D) Overlay of SAs with Tuj-1 immunofluorescence. (E) Transversal section of E6 chick neural tube: unilaterally Hb9MN::mCherry-labelled MEs extending from motor neurons (MNs). Overlay with anti-Isl1 immunofluorescence (grey) to label DRG neurons and MNs. (F) Transversal section of neural E6 chick tube: Isl1DRG::mGFP-labeled SAs extending from DRG neurons, in addition to a subset of non-DRG cells (presumably of neural crest origin) also labeled by the Isl1DRG enhancer (asterisk). (G,H) Transversal section of E5 chick spinal cord: severe reduction of MN numbers, including loss of ventral horn (asterisk), but not DRG neuron numbers, upon unilateral Hb9MN::Cre/PGKneolox2DTAmediated MN ablation (grey: cell nuclei labeled by DAPI). (I) Dorsal wholemount view of E7 chick embryo after unilateral Hb9MN::Cre/PGKneolox2DTA-mediated MN ablation. Innervation of dermis by dorsal cutaneous nerves (arrowheads) visualized by anti-Tuj-1 immunofluorescence (black): intermittent loss of dorsal cutaneous nerves in Hb9MN::Cre/PGKneolox2DTA-transfected (+), but not control (-) side. (J) Visualization of most DRG neurons and SAs by anti-TrkA immunofluorescence (yellow): loss of dorsal cutaneous SA projections (asterisk), but not DRGs (encircled by dotted lines). Scale bars: 300 µm in B,D,J; 50 µm in E,F,H.

Complete and selective ablation of motor neurons in mouse. (A-C) Lateral wholemount view of E10.5 mouse control embryo (upper thoracic to cranial levels): normal appearance of peripheral axon projections (A,C) (grey: anti-Tuj-1 immunofluorescence). Anti-Isl1/2 immunofluorescence (red) visualizes nuclei of DRG neurons (in ganglia lining the neural tube) and motor neurons (MNs) (arrowheads: motor column within ventral neural tube) (A,B). Abbreviations: SCG (sympathetic chain ganglion neurons), BP (brachial plexus). (DF) Lateral wholemount view of E10.5 mouse embryo upon MN ablation (Olig2Cre;Rosa26lxstopDTA): Complete absence of MNs and pure motor nerves (XII, asterisk) (D,E), but normal appearance of DRGs and sympathetic chain ganglia (scg) (D,F). (G,H) Transversal section of E10.5 control spinal cord (sc): extension of MEs from MNs (magenta: anti-vAChT immunofluorescence) (G) and presence of DRG neurons and MNs (red: anti- Isl1/2 immunofluorescence) (H). (I,J) Transversal section of E10.5 Olig2Cre;Rosa26lxstopDTA spinal cord: complete absence of MEs (I, J), but not DRG neurons (J). (K) Dorsal wholemount view of SAs (yellow: Brn3atlz) at sciatic plexus (SP) in E12.5 mouse. Abbreviations: CP (crural plexus), DPN (deep peroneal nerve), PN (peroneal nerve), TN (tibial nerve). (L) Near-total failure of SAs (arrowheads) to innervate limb beyond plexus in the absence of MEs in Olig2Cre;Rosa26lxstopDTA embryo (asterisk). Scale bars: 200 µm in C,F,G,H,I,J,K,L.

Visualizing ME and SA extension and selectively manipulating ME extension in mouse hindlimb. (A-H) Sequential extension of MEs (magenta) and SAs (yellow) beyond the sciatic plexus during development. Arrowheads: forefront SA growth cones extending along pre-extending MEs. Arrows (C-H): routing of MEs and SAs along dorsal and ventral nerve sheets. (I-Q) Dorsal wholemount view of MEs (I,L,O), SAs (J,M,P) or all axons (K, N,Q) in control (I-K) and Olig2Cre;Epha4fx/fx mouse embryos (L-Q) at E12.5. Dotted lines trace peroneal (PN) and solid lines tibial (TN) nerves. (J-P) Relative severity of ventral misrouting of MEs in Olig2Cre;Epha4fx/fx hindlimb (L,O) is faithfully mirrored by reduced/lost dorsal SA extension (M,P), also apparent by corresponding reduction/loss of pan-axon label (N,Q). Scale bars: 100 µm in B,D,F,H; 300 µm in K,N,Q.

Selective ablation of DRG neurons and SAs in mouse. (A-C) Lateral wholemount view of E10.5 mouse control embryo: normal appearance of peripheral SAs (yellow: Brn3atlz), MEs (magenta: Hb9MN::GFP) and pan-axon label (grey: anti-Tuj1 immunofluorescence). (D-F) Near-absence of DRG neurons (D,F), but not MNs or MEs (E,F) in Wnt1Cre;Isl2lxstopDTA embryo. Note: aberrant head structures, likely due to cranial neural crest defects. Abbreviations: br (brachial), th (thoracic), ls (lumbosacral).

Selective manipulation of peripherally projecting neuron types in mouse and relationship between PNs and vasculature. (A-D) Transversal section of E14.5 mouse embryos at trunk levels: anti-Isl1/2 immunofluorescence visualizes nuclei of motor neurons (MNs) in spinal cord, DRG and SCG neurons in control embryo (A), selective ablation of MNs in Olig2Cre;Rosa26lxstopDTA (B), selective ablation of DRG neurons in AdvCre;Isl2lxstopDTA (C), selective ablation of SCG neurons in DbhCre;Rosa26lxstopDTA (D). (E-G) Lateral wholemount view of E14.5 mouse trunk: spatial arrangement of intercostal PNs (IC, grey) (E,G) and intersegmental blood vessels (vi, red) (F,G). (H-J) Lateral wholemount view of E14.5 trunk in Olig2Cre;Rosa26lxstopDTA mouse embryo: intermittent absence of intercostal nerves (IC) (H,J) does not affect formation of intersegmental blood vessels (vi) (I,J). (K-L) Dorsal wholemeount view of AdvCre;Isl2lxstopDTA E18.5 mouse embryo: subdermal cutaneous maximus (cm) ME projections labeled by pan-axon marker anti-Tuj-1/βIII-tubulin (grey) and ME marker Hb9MN::eGFP. Scale bars: 150 µm in A-D; 300 µm in G,J,L.

Acknowledgments
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